Open access article in Acta Crystallographica Section F: Structural Biology and Crystallization Communications

Structure of the hypothetical DUF1811-family protein GK0453 from Geobacillus kaustophilus HTA426

Padmanabhan, B. — 2013-04-01

The crystal structure of a conserved hypothetical protein, GK0453, from Geobacillus kaustophilus has been determined to 2.2 Å resolution. The crystal belonged to space group P43212, with unit-cell parameters a = b = 75.69, c = 64.18 Å. The structure was determined by the molecular-replacement method and was refined to a final R factor of 22.6% (Rfree = 26.3%). Based on structural homology, the GK0453 protein possesses two independent binding sites and hence it may simultaneously interact with two proteins or with a protein and a nucleic acid.

Structure of Pseudomonas aeruginosa inosine 5′-monophosphate dehydrogenase

Rao, V.A. — 2013-03-01

Inosine 5′-monophosphate dehydrogenase (IMPDH) represents a potential antimicrobial drug target. The crystal structure of recombinant Pseudomonas aeruginosa IMPDH has been determined to a resolution of 2.25 Å. The structure is a homotetramer of subunits dominated by a (β/α)8-barrel fold, consistent with other known structures of IMPDH. Also in common with previous work, the cystathionine β-synthase domains, residues 92–204, are not present in the model owing to disorder. However, unlike the majority of available structures, clearly defined electron density exists for a loop that creates part of the active site. This loop, composed of residues 297–315, links α8 and β9 and carries the catalytic Cys304. P. aeruginosa IMPDH shares a high level of sequence identity with bacterial and protozoan homologues, with residues involved in binding substrate and the NAD+ cofactor being conserved. Specific differences that have been proven to contribute to selectivity against the human enzyme in a study of Cryptosporidium parvum IMPDH are also conserved, highlighting the potential value of IMPDH as a drug target.

Crystallization and preliminary structure determination of the transfer protein TraM from the Gram-positive conjugative plasmid pIP501

Goessweiner-Mohr, N. — 2013-02-01

The major means of horizontal gene spread (e.g. of antibiotic resistance) is conjugative plasmid transfer. It presents a serious threat especially for hospitalized and immuno-suppressed patients, as it can lead to the accelerated spread of bacteria with multiple antibiotic resistances. Detailed information about the process is available only for bacteria of Gram-negative (G−) origin and little is known about the corresponding mechanisms in Gram-positive (G+) bacteria. Here we present the purification, biophysical characterization, crystallization and preliminary structure determination of the TraM C-terminal domain (TraMΔ, comprising residues 190–322 of the full-length protein), a putative transfer protein from the G+ conjugative model plasmid pIP501. The crystals diffracted to 2.5 Å resolution and belonged to space group P1, with unit-cell parameters a = 39.21, b = 54.98, c = 93.47 Å, α = 89.91, β = 86.44, γ = 78.63° and six molecules per asymmetric unit. The preliminary structure was solved by selenomethionine single-wavelength anomalous diffraction.

Crystals on the cover 2013

Einspahr, H. — 2013-01-01

Some practical guidelines for UV imaging in the protein crystallization laboratory

Desbois, S. — 2013-02-01

High-throughput imaging of protein crystallization experiments with ultraviolet (UV) light has recently become commercially available and can enable crystallographers to differentiate between crystals of protein and those of salt, as the visualization of protein crystals is based on intrinsic tryptophan fluorescence. Unfortunately, UV imaging is not a panacea, as some protein crystals will not fluoresce under UV excitation and some salt crystals are UV-fluorescently active. As a new technology, there is little experience within the general community on how to use this technology effectively and what caveats to look out for. Here, an attempt is made to identify some of the common problems that may arise using UV-imaging technology by examining test proteins, common crystallization reagents and a range of proteins by assessing their UV–Vis absorbance spectra. Some pointers are offered as to which systems may not be appropriate for this methodology.

Expectation bias and information content

Dauter, Z. — 2013-02-01

The AEROPATH project targeting Pseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery

Moynie, L. — 2013-01-01

Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.

Crystallization of domains involved in self-assembly of the S-layer protein SbsC

Đordić, A. — 2012-12-01

The Gram-positive bacterium Geobacillus stearothermophilus ATCC 12980 is completely covered with a two-dimensional crystalline monolayer composed of the S-layer protein SbsC. In order to complete the structure of the full-length protein, additional soluble constructs containing the crucial domains for self-assembly have been successfully cloned, expressed and purified. Crystals obtained from three different recombinant constructs yielded diffraction to 3.4, 2.8 and 1.5 Å resolution. Native data have been collected.

Crystallization and first data collection of the putative transfer protein TraN from the Gram-positive conjugative plasmid pIP501

Goessweiner-Mohr, N. — 2012-11-01

Conjugative plasmid transfer is the most important route for the spread of resistance and virulence genes among bacteria. Consequently, bacteria carrying conjugative plasmids are a substantial threat to human health, especially hospitalized patients. Whilst detailed information about the process has been obtained for Gram-negative type-4 secretion systems, little is known about the corresponding mechanisms in Gram-positive (G+) bacteria. The successful purification and crystallization of the putative transfer protein TraN from the G+ conjugative model plasmid pIP501 of Enterococcus faecalis are presented. Native crystals diffracted to 1.8 Å resolution on a synchrotron beamline. The crystals belonged to space group P21, with unit-cell parameters a = 32.88, b = 54.94, c = 57.71 Å, β = 91.89° and two molecules per asymmetric unit.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of aspartyl aminopeptidase from the apeB gene of Pseudomonas aeruginosa. Retraction

Natarajan, S. — 2012-11-01

The article by Natarajan & Mathews [(2012) Acta Cryst. F68, 207–210] is retracted.

Solution structure of the cold-shock-like protein from Rickettsia rickettsii

Gerarden, K.P. — 2012-11-01

Rocky Mountain spotted fever is caused by Rickettsia rickettsii infection. R. rickettsii can be transmitted to mammals, including humans, through the bite of an infected hard-bodied tick of the family Ixodidae. Since the R. rickettsii genome contains only one cold-shock-like protein and given the essential nature of cold-shock proteins in other bacteria, the structure of the cold-shock-like protein from R. rickettsii was investigated. With the exception of a short α-helix found between β-strands 3 and 4, the solution structure of the R. rickettsii cold-shock-like protein has the typical Greek-key five-stranded β-barrel structure found in most cold-shock domains. Additionally, the R. rickettsii cold-shock-like protein, with a ΔG of unfolding of 18.4 kJ mol−1, has a similar stability when compared with other bacterial cold-shock proteins.

Recombinant portal protein from Staphylococcus epidermidis bacteriophage CNPH82 is a 13-subunit oligomer

Luan, W. — 2012-10-01

The portal protein cn3 of bacteriophage CNPH82 is predicted to serve as a gateway for translocation of viral genome into preformed pro-capsid, like portal proteins from other double-stranded DNA tailed bacteriophages. The host of bacteriophage CNPH82 is the opportunistic human pathogenic bacterium Staphylococcus epidermidis, a major cause of nosocomial infections. The portal protein of this phage has been cloned, overexpressed and purified. Size-exclusion chromatography–multi-angle laser light scattering analysis has indicated that the portal protein contains ∼13 subunits. Crystals of the portal protein, diffracting to 4.2 Å, have been obtained. These crystals belong to the space group C2221 with the unit-cell parameters of a = 252.4, b = 367.0, c = 175.5 Å. The self-rotation function revealed the presence of a single 13-subunit oligomer in the asymmetric unit.

Crystallization and preliminary X-ray crystallographic analysis of an ice-binding protein (FfIBP) from Flavobacterium frigoris PS1. Addendum

Do, H. — 2012-11-01

An additional acknowledgement is published for the article by Do et al. [(2012) Acta Cryst. F68, 806–809].

Structural insights into ChpT, an essential dimeric histidine phosphotransferase regulating the cell cycle in Caulobacter crescentus

Fioravanti, A. — 2012-09-01

Two-component and phosphorelay signal-transduction proteins are crucial for bacterial cell-cycle regulation in Caulobacter crescentus. ChpT is an essential histidine phosphotransferase that controls the activity of the master cell-cycle regulator CtrA by phosphorylation. Here, the 2.2 Å resolution crystal structure of ChpT is reported. ChpT is a homodimer and adopts the domain architecture of the intracellular part of class I histidine kinases. Each subunit consists of two distinct domains: an N-terminal helical hairpin domain and a C-terminal α/β domain. The two N-terminal domains are adjacent within the dimer, forming a four-helix bundle. The ChpT C-terminal domain adopts an atypical Bergerat ATP-binding fold.

Crystallization and preliminary X-ray diffraction analysis of the high molecular weight ketoacyl reductase FabG4 complexed with NADH. Corrigendum

Dutta, D. — 2012-11-01

In the article by Dutta et al. [(2012) Acta Cryst. F68, 786–789] two citations were given erroneously. These are now corrected.

Anomalous dispersion analysis of inhibitor flexibility: a case study of the kinase inhibitor H-89

Pflug, A. — 2012-08-01

With its ability to show the interactions between drug-target proteins and small-molecule ligands, X-ray crystallography is an essential tool in drug-discovery programmes. However, its usefulness can be limited by crystallization artifacts or by the data resolution, and in particular when assumptions of unimodal binding (and isotropic motion) do not apply. Discrepancies between the modelled crystal structure and the physiological range of structures generally prevent quantitative estimation of binding energies. Improved crystal structure resolution will often not aid energy estimation because the conditions which provide the highest rigidity and resolution are not likely to reflect physiological conditions. Instead, strategies must be employed to measure and model flexibility and multiple binding modes to supplement crystallographic information. One useful tool is the use of anomalous dispersion for small molecules that contain suitable atoms. Here, an analysis of the binding of the kinase inhibitor H-89 to protein kinase A (PKA) is presented. H-89 contains a bromobenzene moiety that apparently binds with multiple conformations in the kinase ATP pocket. Using anomalous dispersion methods, it was possible to resolve these conformations into two distinct binding geometries.

Useable diffraction data from a multiple microdomain-containing crystal of Ascaris suum As-p18 fatty-acid-binding protein using a microfocus beamline

Gabrielsen, M. — 2012-08-01

As-p18 is a fatty-acid-binding protein from the parasitic nematode Ascaris suum. Although it exhibits sequence similarity to mammalian intracellular fatty-acid-binding proteins, it contains features that are unique to nematodes. Crystals were obtained, but initial diffraction data analysis revealed that they were composed of a number of `microdomains'. Interpretable data could only be collected using a microfocus beamline with a beam size of 12 × 8 µm.

Two crystal forms of a helix-rich fatty acid- and retinol-binding protein, Na-FAR-1, from the parasitic nematode Necator americanus

Gabrielsen, M. — 2012-07-01

Na-FAR-1 is an unusual α-helix-rich fatty acid- and retinol-binding protein from Necator americanus, a blood-feeding intestinal parasitic nematode of humans. It belongs to the FAR protein family, which is unique to nematodes; no structural information is available to date for FAR proteins from parasites. Crystals were obtained with two different morphologies that corresponded to different space groups. Crystal form 1 exhibited space group P432 (unit-cell parameters a = b = c = 120.80 Å, α = β = γ = 90°) and diffracted to 2.5 Å resolution, whereas crystal form 2 exhibited space group F23 (unit-cell parameters a = b = c = 240.38 Å, α = β = γ = 90°) and diffracted to 3.2 Å resolution. Crystal form 2 showed signs of significant twinning.

Crystallization of SHARPIN using an automated two-dimensional grid screen for optimization

Stieglitz, B. — 2012-07-01

An N-terminal fragment of human SHARPIN was recombinantly expressed in Escherichia coli, purified and crystallized. Crystals suitable for X-ray diffraction were obtained by a one-step optimization of seed dilution and protein concentration using a two-dimensional grid screen. The crystals belonged to the primitive tetragonal space group P43212, with unit-cell parameters a = b = 61.55, c = 222.81 Å. Complete data sets were collected from native and selenomethionine-substituted protein crystals at 100 K to 2.6 and 2.0 Å resolution, respectively.

Structure of Leishmania major cysteine synthase

Fyfe, P.K. — 2012-07-01

Cysteine biosynthesis is a potential target for drug development against parasitic Leishmania species; these protozoa are responsible for a range of serious diseases. To improve understanding of this aspect of Leishmania biology, a crystallographic and biochemical study of L. major cysteine synthase has been undertaken, seeking to understand its structure, enzyme activity and modes of inhibition. Active enzyme was purified, assayed and crystallized in an orthorhombic form with a dimer in the asymmetric unit. Diffraction data extending to 1.8 Å resolution were measured and the structure was solved by molecular replacement. A fragment of γ-poly-d-glutamic acid, a constituent of the crystallization mixture, was bound in the enzyme active site. Although a d-glutamate tetrapeptide had insignificant inhibitory activity, the enzyme was competitively inhibited (Ki = 4 µM) by DYVI, a peptide based on the C-terminus of the partner serine acetyltransferase with which the enzyme forms a complex. The structure surprisingly revealed that the cofactor pyridoxal phosphate had been lost during crystallization.

Purification, crystallization and preliminary X-ray diffraction data of UDP-galactopyranose mutase from Aspergillus fumigatus

Penman, G.A. — 2012-06-01

Aspergillus fumigatus UDP-galactopyranose mutase (AfUGM) is a potential drug target involved in the synthesis of the cell wall of this fungal pathogen. AfUGM was recombinantly produced in Escherichia coli, purified and crystallized by the sitting-drop method, producing orthorhombic crystals that diffracted to a resolution of 3.25 Å. The crystals contained four molecules per asymmetric unit and belonged to space group P212121, with unit-cell parameters a = 127.72, b = 134.30, c = 173.84 Å. Incorporation of selenomethionine was achieved, but the resulting crystals did not allow solution of the phase problem.

A model for 3-methyladenine recognition by 3-methyladenine DNA glycosylase I (TAG) from Staphylococcus aureus

Zhu, X. — 2012-06-01

The removal of chemically damaged DNA bases such as 3-methyladenine (3-MeA) is an essential process in all living organisms and is catalyzed by the enzyme 3-MeA DNA glycosylase I. A key question is how the enzyme selectively recognizes the alkylated 3-MeA over the much more abundant adenine. The crystal structures of native and Y16F-mutant 3-MeA DNA glycosylase I from Staphylococcus aureus in complex with 3-MeA are reported to 1.8 and 2.2 Å resolution, respectively. Isothermal titration calorimetry shows that protonation of 3-MeA decreases its binding affinity, confirming previous fluorescence studies that show that charge–charge recognition is not critical for the selection of 3-MeA over adenine. It is hypothesized that the hydrogen-bonding pattern of Glu38 and Tyr16 of 3-MeA DNA glycosylase I with a particular tautomer unique to 3-MeA contributes to recognition and selection.

Crystallization and preliminary crystallographic analysis of the major capsid proteins VP16 and VP17 of bacteriophage P23-77

Rissanen, I. — 2012-05-01

Members of the diverse double-β-barrel lineage of viruses are identified by the conserved structure of their major coat protein. New members of this lineage have been discovered based on structural analysis and we are interested in identifying relatives that utilize unusual versions of the double-β-barrel fold. One candidate for such studies is P23-77, an icosahedral dsDNA bacteriophage that infects the extremophile Thermus thermophilus. P23-77 has two major coat proteins, namely VP16 and VP17, of a size consistent with a single-β-barrel core fold. These previously unstudied proteins have now been successfully expressed as recombinant proteins, purified and crystallized using hanging-drop and sitting-drop vapour-diffusion methods. Crystals of coat proteins VP16 and VP17 have been obtained as well as of a putative complex. In addition, virus-derived material has been crystallized. Diffraction data have been collected to beyond 3 Å resolution for five crystal types and structure determinations are in progress.

Another case of fraud in structural biology

Weiss, M.S. — 2012-04-01

Structure of a hexameric form of RadA recombinase from Methanococcus voltae

Du, L. — 2012-05-01

Archaeal RadA proteins are close homologues of eukaryal Rad51 and DMC1 proteins and are remote homologues of bacterial RecA proteins. For the repair of double-stranded breaks in DNA, these recombinases promote a pivotal strand-exchange reaction between homologous single-stranded and double-stranded DNA substrates. This DNA-repair function also plays a key role in the resistance of cancer cells to chemotherapy and radiotherapy and in the resistance of bacterial cells to antibiotics. A hexameric form of a truncated Methanococcus voltae RadA protein devoid of its small N-terminal domain has been crystallized. The RadA hexamers further assemble into two-ringed assemblies. Similar assemblies can be observed in the crystals of Pyrococcus furiosus RadA and Homo sapiens DMC1. In all of these two-ringed assemblies the DNA-interacting L1 region of each protomer points inward towards the centre, creating a highly positively charged locus. The electrostatic characteristics of the central channels can be utilized in the design of novel recombinase inhibitors.

Detection and analysis of unusual features in the structural model and structure-factor data of a birch pollen allergen

Rupp, B. — 2012-04-01

Physically improbable features in the model of the birch pollen structure Bet v 1d (PDB entry 3k78) are faithfully reproduced in electron density generated with the deposited structure factors, but these structure factors themselves exhibit properties that are characteristic of data calculated from a simple model and are inconsistent with the data and error model obtained through experimental measurements. The refinement of the 3k78 model against these structure factors leads to an isomorphous structure different from the deposited model with an implausibly small R value (0.019). The abnormal refinement is compared with normal refinement of an isomorphous variant structure of Bet v 1l (PDB entry 1fm4). A variety of analytical tools, including the application of Diederichs plots, Rσ plots and bulk-solvent analysis are discussed as promising aids in validation. The examination of the Bet v 1d structure also cautions against the practice of indicating poorly defined protein chain residues through zero occupancies. The recommendation to preserve diffraction images is amplified.

Response to Detection and analysis of unusual features in the structural model and structure-factor data of a birch pollen allergen

Zaborsky, N. — 2012-04-01

The authors of J. Immunol. 184, 725–735 respond to the article by Rupp (2012), Acta Cryst. F68, 366–376.

Call for a crystallization ontology

Einspahr, H. — 2012-03-01

On the need for an international effort to capture, share and use crystallization screening data

Newman, J. — 2012-03-01

When crystallization screening is conducted many outcomes are observed but typically the only trial recorded in the literature is the condition that yielded the crystal(s) used for subsequent diffraction studies. The initial hit that was optimized and the results of all the other trials are lost. These missing results contain information that would be useful for an improved general understanding of crystallization. This paper provides a report of a crystallization data exchange (XDX) workshop organized by several international large-scale crystallization screening laboratories to discuss how this information may be captured and utilized. A group that administers a significant fraction of the world's crystallization screening results was convened, together with chemical and structural data informaticians and computational scientists who specialize in creating and analysing large disparate data sets. The development of a crystallization ontology for the crystallization community was proposed. This paper (by the attendees of the workshop) provides the thoughts and rationale leading to this conclusion. This is brought to the attention of the wider audience of crystallographers so that they are aware of these early efforts and can contribute to the process going forward.

Crystals on the cover 2012

Einspahr, H. — 2012-01-01

Expression, crystallization and preliminary X-ray crystallographic analysis of glucose-6-phosphate dehydrogenase from the human pathogen Trypanosoma cruzi in complex with substrate. Corrigendum

Ortíz, C. — 2011-12-01

A figure in the article by Ortíz et al. [(2011), Acta Cryst. F67, 1457–1461] is corrected.

Review panel for Acta Crystallographica Section F

Einspahr, H.M. — 2011-11-01

Seleno-detergent MAD phasing of leukotriene C4 synthase in complex with dodecyl-β-d-selenomaltoside

Saino, H. — 2011-12-01

Dodecyl-β-d-selenomaltoside (SeDDM) is a seleno-detergent with a β-glycosidic seleno-ether in place of the ether moiety in dodecyl-β-d-maltoside. Seleno-detergents are candidates for heavy-atom agents in experimental phasing of membrane proteins in protein crystallography. Crystals of a nuclear membrane-embedded enzyme, leukotriene C4 synthase (LTC4S), in complex with SeDDM were prepared and a multiwavelength anomalous diffraction (MAD) experiment was performed. The SeDDM in the LTC4S crystal exhibited sufficient anomalous diffraction for determination of the structure using MAD phasing.

Therapeutic target-site variability in α1-antitrypsin characterized at high resolution

Patschull, A.O.M. — 2011-12-01

The intrinsic propensity of α1-antitrypsin to undergo conformational transitions from its metastable native state to hyperstable forms provides a motive force for its antiprotease function. However, aberrant conformational change can also occur via an intermolecular linkage that results in polymerization. This has both loss-of-function and gain-of-function effects that lead to deficiency of the protein in human circulation, emphysema and hepatic cirrhosis. One of the most promising therapeutic strategies being developed to treat this disease targets small molecules to an allosteric site in the α1-antitrypsin molecule. Partial filling of this site impedes polymerization without abolishing function. Drug development can be improved by optimizing data on the structure and dynamics of this site. A new 1.8 Å resolution structure of α1-antitrypsin demonstrates structural variability within this site, with associated fluctuations in its upper and lower entrance grooves and ligand-binding characteristics around the innermost stable enclosed hydrophobic recess. These data will allow a broader selection of chemotypes and derivatives to be tested in silico and in vitro when screening and developing compounds to modulate conformational change to block the pathological mechanism while preserving function.

Structure of hyperthermophilic β-glucosidase from Pyrococcus furiosus

Kado, Y. — 2011-12-01

Three categories of cellulases, endoglucanases, cellobiohydrolases and β-glucosidases, are commonly used in the process of cellulose saccharification. In particular, the activity and characteristics of hyperthermophilic β-glucosidase make it promising in industrial applications of biomass. In this paper, the crystal structure of the hyperthermophilic β-glucosidase from Pyrococcus furiosus (BGLPf) was determined at 2.35 Å resolution in a new crystal form. The structure showed that there is one tetramer in the asymmetric unit and that the dimeric molecule exhibits a structure that is stable towards sodium dodecyl sulfate (SDS). The dimeric molecule migrated in reducing SDS polyacrylamide gel electrophoresis (SDS–PAGE) buffer even after boiling at 368 K. Energy calculations demonstrated that one of the two dimer interfaces acquired the largest solvation free energy. Structural comparison and sequence alignment with mesophilic β-glucosidase A from Clostridium cellulovorans (BGLACc) revealed that the elongation at the C-terminal end forms a hydrophobic patch at the dimer interface that might contribute to hyperthermostability.

Wheat germ cell-free expression system as a pathway to improve protein yield and solubility for the SSGCID pipeline

Guild, K. — 2011-09-01

Recombinant expression of proteins of interest in Escherichia coli is an important tool in the determination of protein structure. However, lack of expression and insolubility remain significant challenges to the expression and crystallization of these proteins. The SSGCID program uses a wheat germ cell-free expression system as a rescue pathway for proteins that are either not expressed or insoluble when produced in E. coli. Testing indicates that the system is a valuable tool for these protein targets. Further increases in solubility were obtained by the addition of the NVoy polymer reagent to the reaction mixture. These data indicate that this eukaryotic cell-free expression system has a high success rate and that the addition of specific reagents can increase the yield of soluble protein.

Structure of aldose reductase from Giardia lamblia

Ferrell, M. — 2011-09-01

Giardia lamblia is an anaerobic aerotolerant eukaryotic parasite of the intestines. It is believed to have diverged early from eukarya during evolution and is thus lacking in many of the typical eukaryotic organelles and biochemical pathways. Most conspicuously, mitochondria and the associated machinery of oxidative phosphorylation are absent; instead, energy is derived from substrate-level phosphorylation. Here, the 1.75 Å resolution crystal structure of G. lamblia aldose reductase heterologously expressed in Escherichia coli is reported. As in other oxidoreductases, G. lamblia aldose reductase adopts a TIM-barrel conformation with the NADP+-binding site located within the eight β-strands of the interior.

Structure of 3-ketoacyl-(acyl-carrier-protein) reductase from Rickettsia prowazekii at 2.25 Å resolution

Subramanian, S. — 2011-09-01

Rickettsia prowazekii, a parasitic Gram-negative bacterium, is in the second-highest biodefense category of pathogens of the National Institute of Allergy and Infectious Diseases, but only a handful of structures have been deposited in the PDB for this bacterium; to date, all of these have been solved by the SSGCID. Owing to its small genome (about 800 protein-coding genes), it relies on the host for many basic biosynthetic processes, hindering the identification of potential antipathogenic drug targets. However, like many bacteria and plants, its metabolism does depend upon the type II fatty-acid synthesis (FAS) pathway for lipogenesis, whereas the predominant form of fatty-acid biosynthesis in humans is via the type I pathway. Here, the structure of the third enzyme in the FAS pathway, 3-ketoacyl-(acyl-carrier-protein) reductase, is reported at a resolution of 2.25 Å. Its fold is highly similar to those of the existing structures from some well characterized pathogens, such as Mycobacterium tuberculosis and Burkholderia pseudomallei, but differs significantly from the analogous mammalian structure. Hence, drugs known to target the enzymes of pathogenic bacteria may serve as potential leads against Rickettsia, which is responsible for spotted fever and typhus and is found throughout the world.

An ensemble of structures of Burkholderia pseudomallei 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase

Davies, D.R. — 2011-09-01

Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and Northern Australia. Burkholderia is responsible for melioidosis, a serious infection of the skin. The enzyme 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (PGAM) catalyzes the interconversion of 3-phosphoglycerate and 2-phosphoglycerate, a key step in the glycolytic pathway. As such it is an extensively studied enzyme and X-ray crystal structures of PGAM enzymes from multiple species have been elucidated. Vanadate is a phosphate mimic that is a powerful tool for studying enzymatic mechanisms in phosphoryl-transfer enzymes such as phosphoglycerate mutase. However, to date no X-ray crystal structures of phosphoglycerate mutase have been solved with vanadate acting as a substrate mimic. Here, two vanadate complexes together with an ensemble of substrate and fragment-bound structures that provide a comprehensive picture of the function of the Burkholderia enzyme are reported.

Structure of the cystathionine γ-synthase MetB from Mycobacterium ulcerans

Clifton, M.C. — 2011-09-01

Cystathionine γ-synthase (CGS) is a transulfurication enzyme that catalyzes the first specific step in l-methionine biosynthesis by the reaction of O4-succinyl-l-homoserine and l-cysteine to produce l-cystathionine and succinate. Controlling the first step in l-methionine biosythesis, CGS is an excellent potential drug target. Mycobacterium ulcerans is a slow-growing mycobacterium that is the third most common form of mycobacterial infection, mainly infecting people in Africa, Australia and Southeast Asia. Infected patients display a variety of skin ailments ranging from indolent non-ulcerated lesions as well as ulcerated lesions. Here, the crystal structure of CGS from M. ulcerans covalently linked to the cofactor pyridoxal phosphate (PLP) is reported at 1.9 Å resolution. A second structure contains PLP as well as a highly ordered HEPES molecule in the active site acting as a pseudo-ligand. These results present the first structure of a CGS from a mycobacterium and allow comparison with other CGS enzymes. This is also the first structure reported from the pathogen M. ulcerans.

Structural genomics of infectious disease drug targets: the SSGCID

Stacy, R. — 2011-09-01

The Seattle Structural Genomics Center for Infectious Disease (SSGCID) is a consortium of researchers at Seattle BioMed, Emerald BioStructures, the University of Washington and Pacific Northwest National Laboratory that was established to apply structural genomics approaches to drug targets from infectious disease organisms. The SSGCID is currently funded over a five-year period by the National Institute of Allergy and Infectious Diseases (NIAID) to determine the three-dimensional structures of 400 proteins from a variety of Category A, B and C pathogens. Target selection engages the infectious disease research and drug-therapy communities to identify drug targets, essential enzymes, virulence factors and vaccine candidates of biomedical relevance to combat infectious diseases. The protein-expression systems, purified proteins, ligand screens and three-dimensional structures produced by SSGCID constitute a valuable resource for drug-discovery research, all of which is made freely available to the greater scientific community. This volume of Acta Crystallographica Section F, entirely devoted to the work of the SSGCID, covers the details of the high-throughput pipeline and presents a series of structures from a broad array of pathogenic organisms. Here, a background is provided on the structural genomics of infectious disease, the essential components of the SSGCID pipeline are discussed and a survey of progress to date is presented.

Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase from Babesia bovis

Begley, D.W. — 2011-09-01

Babesiosis is a tick-borne disease caused by eukaryotic Babesia parasites which are morphologically similar to Plasmodium falciparum, the causative agent of malaria in humans. Like Plasmodium, different species of Babesia are tuned to infect different mammalian hosts, including rats, dogs, horses and cattle. Most species of Plasmodium and Babesia possess an essential bifunctional enzyme for nucleotide synthesis and folate metabolism: dihydrofolate reductase-thymidylate synthase. Although thymidylate synthase is highly conserved across organisms, the bifunctional form of this enzyme is relatively uncommon in nature. The structural characterization of dihydrofolate reductase-thymidylate synthase in Babesia bovis, the causative agent of babesiosis in livestock cattle, is reported here. The apo state is compared with structures that contain dUMP, NADP and two different antifolate inhibitors: pemetrexed and raltitrexed. The complexes reveal modes of binding similar to that seen in drug-resistant malaria strains and point to the utility of applying structural studies with proven cancer chemotherapies towards infectious disease research.

The Protein Maker: an automated system for high-throughput parallel purification

Smith, E.R. — 2011-09-01

The Protein Maker is an automated purification system developed by Emerald BioSystems for high-throughput parallel purification of proteins and antibodies. This instrument allows multiple load, wash and elution buffers to be used in parallel along independent lines for up to 24 individual samples. To demonstrate its utility, its use in the purification of five recombinant PB2 C-terminal domains from various subtypes of the influenza A virus is described. Three of these constructs crystallized and one diffracted X-rays to sufficient resolution for structure determination and deposition in the Protein Data Bank. Methods for screening lysis buffers for a cytochrome P450 from a pathogenic fungus prior to upscaling expression and purification are also described. The Protein Maker has become a valuable asset within the Seattle Structural Genomics Center for Infectious Disease (SSGCID) and hence is a potentially valuable tool for a variety of high-throughput protein-purification applications.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1

Park, S.H. — 2011-10-01

Transmembrane protein 16A (TMEM16A, also known as anoctamin 1; ANO1) is a bona fide Ca2+-activated chloride channel that is activated by intracellular Ca2+- and Ca2+-mobilizing stimuli and plays important roles in a variety of physiological functions. To elucidate the structural features of ANO1, structural analysis of the C-terminal cytosolic domain of mouse ANO1 (mANO1-CTD) was initiated. mANO1-CTD was overexpressed in Escherichia coli and was crystallized at 297 K using a reservoir solution consisting of 0.2 M sodium acetate trihydrate, 0.1 M Tris–HCl pH 8.5 and 30%(w/v) PEG 4000. X-ray diffraction data were collected to 2.3 Å resolution. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 73.96, b = 103.73, c = 114.71 Å. If it is assumed that eight copies of a monomer molecule are present in the crystallographic asymmetric unit, the crystal volume per protein mass (VM) is 2.38 Å3 Da−1 and the solvent content is 48.38%. Attempts to solve the structure of mANO1-CTD by the MAD method using selenomethionine-labelled mANO1-CTD or heavy-atom-derivatized crystals are in progress.

Gene Composer in a structural genomics environment

Lorimer, D. — 2011-09-01

The structural genomics effort at the Seattle Structural Genomics Center for Infectious Disease (SSGCID) requires the manipulation of large numbers of amino-acid sequences and the underlying DNA sequences which are to be cloned into expression vectors. To improve efficiency in high-throughput protein structure determination, a database software package, Gene Composer, has been developed which facilitates the information-rich design of protein constructs and their underlying gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bioinformatics steps used in modern structure-guided protein engineering and synthetic gene engineering. An example of the structure determination of H1N1 RNA-dependent RNA polymerase PB2 subunit is given.

Crystallization and preliminary X-ray crystallographic studies of the ice-binding protein from the Antarctic yeast Leucosporidium sp. AY30. Corrigendum

Park, A.K. — 2011-08-01

A correction is made to the title of the article by Park et al. [(2011). Acta Cryst. F67, 800–802].

Structure of fumarate hydratase from Rickettsia prowazekii, the agent of typhus and suspected relative of the mitochondria

Phan, I. — 2011-09-01

Rickettsiae are obligate intracellular parasites of eukaryotic cells that are the causative agents responsible for spotted fever and typhus. Their small genome (about 800 protein-coding genes) is highly conserved across species and has been postulated as the ancestor of the mitochondria. No genes that are required for glycolysis are found in the Rickettsia prowazekii or mitochondrial genomes, but a complete set of genes encoding components of the tricarboxylic acid cycle and the respiratory-chain complex is found in both. A 2.4 Å resolution crystal structure of R. prowazekii fumarate hydratase, an enzyme catalyzing the third step of the tricarboxylic acid cycle pathway that ultimately converts phosphoenolpyruvate into succinyl-CoA, has been solved. A structure alignment with human mitochondrial fumarate hydratase highlights the close similarity between R. prowazekii and mitochondrial enzymes.

Gene design, cloning and protein-expression methods for high-value targets at the Seattle Structural Genomics Center for Infectious Disease

Raymond, A. — 2011-09-01

Any structural genomics endeavor, particularly ambitious ones such as the NIAID-funded Seattle Structural Genomics Center for Infectious Disease (SSGCID) and Center for Structural Genomics of Infectious Disease (CSGID), face technical challenges at all points of the production pipeline. One salvage strategy employed by SSGCID is combined gene engineering and structure-guided construct design to overcome challenges at the levels of protein expression and protein crystallization. Multiple constructs of each target are cloned in parallel using Polymerase Incomplete Primer Extension cloning and small-scale expressions of these are rapidly analyzed by capillary electrophoresis. Using the methods reported here, which have proven particularly useful for high-value targets, otherwise intractable targets can be resolved.

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of glucuronic acid dehydrogenase from Chromohalobacter salexigens. Addendum

Ahn, J.-W. — 2011-08-01

An additional acknowledgment is published for the paper by Ahn et al. [(2011), Acta Cryst. F67, 689–691].

From screen to structure with a harvestable microfluidic device

Stojanoff, V. — 2011-08-01

Advances in automation have facilitated the widespread adoption of high-throughput vapour-diffusion methods for initial crystallization screening. However, for many proteins, screening thousands of crystallization conditions fails to yield crystals of sufficient quality for structural characterization. Here, the rates of crystal identification for thaumatin, catalase and myoglobin using microfluidic Crystal Former devices and sitting-drop vapour-diffusion plates are compared. It is shown that the Crystal Former results in a greater number of identified initial crystallization conditions compared with vapour diffusion. Furthermore, crystals of thaumatin and lysozyme obtained in the Crystal Former were used directly for structure determination both in situ and upon harvesting and cryocooling. On the basis of these results, a crystallization strategy is proposed that uses multiple methods with distinct kinetic trajectories through the protein phase diagram to increase the output of crystallization pipelines.

Salvage and storage of infectious disease protein targets in the SSGCID high-throughput crystallization pathway using microfluidics

Christensen, J. — 2011-09-01

The MPCS Plug Maker is a microcapillary-based protein-crystallization system for generating diffraction-ready crystals from nanovolumes of protein. Crystallization screening using the Plug Maker was used as a salvage pathway for proteins that failed to crystallize during the initial observation period using the traditional sitting-drop vapor-diffusion method. Furthermore, the CrystalCards used to store the crystallization experiments set up by the Plug Maker are shown be a viable container for long-term storage of protein crystals without a discernable loss of diffraction quality with time. Use of the Plug Maker with SSGCID proteins is demonstrated to be an effective crystal-salvage and storage method.

Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline

Hewitt, S.N. — 2011-09-01

Despite recent advances, the expression of heterologous proteins in Escherichia coli for crystallization remains a nontrivial challenge. The present study investigates the efficacy of maltose-binding protein (MBP) fusion as a general strategy for rescuing the expression of target proteins. From a group of sequence-verified clones with undetectable levels of protein expression in an E. coli T7 expression system, 95 clones representing 16 phylogenetically diverse organisms were selected for recloning into a chimeric expression vector with an N-terminal histidine-tagged MBP. PCR-amplified inserts were annealed into an identical ligation-independent cloning region in an MBP-fusion vector and were analyzed for expression and solubility by high-throughput nickel-affinity binding. This approach yielded detectable expression of 72% of the clones; soluble expression was visible in 62%. However, the solubility of most proteins was marginal to poor upon cleavage of the MBP tag. This study offers large-scale evidence that MBP can improve the soluble expression of previously non-expressing proteins from a variety of eukaryotic and prokaryotic organisms. While the behavior of the cleaved proteins was disappointing, further refinements in MBP tagging may permit the more widespread use of MBP-fusion proteins in crystallographic studies.

The structure of fructose bisphosphate aldolase from Encephalitozoon cuniculi

Gardberg, A. — 2011-09-01

Fructose bisphosphate aldolose (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources. Bioinformatic analysis of the genome of the eukaryotic microsporidian parasite Encephalitozoon cuniculi revealed an FBPA homolog. The structures of this enzyme in the presence of the native substrate FBP and also with the partial substrate analog phosphate are reported. The purified enzyme crystallized in 90 mM Bis-Tris propane pH 6.5, 18% PEG 3350, 18 mM NaKHPO4, 10 mM urea for the phosphate-bound form and 100 mM Bis-Tris propane pH 6.5, 20% PEG 3350, 20 mM fructose 1,6-bisphosphate for the FBP-bound form. In both cases protein was present at 25 mg ml−1 and the sitting-drop vapour-diffusion method was used. For the FBP-bound form, a data set to 2.37 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group C2221, with unit-cell parameters a = 121.46, b = 135.82, c = 61.54 Å. The structure was refined to a final free R factor of 20.8%. For the phosphate-bound form, a data set was collected to 2.00 Å resolution. The space group was also C2221 and the unit-cell parameters were a = 121.96, b = 137.61, c = 62.23 Å. The structure shares the typical barrel tertiary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site. The quaternary structure is dimeric. This work provides a direct experimental result for the substrate-binding conformation of the product state of E. cuniculi FBPA.

Conservation of a crystallographic interface suggests a role for β-sheet augmentation in influenza virus NS1 multifunctionality

Kerry, P.S. — 2011-08-01

The effector domain (ED) of the influenza virus virulence factor NS1 is capable of interaction with a variety of cellular and viral targets, although regulation of these events is poorly understood. Introduction of a W187A mutation into the ED abolishes dimer formation; however, strand–strand interactions between mutant NS1 ED monomers have been observed in two previous crystal forms. A new condition for crystallization of this protein [0.1 M Bis-Tris pH 6.0, 0.2 M NaCl, 22%(w/v) PEG 3350, 20 mM xylitol] was discovered using the hanging-drop vapour-diffusion method. Diffraction data extending to 1.8 Å resolution were collected from a crystal grown in the presence of 40 mM thieno[2,3-b]pyridin-2-ylmethanol. It was observed that there is conservation of the strand–strand interface in crystals of this monomeric NS1 ED in three different space groups. This observation, coupled with conformational changes in the interface region, suggests a potential role for β-sheet augmentation in NS1 function.

Structure of triosephosphate isomerase from Cryptosporidium parvum

Nguyen, T.N. — 2011-09-01

Cryptosporidium parvum is one of several Cryptosporidium spp. that cause the parasitic infection cryptosporidiosis. Cryptosporidiosis is a diarrheal infection that is spread via the fecal–oral route and is commonly caused by contaminated drinking water. Triosephosphate isomerase is an enzyme that is ubiquitous to all organisms that perform glycolysis. Triosephosphate isomerase catalyzes the formation of glyceraldehyde 3-phosphate from dihydroxyacetone phosphate, which is a critical step to ensure the maximum ATP production per glucose molecule. In this paper, the 1.55 Å resolution crystal structure of the open-loop form of triosephosphate isomerase from C. parvum Iowa II is presented. An unidentified electron density was found in the active site.

Structure of fructose bisphosphate aldolase from Bartonella henselae bound to fructose 1,6-bisphosphate

Gardberg, A. — 2011-09-01

Fructose bisphosphate aldolase (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources, including the bacterium Brucella melitensis and the protozoan Babesia bovis. Bioinformatic analysis of the Bartonella henselae genome revealed an FBPA homolog. The B. henselae FBPA enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme crystallized in the apo form but failed to diffract; however, well diffracting crystals could be obtained by cocrystallization in the presence of the native substrate fructose 1,6-bisphosphate. A data set to 2.35 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 72.39, b = 127.71, c = 157.63 Å. The structure was refined to a final free R factor of 22.2%. The structure shares the typical barrel tertiary structure and tetrameric quaternary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site.

High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease

Bryan, C.M. — 2011-09-01

The establishment of an efficient and reliable protein-purification pipeline is essential for the success of structural genomic projects. The SSGCID Protein Purification Group at the University of Washington (UW-PPG) has established a robust protein-purification pipeline designed to purify 400 proteins per year at a rate of eight purifications per week. The pipeline was implemented using two ÄKTAexplorer 100s and four ÄKTAprimes to perform immobilized metal-affinity chromatography (IMAC) and size-exclusion chromatography. Purifications were completed in a period of 5 d and yielded an average of 53 mg highly purified protein. This paper provides a detailed description of the methods used to purify, characterize and store SSGCID proteins. Some of the purified proteins were treated with 3C protease, which was expressed and purified by UW-PPG using a similar protocol, to cleave non-native six-histidine tags. The cleavage was successful in 94% of 214 attempts. Cleaved proteins yielded 2.9% more structures than uncleaved six-histidine-tagged proteins. This 2.9% improvement may seem small, but over the course of the project the structure output from UW-PPG is thus predicted to increase from 260 structures to 318 structures. Therefore, the outlined protocol with 3C cleavage and subtractive IMAC has been shown to be a highly efficient method for the standardized purification of recombinant proteins for structure determination via X-ray crystallography.

Structure of a cyclin-dependent kinase from Giardia lamblia

Leibly, D.J. — 2011-09-01

Giardia lamblia is the etiologic agent of giardiasis, a water-borne infection that is prevalent throughout the world. The need for new therapeutics for the treatment of giardiasis is of paramount importance. Owing to the ubiquitous nature of kinases and their vital importance in organisms, they are potential drug targets. In this paper, the first structure of a cyclin-dependent kinase (CDK) from G. lamblia (GlCDK; UniProt A8BZ95) is presented. CDKs are cell-cycle-associated kinases that are actively being pursued as targets for anticancer drugs as well as for antiparasitic chemotherapy. Generally, a CDK forms a complex with its associated cyclin. This CDK–cyclin complex is active and acts as a serine/threonine protein kinase. Typically, CDKs are responsible for the transition to the next phase of the cell cycle. Although the structure of GlCDK with its associated cyclin was not solved, the 1.85 Å resolution structure of apo GlCDK and a 2.0 Å resolution structure of GlCDK in complex with adenosine monophosphate are presented and the structural differences from the orthologous human CDK2 and CDK3 are discussed.

Use of the α-mannosidase I inhibitor kifunensine allows the crystallization of apo CTLA-4 homodimer produced in long-term cultures of Chinese hamster ovary cells

Yu, C. — 2011-07-01

Glycoproteins present problems for structural analysis since they often have to be glycosylated in order to fold correctly and because their chemical and conformational heterogeneity generally inhibits crystallization. It is shown that the α-mannosidase I inhibitor kifunensine, which has previously been used for the purpose of glycoprotein crystallization in short-term (3–5 d) cultures, is apparently stable enough to be used to produce highly endoglycosidase H-sensitive glycoprotein in long-term (3–4 week) cultures of stably transfected Chinese hamster ovary (CHO) cells. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of the extracellular region of the cytotoxic T-lymphocyte antigen 4 (CTLA-4; CD152) homodimer expressed in long-term CHO cell cultures in the presence of kifunensine revealed that the inhibitor restricted CTLA-4 glycan processing to Man9GlcNAc2 and Man5GlcNAc2 structures. Complex-type glycans were undetectable, suggesting that the inhibitor was active for the entire duration of the cultures. Endoglycosidase treatment of the homodimer yielded protein that readily formed orthorhombic crystals with unit-cell parameters a = 43.9, b = 51.5, c = 102.9 Å and space group P212121 that diffracted to Bragg spacings of 1.8 Å. The results indicate that kifunensine will be effective in most, if not all, transient and long-term mammalian cell-based expression systems.

Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success

Choi, R. — 2011-09-01

The recombinant expression of soluble proteins in Escherichia coli continues to be a major bottleneck in structural genomics. The establishment of reliable protocols for the performance of small-scale expression and solubility testing is an essential component of structural genomic pipelines. The SSGCID Protein Production Group at the University of Washington (UW-PPG) has developed a high-throughput screening (HTS) protocol for the measurement of protein recovery from immobilized metal-affinity chromatography (IMAC) which predicts successful purification of hexahistidine-tagged proteins. The protocol is based on manual transfer of samples using multichannel pipettors and 96-well plates and does not depend on the use of robotic platforms. This protocol has been applied to evaluate the expression and solubility of more than 4000 proteins expressed in E. coli. The UW-PPG also screens large-scale preparations for recovery from IMAC prior to purification. Analysis of these results show that our low-cost non-automated approach is a reliable method for the HTS demands typical of large structural genomic projects. This paper provides a detailed description of these protocols and statistical analysis of the SSGCID screening results. The results demonstrate that screening for proteins that yield high recovery after IMAC, both after small-scale and large-scale expression, improves the selection of proteins that can be successfully purified and will yield a crystal structure.

Expression, purification, crystallization and preliminary crystallographic analysis of a putative Clostridium difficile surface protein Cwp19

Kirby, J.M. — 2011-07-01

Cwp19 is a putatively surface-located protein from Clostridium difficile. A recombinant N-terminal protein (residues 27–401) lacking the signal peptide and the C-terminal cell-wall-binding repeats (PFam04122) was crystallized using the sitting-drop vapour-diffusion method and diffracted to 2 Å resolution. The crystal appeared to belong to the primitive monoclinic space group P21, with unit-cell parameters a = 109.1, b = 61.2, c = 109.2 Å, β = 111.85°, and is estimated to contain two molecules of Cwp19 per asymmetric unit.

Structures of the rat complement regulator CrrY

Roversi, P. — 2011-07-01

Complement receptor 1-related protein Y (CrrY) is an important cell-surface regulator of complement that is unique to rodent species. The structure of rat CrrY domains 1–4 has been determined in two distinct crystal forms and reveals a 70° bend between domains 3 and 4. Comparisons of this structure with those of other complement regulators suggests that rearrangement of this interface may occur on forming the regulatory complex with C3b.

Structure of thymidylate kinase from Ehrlichia chaffeensis

Leibly, D.J. — 2011-09-01

The enzyme thymidylate kinase phosphorylates the substrate thymidine 5′-phosphate (dTMP) to form thymidine 5′-diphosphate (dTDP), which is further phosphorylated to dTTP for incorporation into DNA. Ehrlichia chaffeensis is the etiologic agent of human monocytotropic erlichiosis (HME), a potentially life-threatening tick-borne infection. HME is endemic in the United States from the southern states up to the eastern seaboard. HME is transmitted to humans via the lone star tick Amblyomma americanum. Here, the 2.15 Å resolution crystal structure of thymidylate kinase from E. chaffeensis in the apo form is presented.

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

Begley, D.W. — 2011-09-01

Glutaric acidemia type 1 is an inherited metabolic disorder which can cause macrocephaly, muscular rigidity, spastic paralysis and other progressive movement disorders in humans. The defects in glutaryl-CoA dehydrogenase (GCDH) associated with this disease are thought to increase holoenzyme instability and reduce cofactor binding. Here, the first structural analysis of a GCDH enzyme in the absence of the cofactor flavin adenine dinucleotide (FAD) is reported. The apo structure of GCDH from Burkholderia pseudomallei reveals a loss of secondary structure and increased disorder in the FAD-binding pocket relative to the ternary complex of the highly homologous human GCDH. After conducting a fragment-based screen, four small molecules were identified which bind to GCDH from B. pseudomallei. Complex structures were determined for these fragments, which cause backbone and side-chain perturbations to key active-site residues. Structural insights from this investigation highlight differences from apo GCDH and the utility of small-molecular fragments as chemical probes for capturing alternative conformational states of preformed protein crystals.

Structure of nitrilotriacetate monooxygenase component B from Mycobacterium thermoresistibile

Zhang, Y. — 2011-09-01

Mycobacterium tuberculosis belongs to a large family of soil bacteria which can degrade a remarkably broad range of organic compounds and utilize them as carbon, nitrogen and energy sources. It has been proposed that a variety of mycobacteria can subsist on alternative carbon sources during latency within an infected human host, with the help of enzymes such as nitrilotriacetate monooxygenase (NTA-Mo). NTA-Mo is a member of a class of enzymes which consist of two components: A and B. While component A has monooxygenase activity and is responsible for the oxidation of the substrate, component B consumes cofactor to generate reduced flavin mononucleotide, which is required for component A activity. NTA-MoB from M. thermoresistibile, a rare but infectious close relative of M. tuberculosis which can thrive at elevated temperatures, has been expressed, purified and crystallized. The 1.6 Å resolution crystal structure of component B of NTA-Mo presented here is one of the first crystal structures determined from the organism M. thermoresistibile. The NTA-MoB crystal structure reveals a homodimer with the characteristic split-barrel motif typical of flavin reductases. Surprisingly, NTA-MoB from M. thermoresistibile contains a C-terminal tail that is highly conserved among mycobacterial orthologs and resides in the active site of the other protomer. Based on the structure, the C-terminal tail may modulate NTA-MoB activity in mycobacteria by blocking the binding of flavins and NADH.

Comparative analysis of glutaredoxin domains from bacterial opportunistic pathogens

Leeper, T. — 2011-09-01

Glutaredoxin proteins (GLXRs) are essential components of the glutathione system that reductively detoxify substances such as arsenic and peroxides and are important in the synthesis of DNA via ribonucleotide reductases. NMR solution structures of glutaredoxin domains from two Gram-negative opportunistic pathogens, Brucella melitensis and Bartonella henselae, are presented. These domains lack the N-terminal helix that is frequently present in eukaryotic GLXRs. The conserved active-site cysteines adopt canonical proline/tyrosine-stabilized geometries. A difference in the angle of α-helix 2 relative to the β-sheet surface and the presence of an extended loop in the human sequence suggests potential regulatory regions and/or protein–protein interaction motifs. This observation is consistent with mutations in this region that suppress defects in GLXR–ribonucleotide reductase interactions. These differences between the human and bacterial forms are adjacent to the dithiol active site and may permit species-selective drug design.

Structure of a Nudix hydrolase (MutT) in the Mg2+-bound state from Bartonella henselae, the bacterium responsible for cat scratch fever

Buchko, G.W. — 2011-09-01

Cat scratch fever (also known as cat scratch disease and bartonellosis) is an infectious disease caused by the proteobacterium Bartonella henselae following a cat scratch. Although the infection usually resolves spontaneously without treatment in healthy adults, bartonellosis may lead to severe complications in young children and immunocompromised patients, and there is new evidence suggesting that B. henselae may be associated with a broader range of clinical symptoms then previously believed. The genome of B. henselae contains genes for two putative Nudix hydrolases, BH02020 and BH01640 (KEGG). Nudix proteins play an important role in regulating the intracellular concentration of nucleotide cofactors and signaling molecules. The amino-acid sequence of BH02020 is similar to that of the prototypical member of the Nudix superfamily, Escherichia coli MutT, a protein that is best known for its ability to neutralize the promutagenic compound 7,8-dihydro-8-oxoguanosine triphosphate. Here, the crystal structure of BH02020 (Bh-MutT) in the Mg2+-bound state was determined at 2.1 Å resolution (PDB entry 3hhj). As observed in all Nudix hydrolase structures, the α-helix of the highly conserved `Nudix box' in Bh-MutT is one of two helices that sandwich a four-stranded mixed β-sheet with the central two β-strands parallel to each other. The catalytically essential divalent cation observed in the Bh-MutT structure, Mg2+, is coordinated to the side chains of Glu57 and Glu61. The structure is not especially robust; a temperature melt obtained using circular dichroism spectroscopy shows that Bh-MutT irreversibly unfolds and precipitates out of solution upon heating, with a Tm of 333 K.

BrabA.11339.a: anomalous diffraction and ligand binding guide towards the elucidation of the function of a `putative β-lactamase-like protein' from Brucella melitensis

Abendroth, J. — 2011-09-01

The crystal structure of a β-lactamase-like protein from Brucella melitensis was initially solved by SAD phasing from an in-house data set collected on a crystal soaked with iodide. A high-resolution data set was collected at a synchroton at the Se edge wavelength, which also provided an independent source of phasing using a small anomalous signal from metal ions in the active site. Comparisons of anomalous peak heights at various wavelengths allowed the identification of the active-site metal ions as manganese. In the native data set a partially occupied GMP could be identified. When co-crystallized with AMPPNP or GMPPNP, clear density for the hydrolyzed analogs was observed, providing hints to the function of the protein.

Structures of a putative ζ-class glutathione S-transferase from the pathogenic fungus Coccidioides immitis

Edwards, T.E. — 2011-09-01

Coccidioides immitis is a pathogenic fungus populating the southwestern United States and is a causative agent of coccidioidomycosis, sometimes referred to as Valley Fever. Although the genome of this fungus has been sequenced, many operons are not properly annotated. Crystal structures are presented for a putative uncharacterized protein that shares sequence similarity with ζ-class glutathione S-transferases (GSTs) in both apo and glutathione-bound forms. The apo structure reveals a nonsymmetric homodimer with each protomer comprising two subdomains: a C-terminal helical domain and an N-terminal thioredoxin-like domain that is common to all GSTs. Half-site binding is observed in the glutathione-bound form. Considerable movement of some components of the active site relative to the glutathione-free form was observed, indicating an induced-fit mechanism for cofactor binding. The sequence homology, structure and half-site occupancy imply that the protein is a ζ-class glutathione S-transferase, a maleylacetoacetate isomerase (MAAI).

Solution-state NMR structure and biophysical characterization of zinc-substituted rubredoxin B (Rv3250c) from Mycobacterium tuberculosis

Buchko, G.W. — 2011-09-01

Owing to the evolution of multi-drug-resistant and extremely drug-resistant Mycobacterium tuberculosis strains, there is an urgent need to develop new antituberculosis strategies to prevent TB epidemics in the industrial world. Among the potential new drug targets are two small nonheme iron-binding proteins, rubredoxin A (Rv3251c) and rubredoxin B (Rv3250c), which are believed to play a role in electron-transfer processes. Here, the solution structure and biophysical properties of one of these two proteins, rubredoxin B (Mt-RubB), determined in the zinc-substituted form are reported. The zinc-substituted protein was prepared by expressing Mt-RubB in minimal medium containing excess zinc acetate. Size-exclusion chromatography and NMR spectroscopy indicated that Mt-RubB was a monomer in solution. The structure (PDB entry 2kn9) was generally similar to those of other rubredoxins, containing a three-stranded antiparallel β-sheet (β2–β1–β3) and a metal tetrahedrally coordinated to the S atoms of four cysteine residues (Cys9, Cys12, Cys42 and Cys45). The first pair of cysteine residues is at the C-terminal end of the first β-strand and the second pair of cysteine residues is towards the C-terminal end of the loop between β2 and β3. The structure shows the metal buried deeply within the protein, an observation that is supported by the inability to remove the metal with excess EDTA at room temperature. Circular dichroism spectroscopy shows that this stability extends to high temperature, with essentially no change being observed in the CD spectrum of Mt-RubB upon heating to 353 K.

