Automation in biological crystallization

Automation is the response to overcoming the crystallization bottleneck in biological crystallography. This review provides a summary of the current methods and technologies applied in automated platforms for the setup of initial and follow-up crystallization experiments.

Structure of a His170Tyr mutant of thermostable pNPPase from Geobacillus stearothermophilus

The crystal structure of the His170Tyr mutant of thermostable pNPPase from Geobacillus stearothermophilus has been determined at 2.0 Å resolution, with Na⁺ and SO₄²⁻ bound in the active site, showing a half-closed cap conformation.

GlmU (N-acetylglucosamine-1-phosphate uridyltransferase) bound to three magnesium ions and ATP at the active site

The structure of GlmU for M. tuberculosis is determined in complex with GlcNAc-1-P, a non-canonical nucleotide substrate – ATP and three Mg²⁺ ions. ATP is bound at the active site in an unusual conformation.

Structures and binding specificity of galactose- and mannose-binding lectins from champedak: differences from jackfruit lectins

The structures of two lectins utilized as molecular markers are reported.

High-resolution crystal structures of two crystal forms of human cyclophilin D in complex with PEG 400 molecules

High-resolution crystal structures of cyclophilin D in complex with PEG 400 in primitive orthorhombic and primitive tetragonal forms are reported.

Structure of transportin SR2, a karyopherin involved in human disease, in complex with Ran

A 2.9 Å resolution crystal structure of transportin SR2 (TRN-SR2), a β-karyopherin involved in the nuclear import of HIV, in complex with the small GTPase Ran was determined. GTP-loaded Ran binds to the N-terminal part of the α-helical solenoid-like fold of TRN-SR2.

Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones

CsyB from A. oryzae has been overexpressed in E. coli, purified and crystallized. Diffraction data have been collected to a resolution of 1.71 Å.

Crystallization and preliminary X-ray diffraction analysis of AntE, a crotonyl-CoA carboxylase/reductase from Streptomyces sp. NRRL 2288

AntE, a crotonyl-CoA carboxylase/reductase involved in the biosynthesis of antimycin A from Streptomyces sp. NRRL 2288, has been overexpressed in E. coli, purified and crystallized. Diffraction data have been collected to a resolution of 2.29 Å.

Crystallization and preliminary crystallographic studies of PotA, a membrane-associated ATPase of the spermidine-preferential uptake system in Thermotoga maritima

Native and selenomethionine-labelled PotA proteins from T. maritima were expressed, purified and crystallized, and X-ray diffraction data were collected to 2.7 Å resolution.

Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the bovine CD8αα–BoLA-2*02201 complex

The crystallization and preliminary X-ray diffraction analysis of a complex between bovine CD8αα and the bovine MHC class I molecule (BoLA-I, BoLA-2*02201) at 1.7 Å resolution revealed that the crystal belonged to space group P2₁, with unit-cell parameters a = 53.9, b = 103.8, c = 61.8 Å, α = γ = 90, β = 96°, and contained one complex in the asymmetric unit. The Matthews coefficient and solvent content were calculated as 2.41 ų Da⁻¹ and 48.9%, respectively.

Crystallization and preliminary X-ray crystallographic analysis of carboxyl-terminal region 4 of SigR from Streptomyces coelicolor A3(2)

Carboxyl-terminal region 4 of SigR (σ^R₄) from S. coelicolor A3(2) has been crystallized (space group P4₃2₁2, unit-cell parameters a = b = 42.14, c = 102.02 Å). Diffraction data were collected to 2.60 Å resolution using synchrotron radiation.

Expression, purification and preliminary crystallographic analysis of Mycobacterium tuberculosis CysQ, a phosphatase involved in sulfur metabolism

The cysQ gene from Mycobacterium tuberculosis was cloned and the expressed protein, a 3′-phosphoadenosine-5′'-phosphatase, was purified and crystallized. X-ray diffraction data were collected to 1.7 Å resolution.

Overproduction, purification, crystallization and preliminary X-ray characterization of the family 46 carbohydrate-binding module (CBM46) of endo-β-1,4-glucanase B (CelB) from Bacillus halodurans

A family 46 carbohydrate-binding module (BhCBM46) from B. halodurans endo-β-1,4-glucanase B (CelB) was purified and crystallized, and data were collected for the native form and a selenomethionine derivative to 2.46 and 2.3 Å resolution, respectively.

Crystallization and preliminary crystallographic analysis of defective pollen wall (DPW) protein from Oryza sativa

Mutations in the defective pollen wall (dpw) gene cause abnormalities in the development of anthers and pollen grains of O. sativa. In this study, DPWΔ80 protein was cloned and expressed in E. coli. Purified DPWΔ80 in complex with its cofactor NADPH formed crystals. Diffraction data were collected to 3.4 Å resolution.

Crystallization and preliminary X-ray crystallographic studies of the ArsI C–As lyase from Thermomonospora curvata

The ArsI C-As lyase from Thermomonospora curvata was expressed, purified and crystallized. The crystals diffracted to 1.46 Å and belong to space group P4₃2₁2 or its enantiomer P4₁2₁2.

Characterization, crystallization and preliminary X-ray crystallographic analysis of the Uba5 fragment necessary for high-efficiency activation of Ufm1

The minimal Uba5 fragment necessary for high-efficiency activation of Ufm1 was identified, expressed, purified and crystallized, and diffraction data were collected to 2.95 Å resolution.

Crystallization and preliminary X-ray diffraction analysis of proximal thread matrix protein 1 (PTMP1) from Mytilus galloprovincialis

The full-length proximal thread matrix protein 1 (PTMP1) from M. galloprovincialis was recombinantly produced, purified and crystallized. Diffraction analysis of the obtained crystals showed that they belonged to space group P2₁ and diffracted to 1.95 Å resolution.

Crystallization and preliminary X-ray diffraction analysis of Hypocrea jecorina Cel7A in two new crystal forms

The crystallization and preliminary X-ray diffraction analysis of H. jecorina Cel7A in two new crystalline forms are reported.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of a ferredoxin/flavodoxin-NADP(H) oxidoreductase (Bc0385) from Bacillus cereus

A ferredoxin/flavodoxin-NADP(H) oxidoreductase (FNR) from B. cereus (Bc0385) belonging to the thioredoxin reductase-like class of FNRs has been cloned, overexpressed, purified and crystallized. Diffraction data have been collected to 2.5 Å resolution.

Crystallization and preliminary X-ray crystallographic studies of de­hydroascorbate reductase (DHAR) from Oryza sativa L. japonica

A dehydroascorbate reductase (DHAR) from rice (O. sativa L. japonica) was overexpressed, purified and crystallized. The crystals belonged to space group P2₁ and a complete native data set was obtained to a resolution of 1.9 Å.

Expression, purification, crystallization and preliminary X-ray analysis of the HER3–9E12 Fab complex

The crystallization of HER3-ecd in complex with a novel anti-HER3 antibody 9E12 was carried out and data were collected to 2.1 Å.

Crystallization and preliminary