Solution structure of an arsenate reductase-related protein, YffB, from Brucella melitensis, the etiological agent responsible for brucellosis

Buchko, G.W. — 2011-09-01

Brucella melitensis is the etiological agent responsible for brucellosis. Present in the B. melitensis genome is a 116-residue protein related to arsenate reductases (Bm-YffB; BR0369). Arsenate reductases (ArsC) convert arsenate ion (H2AsO4−), a compound that is toxic to bacteria, to arsenite ion (AsO2−), a product that may be efficiently exported out of the cell. Consequently, Bm-YffB is a potential drug target because if arsenate reduction is the protein's major biological function then disabling the cell's ability to reduce arsenate would make these cells more sensitive to the deleterious effects of arsenate. Size-exclusion chromatography and NMR spectroscopy indicate that Bm-YffB is a monomer in solution. The solution structure of Bm-YffB (PDB entry 2kok) shows that the protein consists of two domains: a four-stranded mixed β-sheet flanked by two α-helices on one side and an α-helical bundle. The α/β domain is characteristic of the fold of thioredoxin-like proteins and the overall structure is generally similar to those of known arsenate reductases despite the marginal sequence similarity. Chemical shift perturbation studies with 15N-labeled Bm-YffB show that the protein binds reduced glutathione at a site adjacent to a region similar to the HX3CX3R catalytic sequence motif that is important for arsenic detoxification activity in the classical arsenate-reductase family of proteins. The latter observation supports the hypothesis that the ArsC-YffB family of proteins may function as glutathione-dependent thiol reductases. However, comparison of the structure of Bm-YffB with the structures of proteins from the classical ArsC family suggest that the mechanism and possibly the function of Bm-YffB and other related proteins (ArsC-YffB) may differ from those of the ArsC family of proteins.

NMR structure of an acyl-carrier protein from Borrelia burgdorferi

Barnwal, R.P. — 2011-09-01

Nearly complete resonance assignment and the high-resolution NMR structure of the acyl-carrier protein from Borrelia burgdorferi, a target of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) structure-determination pipeline, are reported. This protein was chosen as a potential target for drug-discovery efforts because of its involvement in fatty-acid biosynthesis, an essential metabolic pathway, in bacteria. It was possible to assign >98% of backbone resonances and >92% of side-chain resonances using multidimensional NMR spectroscopy. The NMR structure was determined to a backbone r.m.s.d. of 0.4 Å and contained four α-helices and two 310-helices. A structure-homology search revealed that this protein is highly similar to the acyl-carrier protein from Aquifex aeolicus.

Structures of phosphopantetheine adenylyltransferase from Burkholderia pseudomallei

Edwards, T.E. — 2011-09-01

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the fourth of five steps in the coenzyme A biosynthetic pathway, reversibly transferring an adenylyl group from ATP onto 4′-phosphopantetheine to yield dephospho-coenzyme A and pyrophosphate. Burkholderia pseudomallei is a soil- and water-borne pathogenic bacterium and the etiologic agent of melioidosis, a potentially fatal systemic disease present in southeast Asia. Two crystal structures are presented of the PPAT from B. pseudomallei with the expectation that, because of the importance of the enzyme in coenzyme A biosynthesis, they will aid in the search for defenses against this pathogen. A crystal grown in ammonium sulfate yielded a 2.1 Å resolution structure that contained dephospho-coenzyme A with partial occupancy. The overall structure and ligand-binding interactions are quite similar to other bacterial PPAT crystal structures. A crystal grown at low pH in the presence of coenzyme A yielded a 1.6 Å resolution structure in the same crystal form. However, the experimental electron density was not reflective of fully ordered coenzyme A, but rather was only reflective of an ordered 4′-diphosphopantetheine moiety.

Refolding, crystallization and preliminary X-ray crystallographic study of the whole extracellular regions of nectins

Narita, H. — 2011-03-01

The nectin family of Ca2+-independent immunoglobulin-like cell–cell adhesion molecules contains four members. Nectins, which have three Ig-like domains in their extracellular region, form cell–cell adherens junctions cooperatively with cadherins. The whole extracellular regions of nectin-1 (nectin-1-EC) and nectin-2 (nectin-2-EC) were expressed in Escherichia coli as inclusion bodies, solubilized in 8 M urea and then refolded by rapid dilution into refolding solution. The refolded proteins were subsequently purified by three chromatographic steps and crystallized using the hanging-drop vapour-diffusion method. The nectin-1-EC crystals belonged to space group P213 and the nectin-2-EC crystals belonged to space group P6122 or P6522.

Editorial

Einspahr, H. — 2011-01-01

Crystallization and preliminary crystallographic analysis of the ADP-ribosyltransferase HopU1. Corrigendum

Lin, Y. — 2010-12-01

A correction is made to the article by Lin et al. [(2010). Acta Cryst. F66, 932–934].

Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active site

Barrack, K.L. — 2011-01-01

Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species, protozoa that are responsible for a range of serious diseases found in tropical and subtropical parts of the world. As part of a structure-based approach to inhibitor development, specifically targeting Leishmania species, well ordered crystals of L. donovani PTR1 were sought to support the characterization of complexes formed with inhibitors. An efficient system for recombinant protein production was prepared and the enzyme was purified and crystallized in an orthorhombic form with ammonium sulfate as the precipitant. Diffraction data were measured to 2.5 Å resolution and the structure was solved by molecular replacement. However, a sulfate occupies a phosphate-binding site used by NADPH and occludes cofactor binding. The nicotinamide moiety is a critical component of the active site and without it this part of the structure is disordered. The crystal form obtained under these conditions is therefore unsuitable for the characterization of inhibitor complexes.

Structure of the C-terminal domain of the surface antigen SpaP from the caries pathogen Streptococcus mutans

Nylander, Å. — 2011-01-01

SpaP is a 1500-residue adhesin expressed on the surface of the caries-implicated bacterium Streptococcus mutans. SpaP is a member of the antigen I/II (AgI/II) family of proteins expressed by oral streptococci. These surface proteins are crucial for the incorporation of streptococci into dental plaque. The structure of the C-terminal domain of SpaP (residues 1136–1489) was solved and refined to 2.2 Å resolution with six molecules in the asymmetric unit. Similar to a related AgI/II structure, SpaP is stabilized by isopeptide bonds between lysine and asparagine side chains.

Publishing structural genomics results: the JCSG Special Issue

Weiss, M.S. — 2010-11-01

Publishing structural genomics results continued: the SSGCID Special Issue

Weiss, M.S. — 2011-10-01

Citations in supplementary material

Weiss, M.S. — 2010-12-01

Crystallization and preliminary X-ray analysis of Na-ASP-1, a multi-domain pathogenesis-related-1 protein from the human hookworm parasite Necator americanus. Corrigendum

Asojo, O.A. — 2010-11-01

Data collection and instrument information reported in the article by Asojo et al. [(2005), Acta Cryst. F61, 391–394] are corrected.

Crystallization and preliminary crystallographic analysis of the central domain of Drosophila Dribble, a protein that is essential for ribosome biogenesis. Addendum

Cheng, T.-C. — 2010-12-01

An author is added to the article by Cheng et al. [(2010), Acta Cryst. F66, 546–548].

Crystallization and preliminary diffraction analysis of Wzi, a member of the capsule export and assembly pathway in Escherichia coli

Bushell, S.R. — 2010-12-01

External polysaccharide capsules provide a physical barrier that is employed by many species of bacteria for the purposes of host evasion and persistence. Wzi is a 53 kDa outer membrane β-barrel protein that is thought to play a role in the attachment of group 1 capsular polysaccharides to the cell surface. The purification and crystallization of an Escherichia coli homologue of Wzi is reported and diffraction data from native and selenomethionine-incorporated protein crystals are presented. Crystals of C-terminally His6-tagged Wzi diffracted to 2.8 Å resolution. Data processing showed that the crystals belonged to the orthorhombic space group C222, with unit-cell parameters a = 128.8, b = 152.8, c = 94.4 Å, α = β = γ = 90°. A His-tagged selenomethionine-containing variant of Wzi has also been crystallized in the same space group and diffraction data have been recorded to 3.8 Å resolution. Data processing shows that the variant crystal has similar unit-cell parameters to the native crystal.

Crystallization and preliminary X-ray diffraction analysis of the protease from Southampton norovirus complexed with a Michael acceptor inhibitor

Hussey, R.J. — 2010-11-01

Noroviruses are the predominant cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional parts. Here, the crystallization of the recombinant protease from the Southampton norovirus is described. Whilst the native crystals were found to diffract only to medium resolution (2.9 Å), cocrystals of an inhibitor complex diffracted X-rays to 1.7 Å resolution. The polypeptide inhibitor (Ac-EFQLQ-propenyl ethyl ester) possesses an amino-acid sequence designed to match the substrate specificity of the enzyme, but was synthesized with a reactive Michael acceptor group at the C-terminal end.

The JCSG high-throughput structural biology pipeline

Elsliger, M.-A. — 2010-10-01

The Joint Center for Structural Genomics high-throughput structural biology pipeline has delivered more than 1000 structures to the community over the past ten years. The JCSG has made a significant contribution to the overall goal of the NIH Protein Structure Initiative (PSI) of expanding structural coverage of the protein universe, as well as making substantial inroads into structural coverage of an entire organism. Targets are processed through an extensive combination of bioinformatics and biophysical analyses to efficiently characterize and optimize each target prior to selection for structure determination. The pipeline uses parallel processing methods at almost every step in the process and can adapt to a wide range of protein targets from bacterial to human. The construction, expansion and optimization of the JCSG gene-to-structure pipeline over the years have resulted in many technological and methodological advances and developments. The vast number of targets and the enormous amounts of associated data processed through the multiple stages of the experimental pipeline required the development of variety of valuable resources that, wherever feasible, have been converted to free-access web-based tools and applications.

Structure of a tryptophanyl-tRNA synthetase containing an iron–sulfur cluster

Han, G.W. — 2010-10-01

A novel aminoacyl-tRNA synthetase that contains an iron–sulfur cluster in the tRNA anticodon-binding region and efficiently charges tRNA with tryptophan has been found in Thermotoga maritima. The crystal structure of TmTrpRS (tryptophanyl-tRNA synthetase; TrpRS; EC 6.1.1.2) reveals an iron–sulfur [4Fe–4S] cluster bound to the tRNA anticodon-binding (TAB) domain and an l-tryptophan ligand in the active site. None of the other T. maritima aminoacyl-tRNA synthetases (AARSs) contain this [4Fe–4S] cluster-binding motif (C-x22-C-x6-C-x2-C). It is speculated that the iron–sulfur cluster contributes to the stability of TmTrpRS and could play a role in the recognition of the anticodon.

Ligands in crystal structures that aid in functional characterization

Speers, A.E. — 2010-10-01

Liganded structures can be instrumental in assigning function to uncharacterized proteins by revealing active sites, conserved residues, binding motifs, and substrate specificity. This introduction provides an overview and commentary on the value of liganded structures emerging from the JCSG structural genomics initiative.

TOPSAN: use of a collaborative environment for annotating, analyzing and disseminating data on JCSG and PSI structures

Krishna, S.S. — 2010-10-01

The NIH Protein Structure Initiative centers, such as the Joint Center for Structural Genomics (JCSG), have developed highly efficient technological platforms that are capable of experimentally determining the three-dimensional structures of hundreds of proteins per year. However, the overwhelming majority of the almost 5000 protein structures determined by these centers have yet to be described in the peer-reviewed literature. In a high-throughput structural genomics environment, the process of structure determination occurs independently of any associated experimental characterization of function, which creates a challenge for the annotation and analysis of structures and the publication of these results. This challenge has been addressed by developing TOPSAN (`The Open Protein Structure Annotation Network'), which enables the generation of knowledge via collaborations among globally distributed contributors supported by automated amalgamation of available information. TOPSAN currently provides annotations for all protein structures determined by the JCSG in addition to preliminary annotations on a large number of structures from the other PSI production centers. TOPSAN-enabled collaborations have resulted in insightful structure–function analysis for many proteins and have led to numerous peer-reviewed publications, as exemplified by the articles included in this issue of Acta Crystallographica Section F.

Comparison of NMR and crystal structures highlights conformational isomerism in protein active sites

Serrano, P. — 2010-10-01

The JCSG has recently developed a protocol for systematic comparisons of high-quality crystal and NMR structures of proteins. In this paper, the extent to which this approach can provide function-related information on the two functionally annotated proteins TM1081, a Thermotoga maritima anti-σ factor antagonist, and A2LD1 (gi:13879369), a mouse γ-glutamylamine cyclotransferase, is explored. The NMR structures of the two proteins have been determined in solution at 313 and 298 K, respectively, using the current JCSG protocol based on the software package UNIO for extensive automation. The corresponding crystal structures were solved by the JCSG at 100 K and 1.6 Å resolution and at 100 K and 1.9 Å resolution, respectively. The NMR and crystal structures of the two proteins share the same overall molecular architectures. However, the precision of the structure determination along the amino-acid sequence varies over a significantly wider range in the NMR structures than in the crystal structures. Thereby, in each of the two NMR structures about 65% of the residues have displacements below the average and in both proteins the less well ordered residues include large parts of the active sites, in addition to some highly solvent-exposed surface areas. Whereas the latter show increased disorder in the crystal and in solution, the active-site regions display increased displacements only in the NMR structures, where they undergo local conformational exchange on the millisecond time scale that appears to be frozen in the crystals. These observations suggest that a search for molecular regions showing increased structural disorder and slow dynamic processes in solution while being well ordered in the corresponding crystal structure might be a valid initial step in the challenge of identifying putative active sites in functionally unannotated proteins with known three-dimensional structure.

Structure of BT_3984, a member of the SusD/RagB family of nutrient-binding molecules

Bakolitsa, C. — 2010-10-01

The crystal structure of the Bacteroides thetaiotaomicron protein BT_3984 was determined to a resolution of 1.7 Å and was the first structure to be determined from the extensive SusD family of polysaccharide-binding proteins. SusD is an essential component of the sus operon that defines the paradigm for glycan utilization in dominant members of the human gut microbiota. Structural analysis of BT_3984 revealed an N-terminal region containing several tetratricopeptide repeats (TPRs), while the signature C-terminal region is less structured and contains extensive loop regions. Sequence and structure analysis of BT_3984 suggests the presence of binding interfaces for other proteins from the polysaccharide-utilization complex.

Mammalian cell expression, purification, crystallization and microcrystal data collection of autotaxin/ENPP2, a secreted mammalian glycoprotein

Hausmann, J. — 2010-09-01

Autotaxin (ATX or ENPP2) is a secreted glycosylated mammalian enzyme that exhibits lysophospholipase D activity, hydrolyzing lysophosphatidylcholine to the signalling lipid lysophosphatidic acid. ATX is an ∼100 kDa multi-domain protein encompassing two N-terminal somatomedin B-like domains, a central catalytic phosphodiesterase domain and a C-terminal nuclease-like domain. Protocols for the efficient expression of ATX from stably transfected mammalian HEK293 cells in amounts sufficient for crystallographic studies are reported. Purification resulted in protein that crystallized readily, but various attempts to grow crystals suitable in size for routine crystallographic structure determination were not successful. However, the available micrometre-thick plates diffracted X-rays beyond 2.0 Å resolution and allowed the collection of complete diffraction data to about 2.6 Å resolution. The problems encountered and the current advantages and limitations of diffraction data collection from thin crystal plates are discussed.

NMR in a crystallography-based high-throughput protein structure-determination environment

Wüthrich, K. — 2010-10-01

An introduction is provided to three papers which compare corresponding protein crystal and NMR solution structures determined by the Joint Center for Structural Genomics (JCSG). Special mention is made of the JCSG strategy for combined use of the two techniques, and of potential applications of the concept of `reference crystal structures', which is introduced in the following three papers.

Viewing the human microbiome through three-dimensional glasses: integrating structural and functional studies to better define the properties of myriad carbohydrate-active enzymes

Turnbaugh, P.J. — 2010-10-01

Recent studies have provided an unprecedented view of the trillions of microbes associated with the human body. The human microbiome harbors tremendous diversity at multiple levels: the species that colonize each individual and each body habitat; the genes that are found in each organism's genome; the expression of these genes and the interactions and activities of their protein products. The sources of this diversity are wide-ranging and reflect both environmental and host factors. A major challenge moving forward is defining the precise functions of members of various families of proteins represented in our microbiomes, including the highly diverse carbohydrate-active enzymes (CAZymes) involved in numerous biologically important chemical transformations, such as the degradation of complex dietary polysaccharides. Coupling metagenomic analyses to structural genomics initiatives and to biochemical and other functional assays of CAZymes will be essential for determining how these as well as other microbiome-encoded proteins operate to shape the properties of microbial communities and their human hosts.

Structure of Bacteroides thetaiotaomicron BT2081 at 2.05 Å resolution: the first structural representative of a new protein family that may play a role in carbohydrate metabolism

Yeh, A.P. — 2010-10-01

BT2081 from Bacteroides thetaiotaomicron (GenBank accession code NP_810994.1) is a member of a novel protein family consisting of over 160 members, most of which are found in the different classes of Bacteroidetes. Genome-context analysis lends support to the involvement of this family in carbohydrate metabolism, which plays a key role in B. thetaiotaomicron as a predominant bacterial symbiont in the human distal gut microbiome. The crystal structure of BT2081 at 2.05 Å resolution represents the first structure from this new protein family. BT2081 consists of an N-terminal domain, which adopts a β-sandwich immunoglobulin-like fold, and a larger C-terminal domain with a β-sandwich jelly-roll fold. Structural analyses reveal that both domains are similar to those found in various carbohydrate-active enzymes. The C-terminal β-jelly-roll domain contains a potential carbohydrate-binding site that is highly conserved among BT2081 homologs and is situated in the same location as the carbohydrate-binding sites that are found in structurally similar glycoside hydrolases (GHs). However, in BT2081 this site is partially occluded by surrounding loops, which results in a deep solvent-accessible pocket rather than a shallower solvent-exposed cleft.

A new crystal form of Lys48-linked diubiquitin

Trempe, J.-F. — 2010-09-01

Lys48-linked polyubiquitin chains are recognized by the proteasome as a tag for the degradation of the attached substrates. Here, a new crystal form of Lys48-linked diubiquitin (Ub2) was obtained and the crystal structure was refined to 1.6 Å resolution. The structure reveals an ordered isopeptide bond in a trans configuration. All three molecules in the asymmetric unit were in the same closed conformation, in which the hydrophobic patches of both the distal and the proximal moieties interact with each other. Despite the different crystallization conditions and different crystal packing, the new crystal structure of Ub2 is similar to the previously published structure of diubiquitin, but differences are observed in the conformation of the flexible isopeptide linkage.

Structure of a putative NTP pyrophosphohydrolase: YP_001813558.1 from Exiguobacterium sibiricum 255-15

Han, G.W. — 2010-10-01

The crystal structure of a putative NTPase, YP_001813558.1 from Exiguobacterium sibiricum 255-15 (PF09934, DUF2166) was determined to 1.78 Å resolution. YP_001813558.1 and its homologs (dimeric dUTPases, MazG proteins and HisE-encoded phosphoribosyl ATP pyrophosphohydrolases) form a superfamily of all-α-helical NTP pyrophosphatases. In dimeric dUTPase-like proteins, a central four-helix bundle forms the active site. However, in YP_001813558.1, an unexpected intertwined swapping of two of the helices that compose the conserved helix bundle results in a `linked dimer' that has not previously been observed for this family. Interestingly, despite this novel mode of dimerization, the metal-binding site for divalent cations, such as magnesium, that are essential for NTPase activity is still conserved. Furthermore, the active-site residues that are involved in sugar binding of the NTPs are also conserved when compared with other α-helical NTPases, but those that recognize the nucleotide bases are not conserved, suggesting a different substrate specificity.

Structure of a membrane-attack complex/perforin (MACPF) family protein from the human gut symbiont Bacteroides thetaiotaomicron

Xu, Q. — 2010-10-01

Membrane-attack complex/perforin (MACPF) proteins are transmembrane pore-forming proteins that are important in both human immunity and the virulence of pathogens. Bacterial MACPFs are found in diverse bacterial species, including most human gut-associated Bacteroides species. The crystal structure of a bacterial MACPF-domain-containing protein BT_3439 (Bth-MACPF) from B. thetaiotaomicron, a predominant member of the mammalian intestinal microbiota, has been determined. Bth-MACPF contains a membrane-attack complex/perforin (MACPF) domain and two novel C-terminal domains that resemble ribonuclease H and interleukin 8, respectively. The entire protein adopts a flat crescent shape, characteristic of other MACPF proteins, that may be important for oligomerization. This Bth-MACPF structure provides new features and insights not observed in two previous MACPF structures. Genomic context analysis infers that Bth-MACPF may be involved in a novel protein-transport or nutrient-uptake system, suggesting an important role for these MACPF proteins, which were likely to have been inherited from eukaryotes via horizontal gene transfer, in the adaptation of commensal bacteria to the host environment.

The structure of Haemophilus influenzae prephenate dehydrogenase suggests unique features of bifunctional TyrA enzymes

Chiu, H.-J. — 2010-10-01

Chorismate mutase/prephenate dehydrogenase from Haemophilus influenzae Rd KW20 is a bifunctional enzyme that catalyzes the rearrangement of chorismate to prephenate and the NAD(P)+-dependent oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate in tyrosine biosynthesis. The crystal structure of the prephenate dehydrogenase component (HinfPDH) of the TyrA protein from H. influenzae Rd KW20 in complex with the inhibitor tyrosine and cofactor NAD+ has been determined to 2.0 Å resolution. HinfPDH is a dimeric enzyme, with each monomer consisting of an N-terminal α/β dinucleotide-binding domain and a C-terminal α-helical dimerization domain. The structure reveals key active-site residues at the domain interface, including His200, Arg297 and Ser179 that are involved in catalysis and/or ligand binding and are highly conserved in TyrA proteins from all three kingdoms of life. Tyrosine is bound directly at the catalytic site, suggesting that it is a competitive inhibitor of HinfPDH. Comparisons with its structural homologues reveal important differences around the active site, including the absence of an α–β motif in HinfPDH that is present in other TyrA proteins, such as Synechocystis sp. arogenate dehydrogenase. Residues from this motif are involved in discrimination between NADP+ and NAD+. The loop between β5 and β6 in the N-terminal domain is much shorter in HinfPDH and an extra helix is present at the C-terminus. Furthermore, HinfPDH adopts a more closed conformation compared with TyrA proteins that do not have tyrosine bound. This conformational change brings the substrate, cofactor and active-site residues into close proximity for catalysis. An ionic network consisting of Arg297 (a key residue for tyrosine binding), a water molecule, Asp206 (from the loop between β5 and β6) and Arg365′ (from the additional C-terminal helix of the adjacent monomer) is observed that might be involved in gating the active site.

Structure of the γ-d-glutamyl-l-diamino acid endopeptidase YkfC from Bacillus cereus in complex with l-Ala-γ-d-Glu: insights into substrate recognition by NlpC/P60 cysteine peptidases

Xu, Q. — 2010-10-01

Dipeptidyl-peptidase VI from Bacillus sphaericus and YkfC from Bacillus subtilis have both previously been characterized as highly specific γ-d-glutamyl-l-diamino acid endopeptidases. The crystal structure of a YkfC ortholog from Bacillus cereus (BcYkfC) at 1.8 Å resolution revealed that it contains two N-terminal bacterial SH3 (SH3b) domains in addition to the C-terminal catalytic NlpC/P60 domain that is ubiquitous in the very large family of cell-wall-related cysteine peptidases. A bound reaction product (l-Ala-γ-d-Glu) enabled the identification of conserved sequence and structural signatures for recognition of l-Ala and γ-d-Glu and, therefore, provides a clear framework for understanding the substrate specificity observed in dipeptidyl-peptidase VI, YkfC and other NlpC/P60 domains in general. The first SH3b domain plays an important role in defining substrate specificity by contributing to the formation of the active site, such that only murein peptides with a free N-terminal alanine are allowed. A conserved tyrosine in the SH3b domain of the YkfC subfamily is correlated with the presence of a conserved acidic residue in the NlpC/P60 domain and both residues interact with the free amine group of the alanine. This structural feature allows the definition of a subfamily of NlpC/P60 enzymes with the same N-terminal substrate requirements, including a previously characterized cyanobacterial l-alanine-γ-d-glutamate endopeptidase that contains the two key components (an NlpC/P60 domain attached to an SH3b domain) for assembly of a YkfC-like active site.

High-quality crystals of human haematopoietic prostaglandin D synthase with novel inhibitors

Takahashi, S. — 2010-07-01

Human haematopoietic prostaglandin D synthase (H-PGDS; EC 5.3.99.2) produces prostaglandin D2, an allergic and inflammatory mediator, in mast cells and Th2 cells. H-PGDS has been crystallized with novel inhibitors with half-maximal inhibitory concentrations (IC50) in the low nanomolar range by the counter-diffusion method onboard the Russian Service Module on the International Space Station. The X-ray diffraction of a microgravity-grown crystal of H-PGDS complexed with an inhibitor with an IC50 value of 50 nM extended to 1.1 Å resolution at 100 K using SPring-8 synchrotron radiation, which is one of the highest resolutions obtained to date for this protein.

Comparison of NMR and crystal structures for the proteins TM1112 and TM1367

Mohanty, B. — 2010-10-01

The NMR structures of the TM1112 and TM1367 proteins from Thermotoga maritima in solution at 298 K were determined following a new protocol which uses the software package UNIO for extensive automation. The results obtained with this novel procedure were evaluated by comparison with the crystal structures solved by the JCSG at 100 K to 1.83 and 1.90 Å resolution, respectively. In addition, the TM1112 solution structure was compared with an NMR structure solved by the NESG using a conventional largely interactive methodology. For both proteins, the newly determined NMR structure could be superimposed with the crystal structure with r.m.s.d. values of <1.0 Å for the backbone heavy atoms, which provided a starting platform to investigate local structure variations, which may arise from either the methods used or from the different chemical environments in solution and in the crystal. Thereby, these comparative studies were further explored with the use of reference NMR and crystal structures, which were computed using the NMR software with input of upper-limit distance constraints derived from the molecular models that represent the results of structure determination by NMR and by X-ray diffraction, respectively. The results thus obtained show that NMR structure calculations with the new automated UNIO software used by the JCSG compare favorably with those from a more labor-intensive and time-intensive interactive procedure. An intriguing observation is that the `bundles' of two TM1112 or three TM1367 molecules in the asymmetric unit of the crystal structures mimic the behavior of the bundles of 20 conformers used to represent the NMR solution structures when comparing global r.m.s.d. values calculated either for the polypeptide backbone, the core residues with solvent accessibility below 15% or all heavy atoms.

Structures of three members of Pfam PF02663 (FmdE) implicated in microbial methanogenesis reveal a conserved α+β core domain and an auxiliary C-terminal treble-clef zinc finger

Axelrod, H.L. — 2010-10-01

Examination of the genomic context for members of the FmdE Pfam family (PF02663), such as the protein encoded by the fmdE gene from the methanogenic archaeon Methanobacterium thermoautotrophicum, indicates that 13 of them are co-transcribed with genes encoding subunits of molybdenum formylmethanofuran dehydrogenase (EC 1.2.99.5), an enzyme that is involved in microbial methane production. Here, the first crystal structures from PF02663 are described, representing two bacterial and one archaeal species: B8FYU2_DESHY from the anaerobic dehalogenating bacterium Desulfitobacterium hafniense DCB-2, Q2LQ23_SYNAS from the syntrophic bacterium Syntrophus aciditrophicus SB and Q9HJ63_THEAC from the thermoacidophilic archaeon Thermoplasma acidophilum. Two of these proteins, Q9HJ63_THEAC and Q2LQ23_SYNAS, contain two domains: an N-terminal thioredoxin-like α+β core domain (NTD) consisting of a five-stranded, mixed β-sheet flanked by several α-helices and a C-terminal zinc-finger domain (CTD). B8FYU2_DESHY, on the other hand, is composed solely of the NTD. The CTD of Q9HJ63_THEAC and Q2LQ23_SYNAS is best characterized as a treble-clef zinc finger. Two significant structural differences between Q9HJ63_THEAC and Q2LQ23_SYNAS involve their metal binding. First, zinc is bound to the putative active site on the NTD of Q9HJ63_THEAC, but is absent from the NTD of Q2LQ23_SYNAS. Second, whereas the structure of the CTD of Q2LQ23_SYNAS shows four Cys side chains within coordination distance of the Zn atom, the structure of Q9HJ63_THEAC is atypical for a treble-cleft zinc finger in that three Cys side chains and an Asp side chain are within coordination distance of the zinc.

New variants of known folds: do they bring new biology?

Koonin, E.V. — 2010-10-01

New distinct versions of known protein folds provide a powerful means of protein-function prediction that complements sequence and genomic context analysis. These structures do not supplant direct biochemical experiments, but are indispensable for the complete characterization of proteins.

Ligands in PSI structures

Kumar, A. — 2010-10-01

Approximately 65% of PSI structures report some type of ligand(s) that is bound in the crystal structure. Here, a description is given of how such ligands are handled and analyzed at the JCSG and a survey of the types, variety and frequency of ligands that are observed in the PSI structures is also compiled and analyzed, including illustrations of how these bound ligands have provided functional clues for annotation of proteins with little or no previous experimental characterization. Furthermore, a web server was developed as a tool to mine and analyze the PSI structures for bound ligands and other identifying features.

Structure of the first representative of Pfam family PF04016 (DUF364) reveals enolase and Rossmann-like folds that combine to form a unique active site with a possible role in heavy-metal chelation

Miller, M.D. — 2010-10-01

The crystal structure of Dhaf4260 from Desulfitobacterium hafniense DCB-2 was determined by single-wavelength anomalous diffraction (SAD) to a resolution of 2.01 Å using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). This protein structure is the first representative of the PF04016 (DUF364) Pfam family and reveals a novel combination of two well known domains (an enolase N-terminal-like fold followed by a Rossmann-like domain). Structural and bioinformatic analyses reveal partial similarities to Rossmann-like methyltransferases, with residues from the enolase-like fold combining to form a unique active site that is likely to be involved in the condensation or hydrolysis of molecules implicated in the synthesis of flavins, pterins or other siderophores. The genome context of Dhaf4260 and homologs additionally supports a role in heavy-metal chelation.

Structural classification of proteins and structural genomics: new insights into protein folding and evolution

Andreeva, A. — 2010-10-01

During the past decade, the Protein Structure Initiative (PSI) centres have become major contributors of new families, superfamilies and folds to the Structural Classification of Proteins (SCOP) database. The PSI results have increased the diversity of protein structural space and accelerated our understanding of it. This review article surveys a selection of protein structures determined by the Joint Center for Structural Genomics (JCSG). It presents previously undescribed β-sheet architectures such as the double barrel and spiral β-roll and discusses new examples of unusual topologies and peculiar structural features observed in proteins characterized by the JCSG and other Structural Genomics centres.

A conserved fold for fimbrial components revealed by the crystal structure of a putative fimbrial assembly protein (BT1062) from Bacteroides thetaiotaomicron at 2.2 Å resolution

Xu, Q. — 2010-10-01

BT1062 from Bacteroides thetaiotaomicron is a homolog of Mfa2 (PGN0288 or PG0179), which is a component of the minor fimbriae in Porphyromonas gingivalis. The crystal structure of BT1062 revealed a conserved fold that is widely adopted by fimbrial components.

NMR structure of the protein NP_247299.1: comparison with the crystal structure

Jaudzems, K. — 2010-10-01

The NMR structure of the protein NP_247299.1 in solution at 313 K has been determined and is compared with the X-ray crystal structure, which was also solved in the Joint Center for Structural Genomics (JCSG) at 100 K and at 1.7 Å resolution. Both structures were obtained using the current largely automated crystallographic and solution NMR methods used by the JCSG. This paper assesses the accuracy and precision of the results from these recently established automated approaches, aiming for quantitative statements about the location of structure variations that may arise from either one of the methods used or from the different environments in solution and in the crystal. To evaluate the possible impact of the different software used for the crystallographic and the NMR structure determinations and analysis, the concept is introduced of reference structures, which are computed using the NMR software with input of upper-limit distance constraints derived from the molecular models representing the results of the two structure determinations. The use of this new approach is explored to quantify global differences that arise from the different methods of structure determination and analysis versus those that represent interesting local variations or dynamics. The near-identity of the protein core in the NMR and crystal structures thus provided a basis for the identification of complementary information from the two different methods. It was thus observed that locally increased crystallographic B values correlate with dynamic structural polymorphisms in solution, including that the solution state of the protein involves a slow dynamic equilibrium on a time scale of milliseconds or slower between two ensembles of rapidly interchanging conformers that contain, respectively, the cis or trans form of the C-terminal proline and represent about 25 and 75% of the total protein.

The structure of SSO2064, the first representative of Pfam family PF01796, reveals a novel two-domain zinc-ribbon OB-fold architecture with a potential acyl-CoA-binding role

Krishna, S.S. — 2010-10-01

SSO2064 is the first structural representative of PF01796 (DUF35), a large prokaryotic family with a wide phylogenetic distribution. The structure reveals a novel two-domain architecture comprising an N-terminal, rubredoxin-like, zinc ribbon and a C-terminal, oligonucleotide/oligosaccharide-binding (OB) fold domain. Additional N-terminal helical segments may be involved in protein–protein interactions. Domain architectures, genomic context analysis and functional evidence from certain bacterial representatives of this family suggest that these proteins form a novel fatty-acid-binding component that is involved in the biosynthesis of lipids and polyketide antibiotics and that they possibly function as acyl-CoA-binding proteins. This structure has led to a re-evaluation of the DUF35 family, which has now been split into two entries in the latest Pfam release (v.24.0).

Evaluating the efficacy of tryptophan fluorescence and absorbance as a selection tool for identifying protein crystals

Gill, H.S. — 2010-03-01

The environment of individual tryptophans in known protein structures and the effectiveness of four commercial robotic UV microscopes to illuminate tryptophan-containing protein crystals by either tryptophan fluorescence (epi-illumination) or absorbance (transmission) are evaluated. In agreement with other studies, tryptophan residues are found on average to be largely buried in protein structures (with ∼84% of their surface area buried) and to be surrounded by partially polar microenvironments (with ∼43% of their surface area covered by polar residues), which suggests an inherent degree of fluorescence signal quenching. In bacterial genomes, up to one-third (∼18.5% on average) of open reading frames are deficient in tryptophan. In the laboratory, because of the attenuation of UV light by the media commonly used in sitting-drop and hanging-drop crystallization trials, it was often necessary to simplify the light path by manually removing or inverting the supporting media. Prolonged exposure (minutes) to UV light precipitates some protein samples. The absorbance spectra of many commercially available media in crystallization trials are presented. The advantages of using tryptophan absorbance over fluorescence for characterizing crystals are discussed.

DUFs: families in search of function

Bateman, A. — 2010-10-01

Domains of unknown function (DUFs) are a large set of uncharacterized protein families that are found in the Pfam database. Here, the scale and growth of functionally uncharacterized families in biological databases are surveyed and the prospects for discovering their function are examined. In particular, the important role that structural genomics can play in identifying potential function is evaluated.

Crystals on the cover and validation reports

Einspahr, H. — 2010-01-01

Expression, purification and preliminary X-ray crystallographic analysis of the chicken MHC class I molecule YF1*7.1. Addendum

Hee, C.S. — 2010-02-01

Additional funding is acknowledged by the authors of Hee et al. [Acta Cryst. (2009), F65, 422–425].

Structures of the first representatives of Pfam family PF06684 (DUF1185) reveal a novel variant of the Bacillus chorismate mutase fold and suggest a role in amino-acid metabolism

Bakolitsa, C. — 2010-10-01

The crystal structures of BB2672 and SPO0826 were determined to resolutions of 1.7 and 2.1 Å by single-wavelength anomalous dispersion and multiple-wavelength anomalous dispersion, respectively, using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). These proteins are the first structural representatives of the PF06684 (DUF1185) Pfam family. Structural analysis revealed that both structures adopt a variant of the Bacillus chorismate mutase fold (BCM). The biological unit of both proteins is a hexamer and analysis of homologs indicates that the oligomer interface residues are highly conserved. The conformation of the critical regions for oligomerization appears to be dependent on pH or salt concentration, suggesting that this protein might be subject to environmental regulation. Structural similarities to BCM and genome-context analysis suggest a function in amino-acid synthesis.

Structures of the first representatives of Pfam family PF06938 (DUF1285) reveal a new fold with repeated structural motifs and possible involvement in signal transduction

Han, G.W. — 2010-10-01

The crystal structures of SPO0140 and Sbal_2486 were determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). The structures revealed a conserved core with domain duplication and a superficial similarity of the C-terminal domain to pleckstrin homology-like folds. The conservation of the domain interface indicates a potential binding site that is likely to involve a nucleotide-based ligand, with genome-context and gene-fusion analyses additionally supporting a role for this family in signal transduction, possibly during oxidative stress.

The structure of BVU2987 from Bacteroides vulgatus reveals a superfamily of bacterial periplasmic proteins with possible inhibitory function

Das, D. — 2010-10-01

Proteins that contain the DUF2874 domain constitute a new Pfam family PF11396. Members of this family have predominantly been identified in microbes found in the human gut and oral cavity. The crystal structure of one member of this family, BVU2987 from Bacteroides vulgatus, has been determined, revealing a β-lactamase inhibitor protein-like structure with a tandem repeat of domains. Sequence analysis and structural comparisons reveal that BVU2987 and other DUF2874 proteins are related to β-lactamase inhibitor protein, PepSY and SmpA_OmlA proteins and hence are likely to function as inhibitory proteins.

Purification, crystallization and preliminary X-ray analysis of a deletion mutant of a major buckwheat allergen

Kezuka, Y. — 2009-12-01

A 16 kDa buckwheat protein (BWp16) is a major allergen responsible for immediate hypersensitivity reactions including anaphylaxis. A deletion mutant of BWp16 (rBWp16ΔN) was overproduced and purified and was shown to be immunologically active. A three-wavelength MAD data set was collected from a crystal of selenomethionine-labelled rBWp16ΔN. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 28.39, b = 31.54, c = 32.20 Å, α = 111.92, β = 108.91, γ = 98.74°. One monomer was expected to be present in the asymmetric unit based on the calculated Matthews coefficient of 1.76 Å3 Da−1.

Open and closed conformations of two SpoIIAA-like proteins (YP_749275.1 and YP_001095227.1) provide insights into membrane association and ligand binding

Kumar, A. — 2010-10-01

The crystal structures of the proteins encoded by the YP_749275.1 and YP_001095227.1 genes from Shewanella frigidimarina and S. loihica, respectively, have been determined at 1.8 and 2.25 Å resolution, respectively. These proteins are members of a novel family of bacterial proteins that adopt the α/β SpoIIAA-like fold found in STAS and CRAL-TRIO domains. Despite sharing 54% sequence identity, these two proteins adopt distinct conformations arising from different dispositions of their α2 and α3 helices. In the `open' conformation (YP_001095227.1), these helices are 15 Å apart, leading to the creation of a deep nonpolar cavity. In the `closed' structure (YP_749275.1), the helices partially unfold and rearrange, occluding the cavity and decreasing the solvent-exposed hydrophobic surface. These two complementary structures are reminiscent of the conformational switch in CRAL-TRIO carriers of hydrophobic compounds. It is suggested that both proteins may associate with the lipid bilayer in their `open' monomeric state by inserting their amphiphilic helices, α2 and α3, into the lipid bilayer. These bacterial proteins may function as carriers of nonpolar substances or as interfacially activated enzymes.

Laboratory Communications in Acta Crystallographica Section F

Weiss, M.S. — 2009-10-01

Structure of the first representative of Pfam family PF09410 (DUF2006) reveals a structural signature of the calycin superfamily that suggests a role in lipid metabolism

Chiu, H.-J. — 2010-10-01

The first structural representative of the domain of unknown function DUF2006 family, also known as Pfam family PF09410, comprises a lipocalin-like fold with domain duplication. The finding of the calycin signature in the N-terminal domain, combined with remote sequence similarity to two other protein families (PF07143 and PF08622) implicated in isoprenoid metabolism and the oxidative stress response, support an involvement in lipid metabolism. Clusters of conserved residues that interact with ligand mimetics suggest that the binding and regulation sites map to the N-terminal domain and to the interdomain interface, respectively.

Crystallization and crystal-packing studies of Chlorella virus deoxyuridine triphosphatase

Homma, K. — 2009-10-01

The 141-amino-acid deoxyuridine triphosphatase (dUTPase) from the algal Chlorella virus IL-3A and its Glu81Ser/Thr84Arg-mutant derivative Mu-22 were crystallized using the hanging-drop vapor-diffusion method at 298 K with polyethylene glycol as the precipitant. An apo IL-3A dUTPase with an amino-terminal T7 epitope tag and a carboxy-terminal histidine tag yielded cubic P213 crystals with unit-cell parameter a = 106.65 Å. In the presence of dUDP, the enzyme produced thin stacked orthorhombic P222 crystals with unit-cell parameters a = 81.0, b = 96.2, c = 132.8 Å. T7-histidine-tagged Mu-22 dUTPase formed thin stacked rectangular crystals. Amino-terminal histidine-tagged dUTPases did not crystallize but formed aggregates. Glycyl-seryl-tagged dUTPases yielded cubic P213 IL-3A crystals with unit-cell parameter a = 105.68 Å and hexagonal P63 Mu-22 crystals with unit-cell parameters a = 132.07, c = 53.45 Å, γ = 120°. Owing to the Thr84Arg mutation, Mu-22 dUTPase had different monomer-to-monomer interactions to those of IL-3A dUTPase.

Overexpression, purification and preliminary X-ray diffraction analysis of the controller protein C.Csp231I from Citrobacter sp. RFL231

Streeter, S.D. — 2009-09-01

Restriction–modification controller proteins play an essential role in regulating the temporal expression of restriction–modification genes. The controller protein C.Csp231I represents a new class of controller proteins. The gene was sublconed to allow overexpression in Escherichia coli. The protein was purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.0 Å resolution and belonged to space group P21. An electrophoretic mobility-shift assay provided evidence of strong binding of C.Csp231I to a sequence located upstream of the csp231IC start codon.

Crystallization and crystallographic analysis of the apo form of the orange protein (ORP) from Desulfovibrio gigas. Corrigendum

Najmudin, S. — 2009-08-01

A correction is made to the name of one of the authors in the article by Najmudin et al. [Acta Cryst. (2009). F65, 730–732].

The structure of Jann_2411 (DUF1470) from Jannaschia sp. at 1.45 Å resolution reveals a new fold (the ABATE domain) and suggests its possible role as a transcription regulator

Bakolitsa, C. — 2010-10-01

The crystal structure of Jann_2411 from Jannaschia sp. strain CCS1, a member of the Pfam PF07336 family classified as a domain of unknown function (DUF1470), was solved to a resolution of 1.45 Å by multiple-wavelength anomalous dispersion (MAD). This protein is the first structural representative of the DUF1470 Pfam family. Structural analysis revealed a two-domain organization, with the N-terminal domain presenting a new fold called the ABATE domain that may bind an as yet unknown ligand. The C-terminal domain forms a treble-clef zinc finger that is likely to be involved in DNA binding. Analysis of the Jann_2411 protein and the broader ABATE-domain family suggests a role as stress-induced transcriptional regulators.

Crystallization and preliminary crystallographic analysis of the bacterial capsule assembly-regulating tyrosine phosphatases Wzb of Escherichia coli and Cps4B of Streptococcus pneumoniae

Huang, H. — 2009-08-01

Bacterial tyrosine kinases and their cognate phosphatases are key players in the regulation of capsule assembly and thus are important virulence determinants of these bacteria. Examples of the kinase/phosphatase pairing are found in Gram-negative bacteria such as Escherichia coli (Wzc and Wzb) and in Gram-positive bacteria such as Streptococcus pneumoniae (CpsCD and CpsB). Although Wzb and Cps4B are both predicted to dephosphorylate the C-terminal tyrosine cluster of their cognate tyrosine kinase, they appear on the basis of protein sequence to belong to quite different enzyme classes. Recombinant purified proteins Cps4B of S. pneumoniae TIGR4 and Wzb of E. coli K-30 have been crystallized. Wzb crystals belonged to space-group family P3x21 and diffracted to 2.7 Å resolution. Crystal form I of Cps4B belonged to space-group family P4x212 and diffracted to 2.8 Å resolution; crystal form II belonged to space group P212121 and diffracted to 1.9 Å resolution.

Structure of LP2179, the first representative of Pfam family PF08866, suggests a new fold with a role in amino-acid metabolism

Bakolitsa, C. — 2010-10-01

The structure of LP2179, a member of the PF08866 (DUF1831) family, suggests a novel α+β fold comprising two β-sheets packed against a single helix. A remote structural similarity to two other uncharacterized protein families specific to the Bacillus genus (PF08868 and PF08968), as well as to prokaryotic S-adenosylmethionine decarboxylases, is consistent with a role in amino-acid metabolism. Genomic neighborhood analysis of LP2179 supports this functional assignment, which might also then be extended to PF08868 and PF08968.

Production, crystallization and preliminary crystallographic analysis of an exosite-containing fragment of human von Willebrand factor-cleaving proteinase ADAMTS13

Akiyama, M. — 2009-07-01

ADAMTS13 is a reprolysin-type metalloproteinase belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motif) family. It specifically cleaves plasma von Willebrand factor (VWF) and regulates platelet adhesion and aggregation. ADAMTS13 is a multi-domain enzyme. In addition to the N-terminal metalloproteinase domain, the ancillary domains, including a disintegrin-like domain, a thrombospondin-1 type 1 repeat, a Cys-rich domain and a spacer domain, are required for VWF recognition and cleavage. In the present study, a fragment of the ADAMTS13 ancillary domains (ADAMTS13-DTCS; residues 287–685) was expressed using CHO Lec cells, purified and crystallized. Diffraction data sets were collected using the SPring-8 beamline. Two ADAMTS13-DTCS crystals with distinct unit-cell parameters generated data sets to 2.6 and 2.8 Å resolution, respectively.

The structure of the first representative of Pfam family PF09836 reveals a two-domain organization and suggests involvement in transcriptional regulation

Das, D. — 2010-10-01

Proteins with the DUF2063 domain constitute a new Pfam family, PF09836. The crystal structure of a member of this family, NGO1945 from Neisseria gonorrhoeae, has been determined and reveals that the N-terminal DUF2063 domain is likely to be a DNA-binding domain. In conjunction with the rest of the protein, NGO1945 is likely to be involved in transcriptional regulation, which is consistent with genomic neighborhood analysis. Of the 216 currently known proteins that contain a DUF2063 domain, the most significant sequence homologs of NGO1945 (∼40–99% sequence identity) are from various Neisseria and Haemophilus species. As these are important human pathogens, NGO1945 represents an interesting candidate for further exploration via biochemical studies and possible therapeutic intervention.

The structure of the first representative of Pfam family PF06475 reveals a new fold with possible involvement in glycolipid metabolism

Bakolitsa, C. — 2010-10-01

The crystal structure of PA1994 from Pseudomonas aeruginosa, a member of the Pfam PF06475 family classified as a domain of unknown function (DUF1089), reveals a novel fold comprising a 15-stranded β-sheet wrapped around a single α-helix that assembles into a tight dimeric arrangement. The remote structural similarity to lipoprotein localization factors, in addition to the presence of an acidic pocket that is conserved in DUF1089 homologs, phospholipid-binding and sugar-binding proteins, indicate a role for PA1994 and the DUF1089 family in glycolipid metabolism. Genome-context analysis lends further support to the involvement of this family of proteins in glycolipid metabolism and indicates possible activation of DUF1089 homologs under conditions of bacterial cell-wall stress or host–pathogen interactions.

Structure of an essential bacterial protein YeaZ (TM0874) from Thermotoga maritima at 2.5 Å resolution

Xu, Q. — 2010-10-01

YeaZ is involved in a protein network that is essential for bacteria. The crystal structure of YeaZ from Thermotoga maritima was determined to 2.5 Å resolution. Although this protein belongs to a family of ancient actin-like ATPases, it appears that it has lost the ability to bind ATP since it lacks some key structural features that are important for interaction with ATP. A conserved surface was identified, supporting its role in the formation of protein complexes.

Conformational changes associated with the binding of zinc acetate at the putative active site of XcTcmJ, a cupin from Xanthomonas campestris pv. campestris

Axelrod, H.L. — 2010-10-01

In the plant pathogen Xanthomonas campestris pv. campestris, the product of the tcmJ gene, XcTcmJ, encodes a protein belonging to the RmlC family of cupins. XcTcmJ was crystallized in a monoclinic space group (C2) in the presence of zinc acetate and the structure was determined to 1.6 Å resolution. Previously, the apo structure has been reported in the absence of any bound metal ion [Chin et al. (2006), Proteins, 65, 1046–1050]. The most significant difference between the apo structure and the structure of XcTcmJ described here is a reorganization of the binding site for zinc acetate, which was most likely acquired from the crystallization solution. This site is located in the conserved metal ion-binding domain at the putative active site of XcTcmJ. In addition, an acetate was also bound within coordination distance of the zinc. In order to accommodate this binding, rearrangement of a conserved histidine ligand is required as well as several nearby residues within and around the putative active site. These observations indicate that binding of zinc serves a functional role in this cupin protein.

The structure of KPN03535 (gi|152972051), a novel putative lipoprotein from Klebsiella pneumoniae, reveals an OB-fold

Das, D. — 2010-10-01

KPN03535 (gi|152972051) is a putative lipoprotein of unknown function that is secreted by Klebsiella pneumoniae MGH 78578. The crystal structure reveals that despite a lack of any detectable sequence similarity to known structures, it is a novel variant of the OB-fold and structurally similar to the bacterial Cpx-pathway protein NlpE, single-stranded DNA-binding (SSB) proteins and toxins. K. pneumoniae MGH 78578 forms part of the normal human skin, mouth and gut flora and is an opportunistic pathogen that is linked to about 8% of all hospital-acquired infections in the USA. This structure provides the foundation for further investigations into this divergent member of the OB-fold family.

Geobacillus stearothermophilus 6-phosphogluconate dehydrogenase complexed with 6-phosphogluconate

Cameron, S. — 2009-05-01

Two crystal structures of recombinant Geobacillus stearothermophilus 6-phosphogluconate dehydrogenase (Gs6PDH) in complex with the substrate 6-phosphogluconate have been determined at medium resolution. Gs6PDH shares significant sequence identity and structural similarity with the enzymes from Lactococcus lactis, sheep liver and the protozoan parasite Trypanosoma brucei, for which a range of structures have previously been reported. Comparisons indicate that amino-acid sequence conservation is more pronounced in the two domains that contribute to the architecture of the active site, namely the N-terminal and C-terminal domains, compared with the central domain, which is primarily involved in the subunit–subunit associations required to form a stable dimer. The active-site residues are highly conserved, as are the interactions with the 6-phosphogluconate. There is interest in 6PDH as a potential drug target for the protozoan parasite T. brucei, the pathogen responsible for African sleeping sickness. The recombinant T. brucei enzyme has proven to be recalcitrant to enzyme–ligand studies and a surrogate protein might offer new opportunities to investigate and characterize 6PDH inhibitors. The high degree of structural similarity, efficient level of expression and straightforward crystallization conditions mean that Gs6PDH may prove to be an appropriate model system for structure-based inhibitor design targeting the enzyme from Trypanosoma species.

Structure of N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) from Mycobacterium tuberculosis in a cubic space group

Verma, S.K. — 2009-05-01

GlmU is a bifunctional enzyme that catalyzes the final two steps in the biosynthesis of UDP-GlcNAc. Crystals of GlmU from Mycobacterium tuberculosis obtained using ammonium sulfate as a precipitant diffracted poorly (to 3.4 Å resolution) and displayed an unusually high solvent content (>80%) with sparse crystal packing that resulted in large solvent channels. With one molecule per asymmetric unit, the monomers from three neighbouring asymmetric units related by the crystal threefold formed a biological trimer. Although this is the first report of the structure of GlmU determined in a cubic crystal form, the trimeric arrangement here is similar to that observed for other GlmU structures determined in hexagonal (H3, H32, P6322) space groups.

The structure of NMB1585, a MarR-family regulator from Neisseria meningitidis

Nichols, C.E. — 2009-03-01

The structure of the MarR-family transcription factor NMB1585 from Neisseria meningitidis has been solved using data extending to a resolution of 2.1 Å. Overall, the dimeric structure resembles those of other MarR proteins, with each subunit comprising a winged helix–turn–helix (wHtH) domain connected to an α-helical dimerization domain. The spacing of the recognition helices of the wHtH domain indicates that NMB1585 is pre-configured for DNA binding, with a putative inducer pocket that is largely occluded by the side chains of two aromatic residues (Tyr29 and Trp53). NMB1585 was shown to bind to its own promoter region in a gel-shift assay, indicating that the protein acts as an auto-repressor.

Crystallization and preliminary X-ray diffraction analyses of several forms of the CfaB major subunit of enterotoxigenic Escherichia coli CFA/I fimbriae

Li, Y.-F. — 2009-03-01

Enterotoxigenic Escherichia coli (ETEC), a major global cause of diarrhea, initiates the pathogenic process via fimbriae-mediated attachment to the small intestinal epithelium. A common prototypic ETEC fimbria, colonization factor antigen I (CFA/I), consists of a tip-localized minor adhesive subunit CfaE and the stalk-forming major subunit CfaB, both of which are necessary for fimbrial assembly. To elucidate the structure of CFA/I at atomic resolution, three recombinant proteins were generated consisting of fusions of the minor and major subunits (CfaEB) and of two (CfaBB) and three (CfaBBB) repeats of the major subunit. Crystals of CfaEB diffracted X-rays to 2.1 Å resolution and displayed the symmetry of space group P21. CfaBB exhibited a crystal diffraction limit of 2.3 Å resolution and had the symmetry of space group P21212. CfaBBB crystallized in the monoclinic space group C2 and diffracted X-rays to 2.3 Å resolution. These structures were determined using the molecular-replacement method.

Substrate binding induces structural changes in cytochrome P450cam

Sakurai, K. — 2009-02-01

The binding of (+)-camphor to cytochrome P450cam (P450cam) expels a cluster of waters at the active site, raising the redox potential of the haem to an extent that allows reduction by the electron-transfer system. This binding was reported to involve no significant structural changes in the protein. Here, two ferric P450cam structures partially complexed with (+)-camphor were determined by X-ray crystallography at 1.30–1.35 Å resolution, revealing the structures of the substrate-free and substrate-bound forms. (+)-Camphor binding induces rotation of Thr101 to form a hydrogen bond that acts as a hydrogen donor to a peripheral haem propionate. This bonding contributes to the redox-potential change.

Crystallization and preliminary X-ray diffraction analysis of the complex between a human anti-interferon antibody fragment and human interferon α-2A

Oganesyan, V. — 2009-01-01

Recombinant human interferon α-2A (rhIFN-α-2A) has been crystallized in complex with the recombinantly produced Fab fragment of a therapeutic monoclonal antibody (MEDI545; IgG1/κ) which targets several human interferon α subtypes. This constitutes the first reported crystals of a human type I interferon bound to an antibody. The orthorhombic crystals belonged to either space group I222 or I212121, with unit-cell parameters a = 134.82, b = 153.26, c = 163.49 Å. The diffraction of the crystals extended to 3.0 Å resolution. The asymmetric unit contained two Fab–rhIFN-α-2A complexes. This corresponded to a crystal volume per protein weight (VM) of 3.02 Å3 Da−1 and a solvent content of 59.3%. The corresponding three-dimensional structure is expected to shed light on the mechanism of action of MEDI545 and the molecular basis of its specificity.

Purification, crystallization and X-ray structures of the two manganese superoxide dismutases from Caenorhabditis elegans

Trinh, C.H. — 2008-12-01

Caenorhabditis elegans expresses two manganese superoxide dismutase enzymes (MnSOD-2 and MnSOD-3) that are targeted to the mitochondrion. MnSOD-2 is constitutively expressed, while synthesis of MnSOD-3 is inducible. The structures of these two mononuclear metalloenzymes have been determined to 1.8 and 1.7 Å resolution, respectively. Pink crystals formed in space group P41212 for each, with unit-cell parameters a = b = 81.0, c = 137.4 Å for MnSOD-2 and a = b = 81.8, c = 136.0 Å for MnSOD-3. The final structure of MnSOD-3 was refined to R = 21.6% and Rfree = 26.2% at 293 K, and R = 18.9% and Rfree = 22.6% at 100 K, while that of MnSOD-2 was refined to R = 16.9% and Rfree = 20.1% at 100 K. The asymmetric unit cell is comprised of two subunits. The resulting structures are very similar to that of human MnSOD and form a tetramer corresponding to a dimer of dimers. The subunit interface between dimers is comprised of two four-helix bundles that stabilize the biologically significant homotetramer.

Structure of a xenon derivative of Escherichia coli copper amine oxidase: confirmation of the proposed oxygen-entry pathway

Pirrat, P. — 2008-12-01

The mechanism of molecular oxygen entry into the buried active site of the copper amine oxidase family has been investigated in several family members using biochemical, structural and in silico methods. These studies have revealed a structurally conserved β-sandwich which acts as a hydrophobic reservoir from which molecular oxygen can take several species-specific preferred pathways to the active site. Escherichia coli copper amine oxidase (ECAO) possesses an extra N-terminal domain that lies close to one entrance to the β-sandwich. In order to investigate whether the presence of this domain alters molecular oxygen entry in this enzyme, xenon was used as a molecular oxygen binding-site probe. The resulting 2.5 Å resolution X-ray crystal structure reveals xenon bound in similar positions to those observed in xenon-derivative crystal structures of other family members, suggesting that the N-terminal domain does not affect oxygen entry and that the E. coli enzyme takes up oxygen in a similar manner to the rest of the copper amine oxidase family.

Crystallization and preliminary diffraction studies of prostaglandin E2-specific monoclonal antibody Fab fragment in the ligand complex

Kurahashi, Y. — 2008-11-01

Prostaglandin E2 is a major lipid mediator that regulates diverse biological processes. To elucidate how prostaglandin E2 is recognized specifically by its antibody, the Fab fragment of a monoclonal anti-prostaglandin E2 antibody was prepared and its complex with prostaglandin E2 was crystallized. The stable Fab–prostaglandin E2 complex was prepared by gel-filtration chromatography. Crystals were obtained by the microbatch method at 277 K using polyethylene glycol 4000 as a precipitant. A diffraction data set was collected to 2.2 Å resolution. The crystals belonged to space group P212121, with unit-cell parameters a = 70.3, b = 81.8, c = 82.2 Å. The asymmetric unit was suggested to contain one molecule of the Fab–prostaglandin E2 complex, with a corresponding crystal volume per protein weight of 2.75 Å3 Da−1.

Structure of the catalytic domain of Streptococcus pneumoniae sialidase NanA

Xu, G. — 2008-09-01

Streptococcus pneumoniae genomes encode three sialidases, NanA, NanB and NanC, which are key virulence factors that remove sialic acids from various glycoconjugates. The enzymes have potential as drug targets and also as vaccine candidates. The 115 kDa NanA is the largest of the three sialidases and is anchored to the bacterial membrane. Although recombinantly expressed full-length NanA was soluble, it failed to crystallize; therefore, a 56.5 kDa domain that retained full enzyme activity was subcloned. The purified enzyme was crystallized in 0.1 M MES pH 6.5, 30%(w/v) PEG 4000 using the sitting-drop vapour-diffusion method. Data were collected at 100 K to 2.5 Å resolution from a crystal grown in the presence of the inhibitor 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid. The crystal belongs to space group P212121, with unit-cell parameters a = 49.2, b = 95.6, c = 226.6 Å. The structure was solved by molecular replacement and refined to final R and Rfree factors of 0.246 and 0.298, respectively.

Crystallization and preliminary X-ray structural studies of human prouroguanylin. Corrigendum

Ito, L. — 2008-08-01

A correction is made to the list of authors for Ito et al. [Acta Cryst. (2008). F64, 531–532].

Preliminary crystallographic data of the three homologues of the thiol–disulfide oxidoreductase DsbA in Neisseria meningitidis. Corrigendum

Lafaye, C. — 2008-08-01

A correction is made to the name one of the authors of Lafaye et al. [Acta Cryst. (2008). F64, 111–114].

Cloning, purification and crystallization of MotB, a stator component of the proton-driven bacterial flagellar motor

O'Neill, J. — 2008-06-01

MotB is an essential component of the proton motive force-driven bacterial flagellar motor. It binds to the stress-bearing layer of peptidoglycan in the periplasm, anchoring the MotA/MotB stator unit to the cell wall. Proton flow through the channel formed by the transmembrane helices of MotA and MotB generates the turning force (torque) applied to the rotor. Crystals of recombinant Helicobacter pylori MotB have been obtained by the sitting-drop vapour-diffusion method using ammonium sulfate as a precipitant. These crystals belong to space group P41212 or its enantiomorph P43212, with unit-cell parameters a = 75.2, b = 75.2, c = 124.7 Å. The asymmetric unit appears to contain one subunit, corresponding to a packing density of 3.4 Å3 Da−1. The crystals diffract X-rays to at least 1.8 Å resolution on a synchrotron-radiation source.

Crystallization of hepatocyte nuclear factor 4α (HNF4α) in complex with the HNF1α promoter element

Lu, P. — 2008-04-01

Hepatocyte nuclear factor 4α (HNF4α) is a member of the nuclear receptor superfamily that plays a central role in organ development and metabolic functions. Mutations on HNF4α cause maturity-onset diabetes of the young (MODY), a dominant monogenic cause of diabetes. In order to understand the molecular mechanism of promoter recognition and the molecular basis of disease-causing mutations, the recombinant HNF4α DNA-binding domain was prepared and used in a study of its binding properties and in crystallization with a 21-mer DNA fragment that contains the promoter element of another MODY gene, HNF1α. The HNF4α protein displays a cooperative and specific DNA-binding activity towards its target gene-recognition elements. Crystals of the complex diffract to 2.0 Å using a synchrotron-radiation source under cryogenic (100 K) conditions and belong to space group C2, with unit-cell parameters a = 121.63, b = 35.43, c = 70.99 Å, β = 119.36°. A molecular-replacement solution has been obtained and structure refinement is in progress. This structure and the binding studies will provide the groundwork for detailed functional and biochemical studies of the MODY mutants.

Cloning, purification and preliminary X-ray analysis of the C-terminal domain of Helicobacter pylori MotB

Roujeinikova, A. — 2008-04-01

The C-terminal domain of MotB (MotB-C) contains a putative peptidoglycan-binding motif and is believed to anchor the MotA/MotB stator unit of the bacterial flagellar motor to the cell wall. Crystals of Helicobacter pylori MotB-C (138 amino-acid residues) were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. These crystals belong to space group P21, with unit-cell parameters a = 50.8, b = 89.5, c = 66.3 Å, β = 112.5°. The crystals diffract X-rays to at least 1.6 Å resolution using a synchrotron-radiation source. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains one tetramer with 222 point-group symmetry. The anomalous difference Patterson maps calculated for an ytterbium-derivative crystal using diffraction data at a wavelength of 1.38 Å showed significant peaks on the v = 1/2 Harker section, suggesting that ab initio phase information could be derived from the MAD data.

Structure of mouse ADP-ribosylhydrolase 3 (mARH3)

Mueller-Dieckmann, C. — 2008-03-01

ADP-ribosylation is a reversible and covalent post-translational modification in which the attachment of ADP-ribose is catalyzed by ADP-ribosyltransferases and the removal of ADP-ribose is catalyzed by ADP-ribosylhydrolases. ADP-ribosylhydrolase 3 from mouse, consisting of 347 amino-acid residues, has been cloned, purified and crystallized. The three-dimensional structure has been resolved at a resolution of 1.8 Å. The structure constitutes a compact all-α-helical protein with two Mg2+ ions located in the active-site crevice. A structural comparison of mouse ADP-ribosylhydrolase 3 with its human orthologue shows a high degree of structural similarity. Furthermore, four prokaryotic proteins deposited in the PDB could be identified as being structurally related.

Structure of HsaD, a steroid-degrading hydrolase, from Mycobacterium tuberculosis

Lack, N. — 2008-01-01

Tuberculosis is a major cause of death worldwide. Understanding of the pathogenicity of Mycobacterium tuberculosis has been advanced by gene analysis and has led to the identification of genes that are important for intracellular survival in macrophages. One of these genes encodes HsaD, a meta-cleavage product (MCP) hydrolase that catalyzes the hydrolytic cleavage of a carbon–carbon bond in cholesterol metabolism. This paper describes the production of HsaD as a recombinant protein and, following crystallization, the determination of its three-dimensional structure to 2.35 Å resolution by X-ray crystallography at the Diamond Light Source in Oxfordshire, England. To the authors' knowledge, this study constitutes the first report of a structure determined at the new synchrotron facility. The volume of the active-site cleft of the HsaD enzyme is more than double the corresponding active-site volumes of related MCP hydrolases involved in the catabolism of aromatic compounds, consistent with the specificity of HsaD for steroids such as cholesterol. Knowledge of the structure of the enzyme facilitates the design of inhibitors.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DapA (Rv2753c) from Mycobacterium tuberculosis. Corrigendum

Kefala, G. — 2008-01-01

A correction is made to the Experimental methods section of the article by Kefala & Weiss [(2006), Acta Cryst. F62, 1116–1119].

Crystallization and preliminary X-ray analysis of ginkbilobin-2 from Ginkgo biloba seeds: a novel antifungal protein with homology to the extracellular domain of plant cysteine-rich receptor-like kinases. Erratum

Miyakawa, T. — 2007-10-01

A correction is made to one of the affiliations of the authors and also to a table heading in Miyakawa et al. (2007), Acta Cryst. F63, 737–739.

Purification, crystallization and preliminary crystallographic characterization of the α2,6-sialyltransferase from Photobacterium sp. JT-ISH-224

Okino, N. — 2007-08-01

Sialyltransferases transfer sialic acid from cytidine-5-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to the nonreducing termini of the oligosaccharyl structures of various glycoproteins and glycolipids. The newly cloned α2,6-sialyltransferase from Photobacterium sp. JT-ISH-224 (from the Vibrionaceae family) is composed of two domains: an unknown N-terminal domain and a catalytic C-terminal domain which shares significant homology with the Pasteurella multocida multifunctional sialyltransferase. The putative mature form of JT-ISH-224 α2,6-sialyltransferase was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method at 293 K. The crystal belonged to space group P3121 or P3221, with unit-cell parameters a = b = 90.29, c = 204.33 Å. X-ray diffraction data were collected to 2.5 Å resolution.

Purification, crystallization and preliminary crystallographic analysis of deoxyuridine triphosphate nucleotidohydrolase from Arabidopsis thaliana

Bajaj, M. — 2007-05-01

The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 Å resolution using Cu Kα radiation. The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 69.90, b = 70.86 Å, c = 75.55 Å. Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a VM of 1.8 Å3 Da−1.

Preliminary crystallographic analysis of l-2-keto-3-deoxyarabonate dehydratase, an enzyme involved in an alternative bacterial pathway of l-arabinose metabolism

Shimada, N. — 2007-05-01

l-2-Keto-3-deoxyarabonate (l-KDA) dehydratase is a novel member of the dihydrodipicolinate synthase (DHDPS)/N-acetylneuraminate lyase (NAL) protein family and catalyzes the hydration of l-KDA to α-ketoglutaric semialdehyde. l-KDA dehydratase was overexpressed, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. The crystal diffracts to 2.0 Å resolution using synchrotron radiation and belongs to the trigonal space group P3121 or its enantiomorph P3221, with unit-cell parameters a = b = 78.91, c = 207.71 Å.

Crystallization and preliminary X-ray characterization of 1,3-propanediol dehydrogenase from the human pathogen Klebsiella pneumoniae

Marçal, D. — 2007-03-01

1,3-Propanediol dehydrogenase (1,3-PD-DH), encoded by the dhaT gene, is a key enzyme in the dissimilation process for converting glycerol to 1,3-propanediol in the human pathogen Klebsiella pneumoniae. Single colourless crystals were obtained from a recombinant preparation of 1,3-propanediol dehydrogenase overexpressed in Escherichia coli. The crystals belong to space group P21, with unit-cell parameters a = 91.9, b = 226.6, c = 232.6 Å, β = 92.9°. The crystals probably contain two decamers in the asymmetric unit, with a VM value of 3.07 Å3 Da−1 and an estimated solvent content of 59%. Diffraction data were collected to 2.7 Å resolution using synchrotron radiation at the ID14-4 beamline of the European Synchrotron Radiation Facility.

Crystallization and preliminary X-ray analysis of the O-methyltransferase NovP from the novobiocin-biosynthetic cluster of Streptomyces spheroides

Stevenson, C.E.M. — 2007-03-01

Crystals of recombinant NovP (subunit MW = 29 967 Da; 262 amino acids), an S-adenosyl-l-methionine-dependent O-methyltransferase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in space group P2, with unit-cell parameters a = 51.81, b = 46.04, c = 61.22 Å, β = 104.97°. Native data to a maximum resolution of 1.4 Å were collected from a single crystal at the synchrotron. NovP is involved in the biosynthesis of the aminocoumarin antibiotic novobiocin that targets the essential bacterial enzyme DNA gyrase.

Crystallization and preliminary X-ray analysis of AbsC, a novel regulator of antibiotic production in Streptomyces coelicolor

Stevenson, C.E.M. — 2007-03-01

Crystals of recombinant AbsC (subunit MW = 18 313 Da; 158 amino acids), a novel regulator of antibiotic production from Streptomyces coelicolor, were grown by vapour diffusion. The protein crystallizes in space group P212121, with unit-cell parameters a = 43.53, b = 121.30, c = 143.75 Å. Native data to a resolution of 2.25 Å were recorded at station PX 14.1 (Daresbury) from a single crystal. Preliminary analysis of these data suggests that the asymmetric unit contains four copies of the AbsC monomer, giving an estimated solvent content of 47.0%. AbsC belongs to the MarR family of proteins that mediate ligand-responsive transcriptional control.

Structure of 5-formyltetrahydrofolate cyclo-ligase from Bacillus anthracis (BA4489)

Meier, C. — 2007-03-01

Bacillus anthracis is a spore-forming bacterium and the causative agent of the disease anthrax. The Oxford Protein Production Facility has been targeting proteins from B. anthracis in order to develop high-throughput technologies within the Structural Proteomics in Europe project. As part of this work, the structure of 5-formyltetrahydrofolate cyclo-ligase (BA4489) has been determined by X-ray crystallography to 1.6 Å resolution. The structure, solved in complex with magnesium-ion-bound ADP and phosphate, gives a detailed picture of the proposed catalytic mechanism of the enzyme. Chemical differences from other cyclo-ligase structures close to the active site that could be exploited to design specific inhibitors are also highlighted.

Monellin (MNEI) at 1.15 Å resolution

Hobbs, J.R. — 2007-03-01

The X-ray crystal structure of a single-chain monellin protein (MNEI) has been determined at 1.15 Å resolution. The model was refined to convergence employing anisotropic displacement parameters and riding H atoms to produce a final model with Rwork and Rfree values of 0.132 and 0.162, respectively. The crystal contains a single MNEI protein in the asymmetric unit and unusually lacks the dimer interface observed in all previous crystal structures of monellin and its single-chain derivatives. The high resolution allowed a more detailed view of MNEI than previously possible, with 38 of the 96 residues modelled with alternative side-chain conformations, including four core residues Thr12, Cys41, Leu62 and Ile75. Four stably bound negative ions were also located, providing new insight into potential electrostatic interactions of MNEI with the largely negatively charged surface of the sweet taste receptor T1R2–T1R3.

Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses. Corrigendum

Mastrangelo, E. — 2007-03-01

A correction is made to the names of two of the authors in Mastrangelo et al. (2006), Acta Cryst. F62, 768–770.

Structure of the T109S mutant of Escherichia coli dihydroorotase complexed with the inhibitor 5-fluoroorotate: catalytic activity is reflected by the crystal form

Lee, M. — 2007-03-01

Crystals of a single-point mutant (T109S) of Escherichia coli dihydroorotase (DHOase) with diminished activity grown in the presence of l-dihydroorotate (l-DHO) are tetragonal, with a monomer in the asymmetric unit. These crystals are extremely unstable and disintegrate shortly after formation, which is followed by the growth of orthorhombic crystals from the remnants of the tetragonal crystals or at new nucleation sites. Orthorhombic crystals, for which a structure has previously been reported [Thoden et al. (2001), Biochemistry, 40, 6989–6997; Lee et al. (2005), J. Mol. Biol. 348, 523–533], contain a dimer of DHOase in the asymmetric unit; the active site of one monomer contains the substrate N-carbamyl-l-aspartate (l-CA-asp) and the active site of the other monomer contains the product of the reaction, l-DHO. In the subunit with l-DHO in the active site, a surface loop (residues 105–115) is `open'. In the other subunit, with l-CA-asp in the active site, the loop folds inwards, forming specific hydrogen bonds from the loop to the l-CA-asp. The tetragonal crystal form can be stabilized by crystallization in the presence of the inhibitor 5-fluoroorotate (FOA), a product (l-DHO) mimic. Crystals of the complex of T109S DHOase with FOA are tetragonal, space group P41212, with unit-cell parameters a = b = 72.6, c = 176.1 Å. The structure has been refined to R and Rfree values of 0.218 and 0.257, despite severe anisotropy of the diffraction. In this structure, the flexible loops are both in the `open' conformation, which is consistent with FOA, like l-DHO, binding at both sites. The behaviour of the T109S mutant crystals of DHOase in the presence of l-DHO is explained by initial binding of l-DHO to both subunits, followed by slow conversion to l-CA-asp, with consequent movement of the flexible loop and dissolution of the crystals. Orthorhombic crystals are then able to grow in the presence of l-DHO and l-CA-asp.

Crystallization and preliminary X-ray diffraction analysis of ω-amino acid:pyruvate transaminase from Chromobacterium violaceum

Sayer, C. — 2007-02-01

The enzyme ω-transaminase catalyses the conversion of chiral ω-amines to ketones. The recombinant enzyme from Chromobacterium violaceum has been purified to homogeneity. The enzyme was crystallized from PEG 4000 using the microbatch method. Data were collected to 1.7 Å resolution from a crystal belonging to the triclinic space group P1, with unit-cell parameters a = 58.9, b = 61.9, c = 63.9 Å, α = 71.9, β = 87.0, γ = 74.6°. Data were also collected to 1.95 Å from a second triclinic crystal form. The structure has been solved using the molecular-replacement method.

Crystallization and X-ray diffraction analysis of the DNA-remodelling protein DnaD from Bacillus subtilis

Schneider, S. — 2007-02-01

The DnaD protein is an essential component of the chromosome-replication machinery of the Gram-positive bacterium Bacillus subtilis and is part of the primosomal cascade that ultimately loads the replicative ring helicase DnaC onto DNA. Moreover, DnaD is a global regulator of DNA architecture, as it forms higher order nucleoprotein structures in order to open supercoiled DNA. Here, the crystallization and preliminary X-ray diffraction analysis of the two domains of DnaD from B. subtilis are reported. Crystals of the N-terminal domain are trigonal, with either P3121 or P3221 space-group symmetry, and diffracted X-rays to 2.0 Å resolution; crystals of the C-terminal domain are hexagonal, with space group P61 or P65, and diffracted X-rays to 2.9 Å resolution in-house. Determination of the structure of the DnaD domains will provide insight into how remodelling of the nucleoid is associated with priming of replication in the model Gram-positive organism B. subtilis.

Crystallization and preliminary X-ray diffraction analysis of two N-terminal fragments of the DNA-cleavage domain of topoisomerase IV from Staphylococcus aureus. Corrigendum

Carr, S.B. — 2007-01-01

A correction is made to a statement in the article by Carr et al. (2006), Acta Cryst. F62, 1164–1167.

Preliminary X-ray diffraction analysis of YcdB from Escherichia coli: a novel haem-containing and Tat-secreted periplasmic protein with a potential role in iron transport

Cartron, M.L. — 2007-01-01

YcdB is a periplasmic haem-containing protein from Escherichia coli that has a potential role in iron transport. It is currently the only reported haem-containing Tat-secreted substrate. Here, the overexpression, purification, crystallization and structure determination at 2.0 Å resolution are reported for the apo form of the protein. The apo-YcdB structure resembles those of members of the haem-dependent peroxidase family and thus confirms that YcdB is also a member of this family. Haem-soaking experiments with preformed apo-YcdB crystals have been optimized to successfully generate haem-containing YcdB crystals that diffract to 2.9 Å. Completion of model building and structure refinement are under way.

Crystallization and preliminary X-ray diffraction analysis of two N-terminal fragments of the DNA-cleavage domain of topoisomerase IV from Staphylococcus aureus

Carr, S.B. — 2006-11-01

DNA topoisomerase IV removes undesirable topological features from DNA molecules in order to help maintain chromosome stability. Two constructs of 56 and 59 kDa spanning the DNA-cleavage domain of the A subunit of topoisomerase IV from Staphylococcus aureus (termed GrlA56 and GrlA59) have been crystallized. Crystals were grown at 291 K using the sitting-drop vapour-diffusion technique with PEG 3350 as a precipitant. Preliminary X-ray analysis revealed that GrlA56 crystals belong to space group P21, diffract to a resolution of 2.9 Å and possess unit-cell parameters a = 83.6, b = 171.5, c = 87.8 Å, β = 90.1°, while crystals of GrlA59 belong to space group P21212, with unit-cell parameters a = 41.5, b = 171.89, c = 87.9 Å. These crystals diffract to a resolution of 2.8 Å. This is the first report of the crystallization and preliminary X-ray analysis of the DNA-cleavage domain of a topoisomerase IV from a Gram-positive organism.

Crystallization and preliminary X-ray analysis of the aromatic prenyltransferase CloQ from the clorobiocin biosynthetic cluster of Streptomyces roseochromogenes

Keller, S. — 2006-11-01

Crystals of recombinant CloQ (subunit MW = 35 626 Da; 324 amino acids), an aromatic prenyltransferase from Streptomyces roseochromogenes, were grown by vapour diffusion. The protein crystallizes in space group I4122, with unit-cell parameters a = b = 135.19, c = 98.13 Å. Native data from a single crystal were recorded to a resolution of 2.2 Å in-house. Preliminary analysis of these data indicated that the asymmetric unit corresponds to a monomer, giving an estimated solvent content of 60.6%. CloQ is involved in the biosynthesis of the aminocoumarin antibiotic clorobiocin, which targets the essential bacterial enzyme DNA gyrase.

The nucleotide-binding site of Aquifex aeolicus LpxC

Buetow, L. — 2006-11-01

The structure of recombinant Aquifex aeolicus UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) in complex with UDP has been determined to a resolution of 2.2 Å. Previous studies have characterized the binding sites of the fatty-acid and sugar moieties of the substrate, UDP-(3-O-hydroxymyristoyl)-N-acetylglucosamine, but not that of the nucleotide. The uracil-binding site is constructed from amino acids that are highly conserved across species. Hydrophobic associations with the Phe155 and Arg250 side chains in combination with hydrogen-bonding interactions with the main chain of Glu154 and the side chains of Tyr151 and Lys227 position the base. The phosphate and ribose groups are directed away from the active site and interact with Arg137, Lys156, Glu186 and Arg250. The orientation of the phosphate-ribose tail is not conducive to catalysis, perhaps owing to the position of an inhibitory Zn2+. However, based on the position of uracil revealed in this study and on the previously reported complex of LpxC with an inhibitor, a model is proposed for substrate binding.

Preliminary crystallographic characterization of PrnB, the second enzyme in the pyrrolnitrin biosynthetic pathway

De Laurentis, W. — 2006-11-01

Pyrrolnitrin is the active ingredient of drugs for the treatment of superficial fungal infections and was used as a lead structure for the development of fludioxonil. It is an effective agent for plant diseases caused by the fungal pathogen Rhizoctonia solani. Pyrrolnitrin is made in four steps, the second of which, catalyzed by PrnB, is a novel chemical rearrangement of 7-chlorotryptophan. PrnB was overproduced in Pseudomonas fluorescens (BL915) and well diffracting crystals were obtained of a triple cysteine-to-serine mutant by sitting-drop vapour diffusion. Crystals grown in the presence of l-7-chlorotryptophan, d-tryptophan and l-tryptophan are reported. Data sets for each are reported with high-resolution limits of 2.0, 1.75 and 1.75 Å, respectively. Two crystals (PrnB in the presence of d-tryptophan and l-7-chlorotryptophan) belong to space group C2 with similar unit-cell parameters (a = 68.6, b = 79.5, c = 92.7 Å, α = γ = 90.0, β = 103.8°). Crystals grown in the presence of l-tryptophan belong to space group C2221 and have unit-cell parameters a = 67.7, b = 80.1, c = 129.5 Å. All crystals contain a monomer in the asymmetric unit.

Crystallization of Ranasmurfin, a blue-coloured protein from Polypedates leucomystax

McMahon, S.A. — 2006-11-01

Ranasmurfin, a previously uncharacterized ∼13 kDa blue protein found in the nests of the frog Polypedates leucomystax, has been purified and crystallized. The crystals are an intense blue colour and diffract to 1.51 Å with P21 symmetry and unit-cell parameters a = 40.9, b = 59.9, c = 45.0 Å, β = 93.3°. Self-rotation function analysis indicates the presence of a dimer in the asymmetric unit. Biochemical data suggest that the blue colour of the protein is related to dimer formation. Sequence data for the protein are incomplete, but thus far have identified no model for molecular replacement. A fluorescence scan shows a peak at 9.676 keV, indicating that the protein binds zinc and suggesting a route for structure solution.

Fortuitous structure determination of `as-isolated' Escherichia coli bacterioferritin in a novel crystal form

van Eerde, A. — 2006-11-01

Escherichia coli bacterioferritin was serendipitously crystallized in a novel cubic crystal form and its structure could be determined to 2.5 Å resolution despite a high degree of merohedral twinning. This is the first report of crystallographic data on `as-isolated' E. coli bacterioferritin. The ferroxidase active site contains positive difference density consistent with two metal ions that had co-purified with the protein. X-ray fluorescence studies suggest that the metal composition is different from that of previous structures and is a mix of zinc and native iron ions. The ferroxidase-centre configuration displays a similar flexibility as previously noted for other bacterioferritins.

Purification, crystallization and preliminary X-ray study of the fungal laccase from Cerrena maxima

Lyashenko, A.V. — 2006-10-01

Laccases are members of the blue multi-copper oxidase family that oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. Crystals of the laccase from Cerrena maxima have been obtained and X-ray data were collected to 1.9 Å resolution using synchrotron radiation. A preliminary analysis shows that the enzyme has the typical laccase structure and several carbohydrate sites have been identified. The carbohydrate chains appear to be involved in stabilization of the intermolecular contacts in the crystal structure, thus promoting the formation of well ordered crystals of the enzyme. Here, the results of an X-ray crystallographic study on the laccase from the fungus Cerrena maxima are reported. Crystals that diffract well to a resolution of at least 1.9 Å (R factor = 18.953%; Rfree = 23.835; r.m.s.d. bond lengths, 0.06 Å; r.m.s.d. bond angles, 1.07°) have been obtained despite the presence of glycan moieties. The overall spatial organization of C. maxima laccase and the structure of its copper-containing active centre have been determined by the molecular-replacement method using the laccase from Trametes versicolor (Piontek et al., 2002) as a structural template. In addition, four glycan-binding sites were identified and the 1.9 Å X-ray data were used to determine the previously unknown primary structure of this protein. The identity (calculated from sequence alignment) between the C. maxima laccase and the T. versicolor laccase is about 87%. Tyr196 and Tyr372 show significant extra density at the ortho positions and this has been interpreted in terms of NO2 substituents.

Structure of Staphylococcus aureus guanylate monophosphate kinase

El Omari, K. — 2006-10-01

Nucleotide monophosphate kinases (NMPKs) are potential antimicrobial drug targets owing to their role in supplying DNA and RNA precursors. The present work reports the crystal structure of Staphylococcus aureus guanylate monophosphate kinase (SaGMK) at 1.9 Å resolution. The structure shows that unlike most GMKs SaGMK is dimeric, confirming the role of the extended C-terminus in dimer formation as first observed for Escherichia coli GMK (EcGMK). One of the two SaGMK dimers within the crystal asymmetric unit has two monomers in different conformations: an open form with a bound sulfate ion (mimicking the β-phosphate of ATP) and a closed form with bound GMP and sulfate ion. GMP-induced domain movements in SaGMK can thus be defined by comparison of these conformational states. Like other GMKs, the binding of GMP firstly triggers a partial closure of the enzyme, diminishing the distance between the GMP-binding and ATP-binding sites. In addition, the closed structure shows the presence of a potassium ion in contact with the guanine ring of GMP. The potassium ion appears to form an integral part of the GMP-binding site, as the Tyr36 side chain has significantly moved to form a metal ion–ligand coordination involving the lone pair of the side-chain O atom. The potassium-binding site might also be exploited in the design of novel inhibitors.

Expression, purification and crystallization of 2-oxo-hept-4-ene-1,7-dioate hydratase (HpcG) from Escherichia coli C

Adachi, T. — 2006-10-01

The gene encoding 2-oxo-hept-3-ene-1,7-dioic acid (OHED) hydratase (HpcG) was cloned into the high-expression plasmid pET26b and overexpressed in Escherichia coli BL21(DE3). The enzyme was purified in three steps to greater than 95% purity prior to crystallization. Crystals were obtained by the hanging-drop vapour-diffusion method at 277 K in a number of screening conditions. Crystals measuring up to 1.5 mm in their longest dimension were grown from solutions containing polyethylene glycol 20 000. The crystals belonged to space group P41212 or P43212, with unit-cell parameters a = 136, b = 136, c = 192 Å. A complete data set was collected to 2.1 Å from a single cryocooled crystal at 100 K using synchrotron radiation.

Cloning, purification and preliminary crystallographic analysis of a putative pyridoxal kinase from Bacillus subtilis

Newman, J.A. — 2006-10-01

Pyridoxal kinases (PdxK) are able to catalyse the phosphorylation of three vitamin B6 precursors, pyridoxal, pyridoxine and pyridoxamine, to their 5′-phosphates and play an important role in the vitamin B6 salvage pathway. Recently, the thiD gene of Bacillus subtilis was found to encode an enzyme which has the activity expected of a pyridoxal kinase despite its previous assignment as an HMPP kinase owing to higher sequence similarity. As such, this enzyme would appear to represent a new class of `HMPP kinase-like' pyridoxal kinases. B. subtilis thiD has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a binary complex with ADP and Mg2+. X-ray diffraction data have been collected from crystals to 2.8 Å resolution at 100 K. The crystals belong to a primitive tetragonal system, point group 422, and analysis of the systematic absences suggest that they belong to one of the enantiomorphic pair of space groups P41212 or P43212. Consideration of the space-group symmetry and unit-cell parameters (a = b = 102.9, c = 252.6 Å, α = β = γ = 90°) suggest that the crystals contain between three and six molecules in the asymmetric unit. A full structure determination is under way to provide insights into aspects of the enzyme mechanism and substrate specificity.

Structure of the heterotrimeric PCNA from Sulfolobus solfataricus

Williams, G.J. — 2006-10-01

PCNA is a ring-shaped protein that encircles DNA, providing a platform for the association of a wide variety of DNA-processing enzymes that utilize the PCNA sliding clamp to maintain proximity to their DNA substrates. PCNA is a homotrimer in eukaryotes, but a heterotrimer in crenarchaea such as Sulfolobus solfataricus. The three proteins are SsoPCNA1 (249 residues), SsoPCNA2 (245 residues) and SsoPCNA3 (259 residues). The heterotrimeric protein crystallizes in space group P21, with unit-cell parameters a = 44.8, b = 78.8, c = 125.6 Å, β = 100.5°. The crystal structure of this heterotrimeric PCNA molecule has been solved using molecular replacement. The resulting structure to 2.3 Å sheds light on the differential stabilities of the interactions observed between the three subunits and the specificity of individual subunits for partner proteins.

Crystallization and X-ray data analysis of the 10 kDa C-terminal lid subdomain from Caenorhabditis elegans Hsp70

Worrall, L. — 2006-09-01

Hsp70 is an important molecular chaperone involved in the regulation of protein folding. Crystals of the C-terminal 10 kDa helical lid domain (residues 542–640) from a Caenorhabditis elegans Hsp70 homologue have been produced that diffract X-rays to ∼3.4 Å. Crystals belong to space group I212121, with unit-cell parameters a = b = 197, c = 200 Å. The Matthews coefficient, self-rotation function and Patterson map indicate 24 monomers in the asymmetric unit, showing non-crystallographic 432 symmetry. Molecular-replacement studies using the corresponding domain from rat, the only eukaryotic homologue with a known structure, failed and a mercury derivative was obtained. Preliminary MAD phasing using SHELXD and SHARP for location and refinement of the heavy-atom substructure and SOLOMON for density modification produced interpretable maps with a clear protein–solvent boundary. Further density-modification, model-building and refinement are currently under way.

Structure of Bacillus halmapalus α-amylase crystallized with and without the substrate analogue acarbose and maltose

Lyhne-Iversen, L. — 2006-09-01

Recombinant Bacillus halmapalus α-amylase (BHA) was studied in two different crystal forms. The first crystal form was obtained by crystallization of BHA at room temperature in the presence of acarbose and maltose; data were collected at cryogenic temperature to a resolution of 1.9 Å. It was found that the crystal belonged to space group P212121, with unit-cell parameters a = 47.0, b = 73.5, c = 151.1 Å. A maltose molecule was observed and found to bind to BHA and previous reports of the binding of a nonasaccharide were confirmed. The second crystal form was obtained by pH-induced crystallization of BHA in a MES–HEPES–boric acid buffer (MHB buffer) at 303 K; the solubility of BHA in MHB has a retrograde temperature dependency and crystallization of BHA was only possible by raising the temperature to at least 298 K. Data were collected at cryogenic temperature to a resolution of 2.0 Å. The crystal belonged to space group P212121, with unit-cell parameters a = 38.6, b = 59.0, c = 209.8 Å. The structure was solved using molecular replacement. The maltose-binding site is described and the two structures are compared. No significant changes were seen in the structure upon binding of the substrates.

Trypanosoma brucei UDP-galactose-4′-epimerase in ternary complex with NAD+ and the substrate analogue UDP-4-deoxy-4-fluoro-α-d-galactose

Alphey, M.S. — 2006-09-01

The structure of the NAD-dependent oxidoreductase UDP-galactose-4′-epimerase from Trypanosoma brucei in complex with cofactor and the substrate analogue UDP-4-deoxy-4-fluoro-α-d-galactose has been determined using diffraction data to 2.7 Å resolution. Despite the high level of sequence and structure conservation between the trypanosomatid enzyme and those from humans, yeast and bacteria, the binding of the 4-fluoro-α-d-galactose moiety is distinct from previously reported structures. Of particular note is the observation that when bound to the T. brucei enzyme, the galactose moiety of this fluoro-derivative is rotated approximately 180° with respect to the orientation of the hexose component of UDP-glucose when in complex with the human enzyme. The architecture of the catalytic centre is designed to effectively bind different orientations of the hexose, a finding that is consistent with a mechanism that requires the sugar to maintain a degree of flexibility within the active site.

Expression, purification and preliminary crystallographic analysis of oligopeptidase B from Trypanosoma brucei

Rea, D. — 2006-08-01

African sleeping sickness, also called trypanosomiasis, is a significant cause of morbidity and mortality in sub-Saharan Africa. Peptidases from Trypanosoma brucei, the causative agent, include the serine peptidase oligopeptidase B, a documented virulence factor and therapeutic target. Determination of the three-dimensional structure of oligopeptidase B is desirable to facilitate the development of novel inhibitors. Oligopeptidase B was overexpressed in Escherichia coli as an N-terminally hexahistidine-tagged fusion protein, purified using metal-affinity chromatography and crystallized using the hanging-drop vapour-diffusion technique in 7%(w/v) polyethylene glycol 6000, 1 M LiCl, 0.1 M bis-tris propane pH 7.5. Diffraction data to 2.7 Å resolution were collected using synchrotron radiation. The crystals belong to space group P3121 or P3221, with unit-cell parameters a = b = 124.5, c = 249.9 Å. A complete data set to 2.7 Å was collected using synchrotron radiation.

Cloning, purification and preliminary crystallographic analysis of a conserved hypothetical protein, SA0961 (YlaN), from Staphylococcus aureus

Xu, L. — 2006-08-01

SA0961 is an unknown hypothetical protein from Staphylococcus aureus that can be identified in the Firmicutes division of Gram-positive bacteria. The gene for the homologue of SA0961 in Bacillus subtilis, ylaN, has been shown to be essential for cell survival, thus identifying the protein encoded by this gene as a potential target for the development of novel antibiotics. SA0961 was cloned and the protein was overexpressed in Escherichia coli, purified and subsequently crystallized. Crystals of selenomethionine-labelled SA0961 diffract to beyond 2.4 Å resolution and belong to the monoclinic space group P21, with unit-cell parameters a = 31.5, b = 42.7, c = 62.7 Å, β = 92.4° and two molecules in the asymmetric unit. A full structure determination is under way to provide insights into the function of this protein.

Expression, purification, crystallization and preliminary crystallographic analysis of BipD, a component of the Burkholderia pseudomallei type III secretion system

Roversi, P. — 2006-09-01

A construct consisting of residues 10–310 of BipD, a component of the Burkholderia pseudomallei type III secretion system (T3SS), has been overexpressed as a GST fusion, cleaved from the GST tag and purified. Crystals were grown of native and selenomethionine-labelled BipD. The crystals grow in two different polymorphs from the same condition. The first polymorph belongs to space group C222, with unit-cell parameters a = 103.98, b = 122.79, c = 49.17 Å, a calculated Matthews coefficient of 2.4 Å3 Da−1 (47% solvent content) and one molecule per asymmetric unit. The second polymorph belongs to space group P21212, with unit-cell parameters a = 136.47, b = 89.84, c = 50.15 Å, and a calculated Matthews coefficient of 2.3 Å3 Da−1 (45% solvent content) for two molecules per asymmetric unit (analysis of the self-rotation function indicates the presence of a weak twofold non-crystallographic symmetry axis in this P21212 form). The native crystals of both forms give diffraction data to 2.7 Å resolution, while the SeMet-labelled P21212 crystals diffract to 3.3 Å resolution. A K2PtCl4 derivative of the P21212 form was also obtained and data were collected to 2.7 Å with radiation of wavelength λ = 0.933 Å. The Pt-derivative anomalous difference Patterson map revealed two self-peaks on the Harker sections.

Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system

Johnson, S. — 2006-09-01

IpaD, the putative needle-tip protein of the Shigella flexneri type III secretion system, has been overexpressed and purified. Crystals were grown of the native protein in space group P212121, with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 Å, and data were collected to 2.9 Å resolution. Analysis of the native Patterson map revealed a peak at 50% of the origin on the Harker section v = 0.5, suggesting twofold non-crystallographic symmetry parallel to the b crystallographic axis. As attempts to derivatize or grow selenomethionine-labelled protein crystals failed, in-drop proteolysis was used to produce new crystal forms. A trace amount of subtilisin Carlsberg was added to IpaD before sparse-matrix screening, resulting in the production of several new crystal forms. This approach produced SeMet-labelled crystals and diffraction data were collected to 3.2 Å resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 Å, β = 107.9°. An anomalous difference Patterson map revealed peaks on the Harker section v = 0, while the self-rotation function indicates the presence of a twofold noncrystallographic symmetry axis, which is consistent with two molecules per asymmetric unit.

Crystallization and preliminary X-ray diffraction analysis of BipD, a virulence factor from Burkholderia pseudomallei

Knight, M.J. — 2006-08-01

Burkholderia pseudomallei, the causative agent of melioidosis, possesses a protein-secretion apparatus that is similar to those found in Salmonella and Shigella. A major function of these secretion systems is to secrete virulence-associated proteins into target cells of the host organism. The BipD gene of B. pseudomallei encodes a secreted virulence factor that is similar in sequence and most likely functionally analogous to IpaD from Shigella and SipD from Salmonella. Thus, the BipD protein is likely to be a component of a type III protein-secretion system (TTSS) in B. pseudomallei. Proteins in the same class as BipD, such as IpaD and SipD, are thought to act as extracellular chaperones to help the hydrophobic translocator proteins enter the target cell membrane, where they form a pore and might even link the translocon pore with the secretion needle. There is evidence that the translocator proteins also bind an integrin which stimulates actin-mediated insertion of the bacterium into the host-cell membrane. Native BipD has been crystallized in a monoclinic crystal form that diffracts X-rays to 2.5 Å resolution. BipD protein which incorporates selenomethionine (SeMet-BipD) has also been expressed and forms crystals which diffract to a higher resolution of 2.1 Å.

Structure of Staphylococcus aureus cytidine monophosphate kinase in complex with cytidine 5′-monophosphate

Dhaliwal, B. — 2006-08-01

The crystal structure of Staphylococcus aureus cytidine monophosphate kinase (CMK) in complex with cytidine 5′-monophosphate (CMP) has been determined at 2.3 Å resolution. The active site reveals novel features when compared with two orthologues of known structure. Compared with the Streptococcus pneumoniae CMK solution structure of the enzyme alone, S. aureus CMK adopts a more closed conformation, with the NMP-binding domain rotating by ∼16° towards the central pocket of the molecule, thereby assembling the active site. Comparing Escherichia coli and S. aureus CMK–CMP complex structures reveals differences within the active site, including a previously unreported indirect interaction of CMP with Asp33, the replacement of a serine residue involved in the binding of CDP by Ala12 in S. aureus CMK and an additional sulfate ion in the E. coli CMK active site. The detailed understanding of the stereochemistry of CMP binding to CMK will assist in the design of novel inhibitors of the enzyme. Inhibitors are required to treat the widespread hospital infection methicillin-resistant S. aureus (MRSA), currently a major public health concern.

Crystallization of DIR1, a LTP2-like resistance signalling protein from Arabidopsis thaliana

Lascombe, M.-B. — 2006-07-01

DIR1, a putative LTP2 protein from Arabidopsis thaliana implicated in systemic acquired resistance in planta, has been crystallized in space group P212121 with one molecule per asymmetric unit. The crystals diffract to a resolution of 1.6 Å.

Purification, crystallization and preliminary X-ray diffraction analysis of RafE, a sugar-binding lipoprotein from Streptococcus pneumoniae

Paterson, N.G. — 2006-07-01

Streptococcus pneumoniae contains a large number of sugar-transport systems and the system responsible for raffinose uptake has recently been identified. The substrate-binding protein component of this system shares strong sequence homology with the multiple sugar metabolism substrate-binding protein MsmE from S. mutans and contains a lipoprotein-attachment site at cysteine residue 23. A truncated form (residues 24–419) of RafE from S. pneumoniae was cloned and overexpressed in Escherichia coli. Native and selenomethionine-labelled protein have been crystallized in the hexagonal space group P6122. Diffraction data have been successfully phased to 2.90 Å using Se SAD data and model building is in progress.

X-ray crystallographic studies reveal that the incorporation of spacer groups in carbonic anhydrase inhibitors causes alternate binding modes

Fisher, S.Z. — 2006-07-01

Human carbonic anhydrases (CAs) are well studied targets for the development of inhibitors for pharmaceutical applications. The crystal structure of human CA II has been determined in complex with two CA inhibitors (CAIs) containing conventional sulfonamide and thiadiazole moieties separated by a —CF2— or —CHNH2— spacer group. The structures presented here reveal that these spacer groups allow novel binding modes for the thiadiazole moiety compared with conventional CAIs.

Expression, purification and preliminary crystallographic analysis of the Toxoplasma gondii enoyl reductase

Muench, S.P. — 2006-06-01

The protozoan parasite Toxoplasma gondii is the causative agent of one of the most widespread parasitic infections of man and is a leading cause of congenital neurological birth defects and the third most common cause of food-borne deaths in the United States. Despite this, to date no drugs are available that provide a fully effective treatment. Recently, the antibacterial agent triclosan was shown to inhibit the fatty-acid biosynthesis pathway in T. gondii and to interact with the enoyl reductase (ENR). In order to analyse the potential of triclosan as a lead compound targeting T. gondii ENR and to explore unique features of the apicomplexan enzyme that could be exploited in future drug development, structural studies have been initiated on T. gondii ENR. Crystals of T. gondii ENR in complex with NAD+ and triclosan were grown using the hanging-drop vapour-diffusion method with PEG 8000 as precipitant. The crystals belong to space group P3221, with approximate unit-cell parameters a = 78.1, b = 78.1, c = 188.5 Å, α = β = 90, γ = 120° and a dimer in the asymmetric unit. Test data were collected to beyond 2.6 Å on cryocooled crystals (100 K) using a Rigaku MM007 rotating-anode X-ray source, revealing that the crystals are suitable for a full structural determination.

Overexpression, purification and crystallization of a choline-binding protein CbpI from Streptococcus pneumoniae

Paterson, N.G. — 2006-07-01

The choline-binding protein CbpI from Streptococcus pneumoniae is a 23.4 kDa protein with no known function. The protein has been successfully purified initially using Ni–NTA chromatography and to homogeneity using Q-Sepharose ion-exchange resin as an affinity column. CbpI was crystallized using PEG 3350 as a precipitant and X-ray crystallographic analysis showed that the crystals belonged to the tetragonal space group P4, with unit-cell parameters a = b = 83.31, c = 80.29 Å, α = β = γ = 90°. The crystal contains two molecules in the asymmetric unit with a solvent content of 55.7% (VM = 2.77 Å3 Da−1) and shows a diffraction limit of 3.5 Å.

Structure of the PII signal transduction protein of Neisseria meningitidis at 1.85 Å resolution

Nichols, C.E. — 2006-06-01

The PII signal transduction proteins GlnB and GlnK are implicated in the regulation of nitrogen assimilation in Escherichia coli and other enteric bacteria. PII-like proteins are widely distributed in bacteria, archaea and plants. In contrast to other bacteria, Neisseria are limited to a single PII protein (NMB 1995), which shows a high level of sequence identity to GlnB and GlnK from Escherichia coli (73 and 62%, respectively). The structure of the PII protein from N. meningitidis (serotype B) has been solved by molecular replacement to a resolution of 1.85 Å. Comparison of the structure with those of other PII proteins shows that the overall fold is tightly conserved across the whole population of related proteins, in particular the positions of the residues implicated in ATP binding. It is proposed that the Neisseria PII protein shares functions with GlnB/GlnK of enteric bacteria.

Crystallization of quinoprotein glucose dehydrogenase variants and homologues by microseeding

Sanchez-Weatherby, J. — 2006-06-01

The soluble quinoprotein glucose dehydrogenase oxidizes glucose, maltose and a variety of other monosaccharides and disaccharides to the corresponding lactones. An efficient microseeding protocol is reported to produce crystals of three variants that display reduced activity towards maltose. Similar cross-seeding protocols to grow crystals of homologues from Escherichia coli and Streptomyces coelicolor are described.

High-resolution complex of papain with remnants of a cysteine protease inhibitor derived from Trypanosoma brucei

Alphey, M.S. — 2006-06-01

Attempts to cocrystallize the cysteine protease papain derived from the latex of Carica papaya with an inhibitor of cysteine proteases (ICP) from Trypanosoma brucei were unsuccessful. However, crystals of papain that diffracted to higher resolution, 1.5 Å, than other crystals of this archetypal cysteine protease were obtained, so the analysis was continued. Surprisingly, the substrate-binding cleft was occupied by two short peptide fragments which have been assigned as remnants of ICP. Comparisons reveal that these peptides bind in the active site in a manner similar to that of the human cysteine protease inhibitor stefin B when it is complexed to papain. The assignment of the fragment sequences is consistent with the specificity of the protease.

Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved

Krengel, U. — 2006-05-01

Chorismate mutase catalyzes the conversion of chorismate to prephenate in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine in bacteria, fungi and plants. Here, the crystallization of the unusual secreted chorismate mutase from Mycobacterium tuberculosis (encoded by Rv1885c), a 37.2 kDa dimeric protein belonging to the AroQγ subclass of mutases, is reported. Crystal optimization was non-trivial and is discussed in detail. To obtain crystals of sufficient quality, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5–15 min, in an adaptation of a previously described technique [Saridakis & Chayen (2000), Protein Sci. 9, 755–757]. As a result of the optimization, diffraction improved from 3.5 to 1.3 Å resolution. The crystals belong to space group P21, with unit-cell parameters a = 42.6, b = 72.6, c = 62.0 Å, β = 104.5°. The asymmetric unit contains one biological dimer, with 167 amino acids per protomer. A soak with a transition-state analogue is also described.

Cloning, purification and preliminary crystallographic analysis of the Bacillus subtilis GTPase YphC–GDP complex

Xu, L. — 2006-05-01

The Bacillus subtilis YphC gene encodes an essential GTPase thought to be involved in ribosome binding and whose protein product may represent a target for the development of a novel antibacterial agent. Sequence analysis reveals that YphC belongs to the EngA family of GTPases, which uniquely contain two adjacent GTP-binding domains. Crystals of a selenomethionine-incorporated YphC–GDP complex have been grown using the hanging-drop vapour-diffusion method and polyethylene glycol as a precipitating agent. The crystals belong to space group P212121, with unit-cell parameters a = 62.71, b = 65.05, c = 110.61 Å, and have one molecule in the asymmetric unit. Data sets at three different wavelengths were collected on a single crystal to 2.5 Å resolution at the Daresbury SRS in order to solve the structure by MAD. Ultimately, analysis of YphC in complex with GDP may allow a greater understanding of the EngA family of essential GTPases.

Purification, crystallization and preliminary X-ray diffraction studies of N-acetylglucosamine-phosphate mutase from Candida albicans

Nishitani, Y. — 2006-04-01

N-acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc) in eukaryotes and belongs to the α-d-phosphohexomutase superfamily. AGM1 from Candida albicans (CaAGM1) was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals obtained belong to the primitive monoclinic space group P21, with unit-cell parameters a = 60.2, b = 130.2, c = 78.0 Å, β = 106.7°. The crystals diffract X-rays to beyond 1.8 Å resolution using synchrotron radiation.

Structure of SAICAR synthase from Thermotoga maritima at 2.2 Å reveals an unusual covalent dimer

Zhang, R. — 2006-04-01

As a part of a structural genomics program, the 2.2 Å resolution crystal structure of the PurC gene product from Thermotoga maritima has been solved. This 26.2 kDa protein belongs to the phophoribosylaminoimidazole-succinocarboxamide or SAICAR synthase family of enzymes, the members of which are involved in de novo purine biosynthesis. SAICAR synthase can be divided into three subdomains: two α+β regions exhibiting structural homology with ATP-binding proteins and a carboxy-terminal subdomain of two α-helices. The asymmetric unit contains two copies of the protein which are covalently linked by a disulfide bond between Cys126(A) and Cys126(B). This 230-amino-acid protein exhibits high structural homology with SAICAR synthase from baker's yeast. The protein structure is described and compared with that of the ATP–SAICAR synthase complex from yeast.

Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle

Deane, J.E. — 2006-03-01

A monodisperse truncation mutant of MxiH, the subunit of the needle from the Shigella flexneri type III secretion system (TTSS), has been overexpressed and purified. Crystals were grown of native and selenomethionine-labelled MxiHCΔ5 and diffraction data were collected to 1.9 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 183.4, b = 28.1, c = 27.8 Å, β = 96.5°. An anomalous difference Patterson map calculated with the data from the SeMet-labelled crystals revealed a single peak on the Harker section v = 0. Inspection of a uranyl derivative also revealed one peak in the isomorphous difference Patterson map on the Harker section v = 0. Analysis of the self-rotation function indicates the presence of a twofold non-crystallographic symmetry axis approximately along a. The calculated Matthews coefficient is 1.9 Å3 Da−1 for two molecules per asymmetric unit, corresponding to a solvent content of 33%.

Overexpression, purification, crystallization and data collection of Sulfolobus solfataricus Sso6206, a novel highly conserved protein

McEwan, A.R. — 2006-03-01

Sso6206, a 10.5 kDa protein from Sulfolobus solfataricus, has been overexpressed, purified and crystallized. The protein crystallizes in space group P61/522, with unit-cell parameters a = b = 157.8, c = 307.3 Å. The crystals are hexagonal bipyramids and a data set has been collected to 2.4 Å resolution. Molecular replacement cannot be attempted as no convincing model can be identified. Crystals of selenomethionine-variant protein have not yet been obtained. Interestingly, crystal packing, gel filtration and mass spectrometry all suggest the native protein forms a multi-subunit oligomer consisting of >9 subunits.

Crystallization of a functionally intact Hsc70 chaperone

Jiang, J. — 2006-01-01

Hsp70s are essential chaperones with roles in a variety of cellular processes and representatives in all kingdoms of life. They are comprised of a nucleotide-binding domain (NBD) and a protein substrate-binding domain (SBD). Structures of isolated NBDs and SBDs have been reported but, until recently, a functionally intact Hsp70 containing both the NBD and SBD has resisted structure determination. Here, it is reported that preparation of diffraction-quality crystals of functionally intact bovine Hsc70 required (i) deletion of part of the protein to reduce oligomerization, (ii) point mutations in the interface between the SBD and NBD and (iii) use of high concentrations of the structure-stabilizing agents glycerol and trimethylamine oxide (TMAO). The introduction of point mutations in interdomain interfaces and the use of the potent structure stabilizer TMAO may be generally useful in crystallization of multidomain proteins that exhibit interdomain motions.

Crystallization and X-ray diffraction analysis of human CLEC-2

Watson, A.A. — 2005-12-01

The human C-type lectin-like protein CLEC-2 has recently been shown to be expressed on the surface of platelets and to function as a receptor for the snake-venom protein rhodocytin. The C-type lectin-like domain (CTLD) of CLEC-2 was expressed in Escherichia coli, refolded and purified. Crystals of this recombinant CLEC-2 were grown by sitting-drop vapour diffusion using polyethylene glycol (PEG) 6000 as a precipitant. After optimization, crystals were grown which diffracted to 2.0 Å using in-house radiation (λ = 1.5418 Å). These crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 35.407, b = 55.143, c = 56.078 Å. The presence of one molecule per asymmetric unit is consistent with a crystal volume per unit weight (VM) of 1.82 Å3 Da−1 and a solvent content of 32.6%. These results suggest that crystals producing diffraction of this quality will be suitable for the structural determination of human CLEC-2.

Crystallization and preliminary crystallographic analysis of porcine acylaminoacyl peptidase

Wright, H. — 2005-10-01

Acylaminoacyl peptidase (also known as acylamino-acid-releasing enzyme or acylpeptide hydrolase; EC 3.4.19.1) is an unusual member of the prolyl oligopeptidase family catalysing the hydrolysis of an N-acylated peptide to an acylamino acid and a peptide with a free N-terminus. Acylaminoacyl peptidase purified from porcine liver has been crystallized in mother liquor containing 0.1 M Tris–HCl pH 7.0, 10%(w/v) polyethylene glycol 8000, 50 mM MgCl2 and 1%(w/v) CHAPS using the hanging-drop vapour-diffusion technique. A full data set to 3.4 Å resolution was collected at ESRF beamline ID14-4 and space group C222 was assigned, with unit-cell parameters a = 84.8, b = 421.1, c = 212.0 Å and four molecules in the asymmetric unit.

Crystallization and preliminary X-ray analysis of the RAD protein from Antirrhinum majus

Stevenson, C.E.M. — 2005-10-01

Crystals of the RADIALIS protein from Antirrhinum majus were grown by vapour diffusion after limited proteolysis. Mass spectrometry indicated that an 8 kDa fragment had been crystallized corresponding to the predicted MYB DNA-binding domain. X-ray data collected at room temperature were consistent with tetragonal symmetry, whereas data collected at 100 K using crystals cryoprotected by supplementing the mother liquor with ethylene glycol conformed to orthorhombic symmetry. It was subsequently shown that crystals soaked in cryoprotectants that were `osmolality-matched' to the mother liquor retained tetragonal symmetry. Using these crystals, X-ray data were collected in-house to a maximum resolution of 2 Å.

Crystallization and preliminary X-ray diffraction analysis of calexcitin from Loligo pealei: a neuronal protein implicated in learning and memory

Beaven, G.D.E. — 2005-10-01

The neuronal protein calexcitin from the long-finned squid Loligo pealei has been expressed in Escherichia coli and purified to homogeneity. Calexcitin is a 22 kDa calcium-binding protein that becomes up-regulated in invertebrates following Pavlovian conditioning and is likely to be involved in signal transduction events associated with learning and memory. Recombinant squid calexcitin has been crystallized using the hanging-drop vapour-diffusion technique in the orthorhombic space group P212121. The unit-cell parameters of a = 46.6, b = 69.2, c = 134.8 Å suggest that the crystals contain two monomers per asymmetric unit and have a solvent content of 49%. This crystal form diffracts X-rays to at least 1.8 Å resolution and yields data of high quality using synchrotron radiation.

Crystallization and preliminary X-ray diffraction analysis of the haem-binding protein HemS from Yersinia enterocolitica

Schneider, S. — 2005-08-01

Bacteria have evolved strategies to acquire iron from their environment. Pathogenic microbes rely on specialized proteins to `steal' haem from their host and use it as an iron source. HemS is the ultimate recipient of a molecular-relay system for haem uptake in Gram-negative species, functioning as the cytosolic carrier of haem. Soluble expression and high-quality diffraction crystals were obtained for HemS from Yersinia enterocolitica. Crystals belong to the orthorhombic space group I222, with unit-cell parameters a = 74.86, b = 77.45, c = 114.09 Å, and diffract X-rays to 2.6 Å spacing in-house. Determination of the structure of the haem–HemS complex will reveal the molecular basis of haem binding.

Expression, purification and preliminary crystallographic analysis of 2,4-dihydroxy-hepta-2-ene-1,7-dioate aldolase (HpcH) from Escherichia coli C

Rea, D. — 2005-09-01

The gene encoding 2,4-dihydroxy-hepta-2-ene-1,7-dioate (HHED) aldolase (HpcH; EC 4.1.2) from Escherichia coli C was cloned into the high-expression plasmid pProEx-HTa and overexpressed in E. coli BL21 (DE3). The 28 kDa enzyme was purified using immobilized metal-affinity and size-exclusion chromatography prior to crystallization. Crystals were obtained by the hanging-drop vapour-diffusion method at 277 K from a number of screening conditions. Type I crystals grown in a solution containing 0.4 M ammonium dihydrogen phosphate belong to space group R32, with unit-cell parameters a = b = 128.92, c = 175.30 Å. Type II crystals grown in a solution containing 0.5 M sodium chloride, 0.1 M sodium citrate pH 5.5 belong to space group I222, with unit-cell parameters a = 133.39, b = 155.39, c = 168.80 Å. Complete data sets were collected to 1.6 and 2.0 Å from type I and type II crystals, respectively, using synchrotron radiation.

Purification, crystallization and preliminary crystallographic analysis of Arabidopsis thaliana imidazoleglycerol-phosphate dehydratase

Glynn, S.E. — 2005-08-01

Imidazoleglycerol-phosphate dehydratase catalyses the sixth step of the histidine-biosynthesis pathway in plants and microorganisms and has been identified as a possible target for the development of novel herbicides. Arabidopsis thaliana IGPD has been cloned and overexpressed in Escherichia coli, purified and subsequently crystallized in the presence of manganese. Under these conditions, the inactive trimeric form of the metal-free enzyme is assembled into a fully active species consisting of a 24-mer exhibiting 432 symmetry. X-ray diffraction data have been collected to 3.0 Å resolution from a single crystal at 293 K. The crystal belongs to space group R3, with approximate unit-cell parameters a = b = 157.9, c = 480.0 Å, α = β = 90, γ = 120° and with either 16 or 24 subunits in the asymmetric unit. A full structure determination is under way in order to provide insights into the mode of subunit assembly and to initiate a programme of rational herbicide design.

High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

Sundara Baalaji, N. — 2005-08-01

Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (VM) of 3.3 Å3 Da−1, corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin.

Crystallization and preliminary X-ray analysis of Leishmania major glyoxalase I

Ariza, A. — 2005-08-01

Glyoxalase I (GLO1) is a putative drug target for trypanosomatids, which are pathogenic protozoa that include the causative agents of leishmaniasis. Significant sequence and functional differences between Leishmania major and human GLO1 suggest that it may make a suitable template for rational inhibitor design. L. major GLO1 was crystallized in two forms: the first is extremely disordered and does not diffract, while the second, an orthorhombic form, produces diffraction to 2.0 Å. Molecular-replacement calculations indicate that there are three GLO1 dimers in the asymmetric unit, which take up a helical arrangement with their molecular dyads arranged approximately perpendicular to the c axis. Further analysis of these data are under way.

Preliminary crystallographic analysis of the major capsid protein P2 of the lipid-containing bacteriophage PM2

Abrescia, N.G.A. — 2005-08-01

PM2 (Corticoviridae) is a dsDNA bacteriophage which contains a lipid membrane beneath its icosahedral capsid. In this respect it resembles bacteriophage PRD1 (Tectiviridae), although it is not known whether the similarity extends to the detailed molecular architecture of the virus, for instance the fold of the major coat protein P2. Structural analysis of PM2 has been initiated and virus-derived P2 has been crystallized by sitting-nanodrop vapour diffusion. Crystals of P2 have been obtained in space group P21212, with two trimers in the asymmetric unit and unit-cell parameters a = 171.1, b = 78.7, c = 130.1 Å. The crystals diffract to 4 Å resolution at the ESRF BM14 beamline (Grenoble, France) and the orientation of the non-crystallographic threefold axes, the spatial relationship between the two trimers and the packing of the trimers within the unit cell have been determined. The trimers form tightly packed layers consistent with the crystal morphology, possibly recapitulating aspects of the arrangement of subunits in the virus.

Crystallization and preliminary X-ray diffraction analysis of a flavoenzyme amine dehydrogenase/oxidase from Pyrococcus furiosus DSM 3638

Monaghan, P.J. — 2005-08-01

A flavoprotein amine dehydrogenase/oxidase with subunit molecular weights of 54.8 kDa (α-subunit) and 42.4 kDa (β-subunit) and specificity for l-proline was cloned from the genomic DNA of the hyperthermophilic marine archaeon Pyrococcus furiosus DSM 3638. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The enzyme was crystallized using the sitting-drop vapour-diffusion technique. Diffraction data from two different crystal forms were collected to 3.3 and 3.6 Å, respectively, using synchrotron radiation. Both crystals belonged to space group P1, with unit-cell parameters a = 91.3, b = 136.3, c = 203.8 Å, α = 94.5, β = 99.4, γ = 102.7° and a = 93.7, b = 116.3, c = 126.9 Å, α = 97.3, β = 99.9, γ = 104.6°.

Crystallization and preliminary X-ray analysis of binary and ternary complexes of Haloferax mediterranei glucose dehydrogenase

Esclapez, J. — 2005-08-01

Haloferax mediterranei glucose dehydrogenase (EC 1.1.1.47) belongs to the medium-chain alcohol dehydrogenase superfamily and requires zinc for catalysis. In the majority of these family members, the catalytic zinc is tetrahedrally coordinated by the side chains of a cysteine, a histidine, a cysteine or glutamate and a water molecule. In H. mediterranei glucose dehydrogenase, sequence analysis indicates that the zinc coordination is different, with the invariant cysteine replaced by an aspartate residue. In order to analyse the significance of this replacement and to contribute to an understanding of the role of the metal ion in catalysis, a range of binary and ternary complexes of the wild-type and a D38C mutant protein have been crystallized. For most of the complexes, crystals belonging to space group I222 were obtained using sodium/potassium citrate as a precipitant. However, for the binary and non-productive ternary complexes with NADPH/Zn, it was necessary to replace the citrate with 2-methyl-2,4-pentanediol. Despite the radical change in conditions, the crystals thus formed were isomorphous.

A double mutation of Escherichia coli 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase disrupts six hydrogen bonds with, yet fails to prevent binding of, an isoprenoid diphosphate

Sgraja, T. — 2005-07-01

The essential enzyme 2C-methyl-d-erythritol-2,4-cyclodiphosphate (MECP) synthase, found in most eubacteria and the apicomplexan parasites, participates in isoprenoid-precursor biosynthesis and is a validated target for the development of broad-spectrum antimicrobial drugs. The structure and mechanism of the enzyme have been elucidated and the recent exciting finding that the enzyme actually binds diphosphate-containing isoprenoids at the interface formed by the three subunits that constitute the active protein suggests the possibility of feedback regulation of MECP synthase. To investigate such a possibility, a form of the enzyme was sought that did not bind these ligands but which would retain the quaternary structure necessary to create the active site. Two amino acids, Arg142 and Glu144, in Escherichia coli MECP synthase were identified as contributing to ligand binding. Glu144 interacts directly with Arg142 and positions the basic residue to form two hydrogen bonds with the terminal phosphate group of the isoprenoid diphosphate ligand. This association occurs at the trimer interface and three of these arginines interact with the ligand phosphate group. A dual mutation was designed (Arg142 to methionine and Glu144 to leucine) to disrupt the electrostatic attractions between the enzyme and the phosphate group to investigate whether an enzyme without isoprenoid diphosphate could be obtained. A low-resolution crystal structure of the mutated MECP synthase Met142/Leu144 revealed that geranyl diphosphate was retained despite the removal of six hydrogen bonds normally formed with the enzyme. This indicates that these two hydrophilic residues on the surface of the enzyme are not major determinants of isoprenoid binding at the trimer interface but rather that hydrophobic interactions between the hydrocarbon tail and the core of the enzyme trimer dominate ligand binding.

Cloning, purification, crystallization and preliminary structural studies of penicillin V acylase from Bacillus subtilis

Rathinaswamy, P. — 2005-07-01

Penicillin acylase proteins are amidohydrolase enzymes that cleave penicillins at the amide bond connecting the side chain to their β-lactam nucleus. An unannotated protein from Bacillus subtilis has been expressed in Escherichia coli, purified and confirmed to possess penicillin V acylase activity. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 4 M sodium formate in 100 mM Tris–HCl buffer pH 8.2. Diffraction data were collected under cryogenic conditions to a spacing of 2.5 Å. The crystals belonged to the orthorhombic space group C2221, with unit-cell parameters a = 111.0, b = 308.0, c = 56.0 Å. The estimated Matthews coefficient was 3.23 Å3 Da−1, corresponding to 62% solvent content. The structure has been solved using molecular-replacement methods with B. sphaericus penicillin V acylase (PDB code 2pva) as the search model.

Structures of two superoxide dismutases from Bacillus anthracis reveal a novel active centre

Boucher, I.W. — 2005-07-01

The BA4499 and BA5696 genes of Bacillus anthracis encode proteins homologous to manganese superoxide dismutase, suggesting that this organism has an expanded repertoire of antioxidant proteins. Differences in metal specificity and quaternary structure between the dismutases of prokaryotes and higher eukaryotes may be exploited in the development of therapeutic antibacterial compounds. Here, the crystal structure of two Mn superoxide dismutases from B. anthracis solved to high resolution are reported. Comparison of their structures reveals that a highly conserved residue near the active centre is substituted in one of the proteins and that this is a characteristic feature of superoxide dismutases from the B. cereus/B. anthracis/B. thuringiensis group of organisms.

Complex assembly, crystallization and preliminary X-ray crystallographic studies of rhesus macaque MHC Mamu-A*01 complexed with an immunodominant SIV-Gag nonapeptide

Chu, F. — 2005-06-01

Simian immunodeficiency virus (SIV) infection in rhesus macaques has been used as the best model for the study of human immunodeficiency virus (HIV) infection in humans, especially in the cytotoxic T-lymphocyte (CTL) response. However, the structure of rhesus macaque (or any other monkey model) major histocompatibility complex class I (MHC I) presenting a specific peptide (the ligand for CTL) has not yet been elucidated. Here, using in vitro refolding, the preparation of the complex of the rhesus macaque MHC I allele (Mamu-A*01) with human β2m and an immunodominant peptide, CTPYDINQM (Gag_CM9), derived from SIV Gag protein is reported. The complex (45 kDa) was crystallized; the crystal belongs to space group I422, with unit-cell parameters a = b = 183.8, c = 155.2 Å. The crystal contains two molecules in the asymmetric unit and diffracts X-rays to 2.8 Å resolution. The structure is being solved by molecular replacement and this is the first attempt to determined the crystal structure of a peptide–nonhuman primate MHC complex

A preliminary crystallographic analysis of the putative mevalonate diphosphate decarboxylase from Trypanosoma brucei

Byres, E. — 2005-06-01

Mevalonate diphosphate decarboxylase catalyses the last and least well characterized step in the mevalonate pathway for the biosynthesis of isopentenyl pyrophosphate, an isoprenoid precursor. A gene predicted to encode the enzyme from Trypanosoma brucei has been cloned, a highly efficient expression system established and a purification protocol determined. The enzyme gives monoclinic crystals in space group P21, with unit-cell parameters a = 51.5, b = 168.7, c = 54.9 Å, β = 118.8°. A Matthews coefficient VM of 2.5 Å3 Da−1 corresponds to two monomers, each approximately 42 kDa (385 residues), in the asymmetric unit with 50% solvent content. These crystals are well ordered and data to high resolution have been recorded using synchrotron radiation.

High-resolution structure of the native histone octamer

Wood, C.M. — 2005-06-01

Crystals of native histone octamers (H2A–H2B)–(H4–H3)–(H3′–H4′)–(H2B′–H2A′) from chick erythrocytes in 2 M KCl, 1.35 M potassium phosphate pH 6.9 diffract X-rays to 1.90 Å resolution, yielding a structure with an Rwork value of 18.7% and an Rfree of 22.2%. The crystal space group is P65, the asymmetric unit of which contains one complete octamer. This high-resolution model of the histone-core octamer allows further insight into intermolecular interactions, including water molecules, that dock the histone dimers to the tetramer in the nucleosome-core particle and have relevance to nucleosome remodelling. The three key areas analysed are the H2A′–H3–H4 molecular cluster (also H2A–H3′–H4′), the H4–H2B′ interaction (also H4′–H2B) and the H2A′–H4 β-sheet interaction (also H2A–H4′). The latter of these three regions is important to nucleosome remodelling by RNA polymerase II, as it is shown to be a likely core-histone binding site, and its disruption creates an instability in the nucleosome-core particle. A majority of the water molecules in the high-resolution octamer have positions that correlate to similar positions in the high-resolution nucleosome-core particle structure, suggesting that the high-resolution octamer model can be used for comparative studies with the high-resolution nucleosome-core particle.

Initiating a crystallographic analysis of recombinant (S)-2-hydroxypropylphosphonic acid epoxidase from Streptomyces wedmorensis

Cameron, S. — 2005-05-01

The oxirane (1R,2S)-1,2-epoxypropylphosphonic acid (fosfomycin) is a natural product antibiotic produced in Streptomyces wedmorensis by the metal-ion-dependent (S)-2-hydroxypropylphosphonic acid epoxidase. This epoxidase is highly unusual since it has no requirement for a haem prosthetic group. The gene encoding the enzyme, fom4, has been cloned and a highly efficient recombinant source of the enzyme established. Two different crystal forms, tetragonal and hexagonal, have been obtained. The hexagonal form displays symmetry consistent with space group P61/522 and unit-cell parameters a = 86.44, c = 221.56 Å, γ = 120°. The Matthews coefficient, VM, of 2.7 Å3 Da−1 corresponds to two subunits, each of approximate weight 21.4 kDa, in the asymmetric unit with 55% solvent content. These crystals diffract to high resolution and experimental phases are being sought to determine the structure.

Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis

Grenha, R. — 2005-05-01

Protein structures from the causative agent of anthrax (Bacillus anthracis) are being determined as part of a structural genomics programme. Amongst initial candidates for crystallographic analysis are enzymes involved in nucleotide biosynthesis, since these are recognized as potential targets in antibacterial therapy. Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway. The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis has been solved by molecular replacement at 2.24 Å resolution and refined to an R factor of 18.4%. This is the first report of a DeoD structure from a Gram-positive bacterium.

Refolding, crystallization and preliminary X-ray structural studies of the West Nile virus envelope (E) protein domain III

Yuan, F. — 2005-04-01

Domain III of the West Nile virus envelope protein, the putative receptor-binding domain, is a major virion-surface determinant for virulence. This protein was reported to be intrinsically unstable and has defied previous crystallization attempts. It has now been purified from inclusion bodies by protein refolding and was crystallized using the hanging-drop vapour-diffusion method at 291 K. The crystals belong to space group P2221, with unit-cell parameters a = 52.6, b = 59.7, c = 95.0 Å. A complete data set was collected to 2.8 Å at 100 K with Cu Kα X-rays from a rotating-anode generator.

Crystallization and preliminary structure analysis of CobE, an essential protein of cobalamin (vitamin B12) biosynthesis

Vévodová, J. — 2005-04-01

CobE, a protein implicated in vitamin B12 biosynthesis, from Pseudomonas aeruginosa has been overexpressed in Escherichia coli, purified and crystallized using hanging-drop vapour diffusion. The crystals belong to the primitive orthorhombic space group P212121, with unit-cell parameters a = 31.86, b = 41.07, c = 87.41 Å. The diffraction extends to a resolution of 1.9 Å. There is one molecule per asymmetric unit and the estimated solvent content is 35%. SeMet-labelled CobE has been prepared and crystallizes under the same conditions as the native protein with diffraction to 1.7 Å. The anomalous measurements will be used for phasing.

Expression, purification and crystallization of the cell-division protein YgfE from Escherichia coli

Addinall, S.G. — 2005-03-01

An open reading frame designated b2910 (ygfE) in the Escherichia coli K12-MG1655 genome sequence, identified as a possible homologue to the cell-division protein ZapA, was cloned into the high-expression plasmid pETDuet-1 and overexpressed in E. coli BL21 (DE3)-AI. The protein was purified in three steps to 99% purity. Crystals were obtained by the hanging-drop vapour-diffusion method at 291 K from a wide range of screened conditions, but principally from solutions containing 0.1 M HEPES pH 7.0, 18% PEG 6000, 5 mM CaCl2. Diffraction data to 1.8 Å were collected at the European Synchrotron Radiation Facility (ESRF). The crystals belong to space group P6122 or P6522, with unit-cell parameters a = 53.8, b = 53.8, c = 329.7 Å, α = β = 90, γ = 120°.

Crystallization and preliminary X-ray structural studies of a high-affinity CD8αα co-receptor to pMHC

Cole, D.K. — 2005-03-01

The class I CD8 positive T-cell response is involved in a number of conditions in which artificial down-regulation and control would be therapeutically beneficial. Such conditions include a number of autoimmune diseases and graft rejection in transplant patients. Although the CD8 T-cell response is dominated by the TCR–pMHC interaction, activation of T cells is in most cases also dependent on a number of associated signalling molecules. Previous work has demonstrated the ability of one such molecule (CD8) to act as an antagonist to T-cell activation if added in soluble form. Therefore, a high-affinity mutant CD8 (haCD8) has been developed with the aim of developing a therapeutic immunosuppressor. In order to fully understand the nature of the haCD8 interaction, this protein was crystallized using the sitting-drop vapour-diffusion method. Single haCD8 crystals were cryocooled and used for data collection. These crystals belonged to space group P6422 (assumed by similarity to the wild type), with unit-cell parameters a = 101.08, c = 56.54 Å. VM calculations indicated one molecule per asymmetric unit. A 2 Å data set was collected and the structure is currently being determined using molecular replacement.

Crystallographic analysis of murine constitutive androstane receptor ligand-binding domain complexed with 5α-androst-16-en-3α-ol

Vincent, J. — 2005-01-01

The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily. In contrast to classical nuclear receptors, which possess small-molecule ligand-inducible activity, CAR exhibits constitutive transcriptional activity in the apparent absence of ligand. CAR is among the most important transcription factors; it coordinately regulates the expression of microsomal cytochrome P450 genes and other drug-metabolizing enzymes. The murine CAR ligand-binding domain (LBD) was coexpressed with the steroid receptor coactivator protein (SRC-1) receptor-interacting domain (RID) in Escherichia coli. The mCAR LBD subunit was purified away from SRC-1 by affinity, anion-exchange and size-exclusion chromatography, crystallized with androstenol and the structure of the complex determined by molecular replacement.

Structure of a putative trans-editing enzyme for prolyl-tRNA synthetase from Aeropyrum pernix K1 at 1.7 Å resolution

Murayama, K. — 2005-01-01

The crystal structure of APE2540, the putative trans-editing enzyme ProX from Aeropyrum pernix K1, was determined in a high-throughput manner. The crystal belongs to the monoclinic space group P21, with unit-cell parameters a = 47.4, b = 58.9, c = 53.6 Å, β = 106.8°. The structure was solved by the multiwavelength anomalous dispersion method at 1.7 Å and refined to an R factor of 16.8% (Rfree = 20.5%). The crystal structure includes two protein molecules in the asymmetric unit. Each monomer consists of eight β-strands and seven α-helices. A structure-homology search revealed similarity between the trans-editing enzyme YbaK (or cysteinyl-tRNAPro deacylase) from Haemophilus influenzae (HI1434; 22% sequence identity) and putative ProX proteins from Caulobacter crescentus (16%) and Agrobacterium tumefaciens (21%).

Crystallization and preliminary X-ray analysis of coagulation factor IX-binding protein from habu snake venom at pH 6.5 and 4.6

Suzuki, N. — 2005-01-01

Coagulation factor IX-binding protein isolated from Trimeresurus flavoviridis (IX-bp) is a C-type lectin-like protein. It is an anticoagulant protein consisting of homologous subunits A and B. The subunits both contain a Ca2+-binding site with differing affinity (Kd values of 14 and 130 µM at pH 7.5). These binding characteristics are pH-dependent; under acidic conditions, the affinity of the low-affinity site was reduced considerably. In order to identify which site has high affinity and also to investigate the Ca2+-releasing mechanism, IX-bp was crystallized at pH 6.5 and 4.6. The crystals at pH 6.5 and 4.6 diffracted to 1.72 and 2.29 Å resolution, respectively; the former crystals belong to the monoclinic space group P21, with unit-cell parameters a = 60.7, b = 63.5, c = 66.9 Å, β = 117.0°, while the latter belong to the monoclinic space group C2, with a = 134.1, b = 37.8, c = 55.8 Å, β = 110.4°.

Crystallization and preliminary X-ray crystallographic analysis of peptide deformylase (PDF) from Bacillus cereus in ligand-free and actinonin-bound forms

Park, J.K. — 2005-01-01

In bacteria, protein expression initiates with an N-formyl group and this needs to be removed in order to ensure proper bacterial growth. These formylation and deformylation processes are unique to eubacteria; therefore, inhibition of these would provide a novel antibacterial therapy. Deformylation is carried out by peptide deformylase (PDF). PDF from Bacillus cereus, one of the major pathogenic bacteria, was cloned into expression plasmid pET-28a (Novagen), overexpressed in Escherichia coli BL21 (DE3) and purified to high quality. Crystals have been obtained of both ligand-free PDF and PDF to which actinonin, a highly potent naturally occurring inhibitor, is bound. Both crystals belong to space group P212121, with unit-cell parameters a = 42.72, b = 44.04, c = 85.19 Å and a = 41.31, b = 44.56, c = 84.47 Å, respectively. Diffraction data were collected to 1.7 Å resolution for the inhibitor-free crystals and to 2.0 Å resolution for the actinonin-bound crystals.

Purification, crystallization and preliminary X-ray diffraction analysis of the Kelch-like motif region of mouse Keap1

Padmanabhan, B. — 2005-01-01

Keap1 (Kelch-like ECH-associating protein 1) is a negative regulator of the Nrf2 transcription factor in the cytoplasm. The Kelch/DGR (double-glycine repeat) domain of Keap1 associates with Nrf2 as well as with actin filaments. A recombinant protein containing both the Kelch/DGR domain and the C-terminal region of mouse Keap1 was expressed in Escherichia coli, purified to near-homogeneity and crystallized by the sitting-drop vapour-diffusion method. The crystal belongs to space group P61 or P65, with unit-cell parameters a = b = 102.95, c = 55.21 Å, and contains one molecule in the asymmetric unit. A complete diffraction data was collected to 2.25 Å resolution using an R-AXIS IV++ imaging plate mounted on an RA-Micro7 Cu Kα rotating-anode X-ray generator.

Preliminary characterization of two different crystal forms of acylphosphatase from the hyperthermophile archaeon Sulfolobus solfataricus

Zuccotti, S. — 2005-01-01

Acylphosphatase is a ubiquitous small enzyme that was first characterized in mammals. It is involved in the hydrolysis of carboxyl-phosphate bonds in several acylphosphate substrates, such as carbamoylphosphate and 1,3-biphosphoglycerate; however, a consensus on acylphosphatase action in vivo has not yet been reached. Recent investigations have focused on acylphosphatases from lower phyla, such as Drosophila melanogaster and Escherichia coli, in view of the application of these small proteins as models in the study of folding, misfolding and aggregation processes. An acylphosphatase from the hyperthermophilic archaeon Sulfolobus solfataricus has been cloned, expressed and purified. Here, the growth and characterization of a triclinic and a monoclinic crystal form of the hyperthermophilic enzyme are reported; X-ray diffraction data have been collected to 1.27 and 1.90 Å resolution, respectively.

Crystallization and preliminary X-ray characterization of a lectin from Cicer arietinum (chickpea)

Katre, U.V. — 2005-01-01

The lectin isolated from mature seeds of Cicer arietinum (CAL) agglutinates pronase-treated rabbit and human erythrocytes and its haemagglutination activity is inhibited by fetuin and desialated fetuin but not by simple monosaccharides or oligosaccharides. The purified lectin is a dimer of molecular weight 43 000 Da composed of two identical subunits (MW 21 500), as confirmed by SDS–PAGE. The lectin has been crystallized using the hanging-drop vapour-diffusion method at 295 K over a well solution containing 0.2 M sodium acetate, 0.1 M sodium phosphate buffer pH 6.5 and 14%(w/v) polyethylene glycol 8000. The triangular prism-shaped crystals belong to space group R3 and have unit-cell parameters a = b = 81.2, c = 69.4 Å. The diffraction data are 93.8% complete to 2.3 Å Bragg spacing with an Rmerge of 0.103.

On the purification and preliminary crystallographic analysis of isoquinoline 1-oxidoreductase from Brevundimonas diminuta 7

Boer, D.R. — 2005-01-01

Isoquinoline 1-oxidoreductase (IOR) from Brevundimonas diminuta is a mononuclear molybdoenzyme of the xanthine-dehydrogenase family of proteins and catalyzes the conversion of isoquinoline to isoquinoline-1-one. Its primary sequence and behaviour, specifically in its substrate specificity and lipophilicity, differ from other members of the family. A crystal structure of the enzyme is expected to provide an explanation for these differences. This paper describes the crystallization and preliminary X-ray diffraction experiments as well as an optimized purification protocol for IOR. Crystallization of IOR was achieved using two different crystallization buffers. Streak-seeding and cross-linking were essential to obtain well diffracting crystals. Suitable cryo-conditions were found and a structure solution was obtained by molecular replacement. However, phases need to be improved in order to obtain a more interpretable electron-density map.

Preliminary crystallographic analysis of the antibiotic discharge outer membrane lipoprotein OprM of Pseudomonas aeruginosa with an exceptionally long unit cell and complex lattice structure

Akama, H. — 2005-01-01

Crystals of the drug-discharge outer membrane protein OprM (MW = 50.9 kDa) of the MexAB-OprM multidrug transporter of Pseudomonas aeruginosa have been grown at 293 K in the presence of 2-methyl-2,4-propanediol and a combination of surfactants. The crystal belonged to space group R32, with unit-cell parameters a = b = 85.43, c = 1044.3 Å. Diffraction data for OprM were obtained using the undulator synchrotron-radiation beamline at SPring-8 (BL44XU, Osaka University), which allowed an extra-long specimen-to-detector distance with a wide detector area. The crystal diffracted to 2.56 Å resolution using 0.9 Å X-rays from the synchrotron-radiation source. A heavy-atom derivative for isomorphous replacement phasing was obtained using iridium chloride.

Crystallization and preliminary X-ray diffraction analysis of human growth and differentiation factor 5 (GDF-5)

Mueller, T.D. — 2005-01-01

Growth and differentiation factor 5 (GDF-5) belongs to the large TGF-β superfamily of secreted signalling proteins and plays a pivotal role in skeletal development during embryogenesis. The gene for human GDF-5 was cloned, expressed in Escherichia coli and purified to homogeneity. Crystals were obtained that diffracted to 2.2 Å resolution. A native data set was acquired, showing that the crystals belong to a trigonal space group, i.e. P3121 or P3221, with unit-cell parameters a = b = 97.1, c = 48.3 Å. Initial analysis suggest the presence of only one monomer in the asymmetric unit, resulting in a high solvent content of 72% in the crystal.

Crystallization and preliminary X-ray crystallographic analysis of osteoclast-stimulating factor

Li, M. — 2005-01-01

Osteoclast-stimulating factor increases osteoclast formation and bone resorption through a cellular signal transduction cascade, possibly by its interaction with c-Src or related family members. Crystals of human osteoclast-stimulating factor were obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. The crystals are primitive orthorhombic and belong to P222 or a related space group, with unit-cell parameters a = 38.1, b = 54.9, c = 64.7 Å.

Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants

Chandra, P.M. — 2005-01-01

The crystallization of three catalytically inactive mutants of penicillin V acylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide three-dimensional structures of precursor PVA forms, plus open a route to the study of enzyme–substrate complexes for this industrially important enzyme.

Mistletoe lectin I in complex with galactose and lactose reveals distinct sugar-binding properties

Mikeska, R. — 2005-01-01

The structures of mistletoe lectin I (ML-I) from Viscum album complexed with lactose and galactose have been determined at 2.3 Å resolution and refined to R factors of 20.9% (Rfree = 23.6%) and 20.9 (Rfree = 24.6%), respectively. ML-I is a heterodimer and belongs to the class of ribosome-inactivating proteins of type II, which consist of two chains. The A-chain has rRNA N-glycosidase activity and irreversibly inhibits eukaryotic ribosomes. The B-chain is a lectin and preferentially binds to galactose-terminated glycolipids and glycoproteins on cell membranes. Saccharide binding is performed by two binding sites in subdomains α1 and γ2 of the ML-I B-chain separated by ∼62 Å from each other. The favoured binding of galactose in subdomain α1 is achieved via hydrogen bonds connecting the 4-hydroxyl and 3-hydroxyl groups of the sugar moiety with the side chains of Asp23B, Gln36B and Lys41B and the main chain of 26B. The aromatic ring of Trp38B on top of the preferred binding pocket supports van der Waals packing of the apolar face of galactose and stabilizes the sugar–lectin complex. In the galactose-binding site II of subdomain γ2, Tyr249B provides the hydrophobic stacking and the side chains of Asp235B, Gln238B and Asn256B are hydrogen-bonding partners for galactose. In the case of the galactose-binding site I, the 2-hydroxyl group also stabilizes the sugar–protein complex, an interaction thus far rarely detected in galactose-specific lectins. Finally, a potential third low-affinity galactose-binding site in subunit β1 was identified in the present ML-I structures, in which a glycerol molecule from the cryoprotectant buffer has bound, mimicking the sugar compound.

Crystallization and preliminary X-ray crystallographic analysis of MbtI, a protein essential for siderophore biosynthesis in Mycobacterium tuberculosis

Harrison, A.J. — 2005-01-01

Mycobacterium tuberculosis, the causative agent of tuberculosis, depends on the secretion of salicylate-based siderophores called mycobactins for the acquisition of extracellular iron, which is essential for the growth and virulence of the bacterium. The protein MbtI is thought to be the isochorismate synthase enzyme responsible for the conversion of chorismate to isochorismate, the first step in the salicylate production required for mycobactin biosynthesis. MbtI has been overexpressed in Escherichia coli, purified and crystallized. The crystals diffract to a maximum resolution of 1.8 Å. They belong to space group P212121, with unit-cell parameters a = 51.8, b = 163.4, c = 194.9 Å, consistent with the presence of either two, three or four molecules in the asymmetric unit.

Crystallization and preliminary X-ray diffraction studies of a catechol-O-methyltransferase/inhibitor complex

Rodrigues, M.L. — 2005-01-01

Inhibitors of the enzyme catechol-O-methyltransferase (COMT) are used as co-adjuvants in the therapy of Parkinson's disease. A recombinant form of the soluble cytosolic COMT from rat has been co-crystallized with a new potent inhibitor, BIA 8-176 [(3,4-dihydroxy-2-nitrophenyl)phenylmethanone], by the vapour-diffusion method using PEG 6K as precipitant. Crystals diffract to 1.6 Å resolution on a synchrotron-radiation source and belong to the monoclinic space group P21, with unit-cell parameters a = 52.77, b = 79.63, c = 61.54 Å, β = 91.14°.

Preliminary crystallographic studies of glucose dehydrogenase from the promiscuous Entner–Doudoroff pathway in the hyperthermophilic archaeon Sulfolobus solfataricus

Theodossis, A. — 2005-01-01

The hyperthermophilic archaeon Sulfolobus solfataricus grows optimally above 353 K and can metabolize glucose and its C4 epimer galactose via a non-phosphorylative variant of the Entner–Doudoroff pathway involving catalytically promiscuous enzymes that can operate with both sugars. The initial oxidation step is catalysed by glucose dehydrogenase (SsGDH), which can utilize both NAD and NADP as cofactors. The enzyme operates with glucose and galactose at similar catalytic efficiency, while its substrate profile also includes a range of other five- and six-carbon sugars. Crystals of the 164 kDa SsGDH homotetramer have been grown under a variety of conditions. The best crystals to date diffract to 1.8 Å on a synchrotron source, have orthorhombic symmetry and belong to space group P21212. Attempts are being made to solve the structure by MAD and MR.

Expression, purification and preliminary crystallographic analysis of sucrose phosphate synthase (SPS) from Halothermothrix orenii

Huynh, F. — 2005-01-01

This is the first report of the crystallization of a sucrose phosphate synthase (SPS; EC 2.4.1.14). It also constitutes the first study of a sucrose phosphate synthase from a non-photosynthetic thermohalophilic anaerobic bacterium, Halothermothrix orenii. The purified recombinant spsA protein has been crystallized in the monoclinic space group C2, with unit-cell parameters a = 154.2, b = 47.9, c = 72.3 Å, β = 103.16°, using the hanging-drop vapour-diffusion method. The crystal diffracts X-rays to a resolution limit of 3.01 Å. Heavy-metal and halide-soaking trials are currently in progress to solve the structure.

Crystallization and preliminary X-ray crystallographic study of disproportionating enzyme from potato

Imamura, K. — 2005-01-01

Disproportionating enzyme (D-enzyme; EC 2.4.1.25) is a 59 kDa protein that belongs to the α-amylase family. D-enzyme catalyses intramolecular and intermolecular transglycosylation reactions of α-1,4 glucan. A crystal of the D-enzyme from potato was obtained by the hanging-drop vapour-diffusion method. Preliminary X-ray data showed that the crystal diffracts to 2.0 Å resolution and belongs to space group C2221, with unit-cell parameters a = 69.7, b = 120.3, c = 174.2 Å.

Structure of Mesorhizobium loti arylamine N-acetyltransferase 1

Holton, S.J. — 2005-01-01

The arylamine N-acetyltransferase (NAT) enzymes have been found in a broad range of both eukaryotic and prokaryotic organisms. The NAT enzymes catalyse the transfer of an acetyl group from acetyl Co-enzyme A onto the terminal nitrogen of a range of arylamine, hydrazine and arylhydrazine compounds. Recently, several NAT structures have been reported from different prokaryotic sources including Salmonella typhimurium, Mycobacterium smegmatis and Pseudomonas aeruginosa. Bioinformatics analysis of the Mesorhizobium loti genome revealed two NAT paralogues, the first example of multiple NAT isoenzymes in a eubacterial organism. The M. loti NAT 1 enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme was crystallized in 0.5 M Ca(OAc)2, 16% PEG 3350, 0.1 M Tris–HCl pH 8.5 using the sitting-drop vapour-diffusion method. A data set diffracting to 2.0 Å was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic spacegroup P212121, with unit-cell parameters a = 53.2, b = 97.3, c = 114.3 Å. The structure was refined to a final free-R factor of 24.8%. The structure reveals that despite low sequence homology, M. loti NAT1 shares the common fold as reported in previous NAT structures and exhibits the same catalytic triad of residues (Cys-His-Asp) in the active site.

Expression, purification, crystallization and preliminary X-ray crystallographic studies of the trehalulose synthase MutB from Pseudomonas mesoacidophila MX-45

Ravaud, S. — 2005-01-01

The trehalulose synthase (MutB) from Pseudomonas mesoacidophila MX-45, belonging to glycoside hydrolase family 13, catalyses the isomerization of sucrose to trehalulose (α-d-glucosylpyranosyl-1,1-d-fructofuranose) and isomaltulose (α-d-glucosylpyranosyl-1,6-d-fructofuranose) as main products and glucose and fructose in residual amounts from the hydrolytic reaction. To date, a three-dimensional structure of a sucrose isomerase that produces mainly trehalulose, as is the case for MutB, has been lacking. Crystallographic studies of this 64 kDa enzyme have therefore been initiated in order to contribute to the understanding of the molecular basis of sucrose decomposition, isomerization and of the selectivity of this enzyme that leads to the formation of different products. The MutB protein has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. Two different crystal forms have been obtained: one diffracts X-rays to 1.6 Å resolution using synchrotron radiation and belongs to space group P1, with unit-cell parameters a = 63.8, b = 72.0, c = 82.2 Å, α = 67.5, β = 73.1, γ = 70.8°, while the other form diffracts to 1.8 Å resolution using synchrotron radiation and belongs to space group P21, with unit-cell parameters a = 63.7, b = 85.9, c = 119.7 Å, β = 97.7°. A molecular-replacement solution has been found using the structure of the isomaltulose synthase (PalI) from Klebsiella sp. LX3 as a search model.

Crystallization and preliminary crystallographic study of a recombinant predicted acetamidase/formamidase from the thermophile Thermoanaerobacter tengcongensis

Wu, G. — 2005-01-01

No crystal structures are yet available for homologues of a predicted acetamidase/formamidase (Amds/Fmds) from the archaeon Thermoanaerobacter tengcongensis. The Amds/Fmds gene was cloned and expressed as a soluble protein in Escherichia coli. Native Amds/Fmds and its SeMet-substituted form were purified and crystallized by vapour diffusion in hanging drops at 296 K. The native crystals, which were grown in PEG 8000, belong to the monoclinic space group P21, with unit-cell parameters a = 41.23 (3), b = 152.88 (6), c = 100.26 (7) Å, β = 99.49 (3)°. The diffraction data were collected to 2.00 Å resolution using synchrotron radiation. Based on a predicted solvent content of 50%, a Matthews coefficient of 2.44 Å3 Da−1 and two main peaks in the self-rotation function, the asymmetric unit is predicted to contain two dimers of the 32 kDa native protein. MAD data were collected for the SeMet protein, but the corresponding crystals display different unit-cell parameters and appear to contain four dimers in the asymmetric unit.

Expression, purification, crystallization and preliminary crystallographic analysis of human Pim-1 kinase

Qian, K.C. — 2005-01-01

Pim kinases, including Pim-1, Pim-2 and Pim-3, belong to a distinctive serine/threonine protein-kinase family. They are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Their kinase domains are highly homologous to one another, but share low sequence identity to other kinases. Specifically, there are two proline residues in the conserved hinge-region sequence ERPXPX separated by a residue that is non-conserved among Pim kinases. Full-length human Pim-1 kinase (1–313) was cloned and expressed in Escherichia coli as a GST-fusion protein and truncated to Pim-1 (14–313) by thrombin digestion during purification. The Pim-1 (14–313) protein was purified to high homogeneity and monodispersity. This protein preparation yielded small crystals in the initial screening and large crystals after optimization. The large crystals of apo Pim-1 enzyme diffracted to 2.1 Å resolution and belong to space group P65, with unit-cell parameters a = b = 95.9, c = 80.0 Å, β = 120° and one molecule per asymmetric unit.

Crystallization and preliminary crystallographic studies of the copper-binding domain of the amyloid precursor protein of Alzheimer's disease

Kong, G.K.-W. — 2005-01-01

Alzheimer's disease is thought to be triggered by production of the amyloid β (Aβ) peptide through proteolytic cleavage of the amyloid precursor protein (APP). The binding of Cu2+ to the copper-binding domain (CuBD) of APP reduces the production of Aβ in cell-culture and animal studies. It is expected that structural studies of the CuBD will lead to a better understanding of how copper binding causes Aβ depletion and will define a potential drug target. The crystallization of CuBD in two different forms suitable for structure determination is reported here.

Crystallization and preliminary X-ray analysis of gene product 44 from bacteriophage Mu

Kondou, Y. — 2005-01-01

Bacteriophage Mu baseplate protein gene product 44 (gp44) is an essential protein required for the assembly of viable phages. To investigate the roles of gp44 in baseplate assembly and infection, gp44 was crystallized at pH 6.0 in the presence of 20% 2-methyl-2,4-pentanediol. The crystals belong to space group R3, with unit-cell parameters a = b = 127.47, c = 63.97 Å. The crystals diffract X-rays to at least 2.1 Å resolution and are stable in the X-ray beam and are therefore appropriate for structure determination. Native data have been collected to 2.1 Å resolution using a DIP6040 image-plate system at beamline BL44XU at the SPring-8 facility in Japan.

Expression, purification, crystallization and preliminary crystallographic study of a potential metal-dependent hydrolase with cyclase activity from Thermoanaerobacter tengcongensis

Liu, S. — 2005-01-01

The putative metal-dependent hydrolase gene TTE1006 from Thermoanaerobacter tengcongensis strain MB4T (T = type strain; Genbank accession No. AE008691) was heterologously expressed in Escherichia coli. The 205-amino-acid gene product was purified and crystallized. The crystal used for data collection belongs to space group P21, with unit-cell parameters a = 85.2, b = 62.1, c = 172.4 Å, β = 104.2°. Using a synchrotron-radiation source, the resolution limit of the data reached 1.87 Å. Eight molecules were estimated to be present in the asymmetric unit, with a solvent content of 48%. Structure determination is ongoing using the multiple-wavelength anomalous diffraction (MAD) method and also the molecular-replacement (MR) method.

Crystallization and preliminary X-ray diffraction analysis of a lectin from Canavalia maritima seeds

Gadelha, C.A.A. — 2005-01-01

A lectin from Canavalia maritima seeds (ConM) was purified and submitted to crystallization experiments. The best crystals were obtained using the vapour-diffusion method at a constant temperature of 293 K and grew in 7 d. A complete structural data set was collected to 2.1 Å resolution using a synchrotron-radiation source. The ConM crystal belongs to the orthorhombic space group P21212, with unit-cell parameters a = 67.15, b = 70.90, c = 97.37 Å. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%. Crystallographic refinement is under way.

Crystallization and preliminary X-ray studies on the reaction center–light-harvesting 1 core complex from Rhodopseudomonas viridis

Saijo, S. — 2005-01-01

The reaction center–light-harvesting 1 (RC–LH1) core complex is the photosynthetic apparatus in the membrane of the purple photosynthetic bacterium Rhodopseudomonas viridis. The RC is surrounded by an LH1 complex that is constituted of oligomers of three types of apoproteins (α, β and γ chains) with associated bacteriochlorophyll bs and carotenoid. It has been crystallized by the sitting-drop vapour-diffusion method. A promising crystal diffracted to beyond 8.0 Å resolution. It belonged to space group P1, with unit-cell parameters a = 141.4, b = 136.9, c = 185.3 Å, α = 104.6, β = 94.0, γ = 110.7°. A Patterson function calculated using data between 15.0 and 8.0 Å resolution suggested that the LH1 complex is distributed with quasi-16-fold rotational symmetry around the RC.

Four crystal forms of a Bence-Jones protein

Makino, D.L. — 2005-01-01

Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 years ago. Closely related tetragonal and orthorhombic forms belonging to space groups P43212 and P212121, with unit-cell parameters a = b = 68.7, c = 182.1 and a = 67.7, b = 69.4, c = 87.3 Å, diffract to 1.5 and 1.9 Å, respectively. Two closely related trigonal forms, both belonging to space group P3121 with unit-cell parameters a = b = 154.3 Å but differing by a doubling of the c axis, one 46.9 Å and the other 94.0 Å, diffract to 2.9 and 2.6 Å resolution, respectively. The trigonal crystal of short c-axis length shows a positive indication of twinning. The trigonal crystal of longer c axis, which appeared only after eight months of incubation at room temperature, is likely to be composed of proteolytically degraded molecules and unlike the other crystal forms contains two entire Bence-Jones dimers in the asymmetric unit. This latter crystal form may shed some light on the formation of fibrils common to certain storage diseases.

Crystallization and preliminary diffraction analysis of a group I ribozyme from bacteriophage Twort

Chase, E. — 2005-01-01

Group I introns are catalytic RNAs that are capable of performing a variety of phosphotransesterification reactions including self-splicing and RNA cleavage. The reactions are efficient, accurate and dependent only on the presence of guanosine-nucleotide substrate and sufficient magnesium ion to stabilize the structure of the RNA. To understand how the group I intron active-site facilitates catalysis, crystals of a 242-nucleotide ribozyme bound to a four-nucleotide product RNA have been produced that diffract to 3.6 Å resolution. The space group of these crystals is I212121 and the unit-cell parameters are a = 94.6, b = 141.0, c = 210.9 Å. A single heavy-atom derivative has been synthesized by covalent modification of the product RNA with iodine.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of pantothenate kinase from Mycobacterium tuberculosis

Das, S. — 2005-01-01

Pantothenate kinase is an essential enzyme in the bacterial life cycle. It catalyzes the phosphorylation of pantothenate (vitamin B5) to 4′-phosphopantothenate, the first step in the coenzyme A biosynthetic pathway. The enzyme from Mycobacterium tuberculosis, MW 35.7 kDa, has been cloned, expressed, purified and crystallized in two different trigonal crystal forms, both belonging to space group P3121. Two complete data sets of resolution 2.5 Å (form I) and 2.9 Å (form II) from crystals with unit-cell parameters a = b = 78.3, c = 115.45 Å and a = b = 107.63, c = 89.85 Å, respectively, were collected at room temperature on a home X-ray source. Structures of both crystal forms were solved for one subunit in the asymmetric unit by molecular replacement.

Crystallization and preliminary crystallographic analysis of endo-1,3-β-glucanase from Arthrobacter sp.

Pang, Z. — 2005-01-01

Endo-1,3-β-glucanases hydrolyze internal 1,3-β-glucosyl linkages. The endo-1,3-β-glucanase from Arthrobacter sp. was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to space group P41, with unit-cell parameters a = 71.31, c = 60.07 Å, and contained one molecule per asymmetric unit. The Matthews coefficient (VM) and the solvent content were 2.35 Å3 Da−1 and 47.63%, respectively. Diffraction data were collected to a resolution of 1.66 Å at SPring-8 using a MAR CCD area detector and gave a data set with an overall Rmerge of 5.4% and a completeness of 99.4%.

Crystallization and preliminary X-ray crystallographic analysis of agkicetin-C from Deinagkistrodon acutus venom

Xu, G. — 2005-01-01

The crystallization and preliminary crystallographic analysis of agkicetin-C, a well known platelet glycoprotein Ib (GPIb) antagonist from the venom of Deinagkistrodon acutus found in Anhui Province, China is reported. Crystals of agkicetin-C suitable for structure determination were obtained from 1.8 M ammonium sulfate, 40 mM MES pH 6.5 with 2%(v/v) PEG 400. Interestingly, low buffer concentrations of MES seem to be necessary for crystal growth. The crystals of agkicetin-C belong to space group C2, with unit-cell parameters a = 177.5, b = 97.7, c = 106.8 Å, β = 118.5°, and diffract to 2.4 Å resolution. Solution of the phase problem by the molecular-replacement method shows that there are four agkicetin-C molecules in the asymmetric unit, with a VM value of 3.4 Å3 Da−1, which corresponds to a high solvent content of approximately 64%. Self-rotation function calculations show a single well defined non-crystallographic twofold axis with features that may represent additional elements of non-crystallographic symmetry.

Crystallization and preliminary X-ray analysis of the GST-fused human Bri3 N-terminal domain

Ye, Q. — 2005-01-01

Bri3 is a recently identified proline-rich transmembrane polypeptide up-regulated during TNF-mediated inflammation and immunity. The polyproline-rich N-terminal (residues 1–60) domain of Bri3 was affinity-purified to homogeneity as a glutathione-S-transferase (GST) fusion protein. Crystals were obtained in ∼3 d by the equilibrium vapour-diffusion method from a solution containing 1.5–2.2 M ammonium sulfate and 0.1 M bis-tris pH 6.0. The crystals belong to space group P43212, with unit-cell parameters a = b = 91.66, c = 57.53 Å. An X-ray data set was collected to 1.6 Å resolution using synchrotron radiation, with an Rsym of 0.058 and a completeness of 95.3%. There is one molecule of the fusion protein in the asymmetric unit, which corresponds to ∼35% solvent content.

Purification, crystallization and preliminary crystallographic analysis of the vacuole-type ATPase subunit E from Pyrococcus horikoshii OT3

Lokanath, N.K. — 2005-01-01

The vacuole-type ATPases in eukaryotic cells translocate protons across various biological membranes including the vacuolar membrane by consuming ATP molecules. The E subunit of the multisubunit complex V-ATPase from Pyrococcus horikoshii OT3, which has a molecular weight of 22.88 kDa, has been cloned, overexpressed in Escherichia coli, purified and crystallized by the microbatch method using PEG 4000 as a precipitant at 296 K. A data set to 1.85 Å resolution with 98.8% completeness and an Rmerge of 6.5% was collected from a single flash-cooled crystal using synchrotron radiation. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 52.196, b = 55.317, c = 77.481 Å, and is most likely to contain one molecule per asymmetric unit.

Open access article in Acta Crystallographica Section F: Structural Biology and Crystallization Communications

Structure of the hypothetical DUF1811-family protein GK0453 from Geobacillus kaustophilus HTA426

Structure of Pseudomonas aeruginosa inosine 5′-monophosphate dehydrogenase

Crystallization and preliminary structure determination of the transfer protein TraM from the Gram-positive conjugative plasmid pIP501

Crystals on the cover 2013

Some practical guidelines for UV imaging in the protein crystallization laboratory

Expectation bias and information content

The AEROPATH project targeting Pseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery

Crystallization of domains involved in self-assembly of the S-layer protein SbsC

Crystallization and first data collection of the putative transfer protein TraN from the Gram-positive conjugative plasmid pIP501

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of aspartyl aminopeptidase from the apeB gene of Pseudomonas aeruginosa. Retraction

Solution structure of the cold-shock-like protein from Rickettsia rickettsii

Recombinant portal protein from Staphylococcus epidermidis bacteriophage CNPH82 is a 13-subunit oligomer

Crystallization and preliminary X-ray crystallographic analysis of an ice-binding protein (FfIBP) from Flavobacterium frigoris PS1. Addendum

Structural insights into ChpT, an essential dimeric histidine phosphotransferase regulating the cell cycle in Caulobacter crescentus

Crystallization and preliminary X-ray diffraction analysis of the high molecular weight ketoacyl reductase FabG4 complexed with NADH. Corrigendum

Anomalous dispersion analysis of inhibitor flexibility: a case study of the kinase inhibitor H-89

Useable diffraction data from a multiple microdomain-containing crystal of Ascaris suum As-p18 fatty-acid-binding protein using a microfocus beamline

Two crystal forms of a helix-rich fatty acid- and retinol-binding protein, Na-FAR-1, from the parasitic nematode Necator americanus

Crystallization of SHARPIN using an automated two-dimensional grid screen for optimization

Structure of Leishmania major cysteine synthase

Purification, crystallization and preliminary X-ray diffraction data of UDP-galactopyranose mutase from Aspergillus fumigatus

A model for 3-methyladenine recognition by 3-methyladenine DNA glycosylase I (TAG) from Staphylococcus aureus

Crystallization and preliminary crystallographic analysis of the major capsid proteins VP16 and VP17 of bacteriophage P23-77

Another case of fraud in structural biology

Structure of a hexameric form of RadA recombinase from Methanococcus voltae

Detection and analysis of unusual features in the structural model and structure-factor data of a birch pollen allergen

Response to Detection and analysis of unusual features in the structural model and structure-factor data of a birch pollen allergen

Call for a crystallization ontology

On the need for an international effort to capture, share and use crystallization screening data

Crystals on the cover 2012

Expression, crystallization and preliminary X-ray crystallographic analysis of glucose-6-phosphate dehydrogenase from the human pathogen Trypanosoma cruzi in complex with substrate. Corrigendum

Review panel for Acta Crystallographica Section F

Seleno-detergent MAD phasing of leukotriene C4 synthase in complex with dodecyl-β-d-selenomaltoside

Therapeutic target-site variability in α1-antitrypsin characterized at high resolution

Structure of hyperthermophilic β-glucosidase from Pyrococcus furiosus

Wheat germ cell-free expression system as a pathway to improve protein yield and solubility for the SSGCID pipeline

Structure of aldose reductase from Giardia lamblia

Structure of 3-ketoacyl-(acyl-carrier-protein) reductase from Rickettsia prowazekii at 2.25 Å resolution

An ensemble of structures of Burkholderia pseudomallei 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase

Structure of the cystathionine γ-synthase MetB from Mycobacterium ulcerans

Structural genomics of infectious disease drug targets: the SSGCID

Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase from Babesia bovis

The Protein Maker: an automated system for high-throughput parallel purification

Overexpression, crystallization and preliminary X-­ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1

Gene Composer in a structural genomics environment

Crystallization and preliminary X-ray crystallographic studies of the ice-binding protein from the Antarctic yeast Leucosporidium sp. AY30. Corrigendum

Structure of fumarate hydratase from Rickettsia prowazekii, the agent of typhus and suspected relative of the mitochondria

Gene design, cloning and protein-expression methods for high-value targets at the Seattle Structural Genomics Center for Infectious Disease

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of glucuronic acid dehy­drogenase from Chromohalobacter salexigens. Addendum

From screen to structure with a harvestable microfluidic device

Salvage and storage of infectious disease protein targets in the SSGCID high-throughput crystallization pathway using microfluidics

Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline

The structure of fructose bisphosphate aldolase from Encephalitozoon cuniculi

Conservation of a crystallographic interface suggests a role for β-sheet augmentation in influenza virus NS1 multifunctionality

Structure of triosephosphate isomerase from Cryptosporidium parvum

Structure of fructose bisphosphate aldolase from Bartonella henselae bound to fructose 1,6-bisphosphate

High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease

Structure of a cyclin-dependent kinase from Giardia lamblia

Use of the α-mannosidase I inhibitor kifunensine allows the crystallization of apo CTLA-4 homodimer produced in long-term cultures of Chinese hamster ovary cells

Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success

Expression, purification, crystallization and preliminary crystallographic analysis of a putative Clostridium difficile surface protein Cwp19

Structures of the rat complement regulator CrrY

Structure of thymidylate kinase from Ehrlichia chaffeensis

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

Structure of nitrilotriacetate monooxygenase component B from Mycobacterium thermoresistibile

Comparative analysis of glutaredoxin domains from bacterial opportunistic pathogens

Structure of a Nudix hydrolase (MutT) in the Mg2+-­bound state from Bartonella henselae, the bacterium responsible for cat scratch fever

BrabA.11339.a: anomalous diffraction and ligand binding guide towards the elucidation of the function of a `putative β-lactamase-like protein' from Brucella melitensis

Structures of a putative ζ-class glutathione S-transferase from the pathogenic fungus Coccidioides immitis

Solution-state NMR structure and biophysical characterization of zinc-substituted rubredoxin B (Rv3250c) from Mycobacterium tuberculosis

Solution structure of an arsenate reductase-related protein, YffB, from Brucella melitensis, the etiological agent responsible for brucellosis

NMR structure of an acyl-carrier protein from Borrelia burgdorferi

Structures of phosphopantetheine adenylyltransferase from Burkholderia pseudomallei

Refolding, crystallization and preliminary X-ray crystallographic study of the whole extracellular regions of nectins

Editorial

Crystallization and preliminary crystallographic analysis of the ADP-ribosyltransferase HopU1. Corrigendum

Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active site

Structure of the C-terminal domain of the surface antigen SpaP from the caries pathogen Streptococcus mutans

Publishing structural genomics results: the JCSG Special Issue

Overexpression, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of glucuronic acid dehydrogenase from Chromohalobacter salexigens. Addendum

Structure of a Nudix hydrolase (MutT) in the Mg2+-bound state from Bartonella henselae, the bacterium responsible for cat scratch fever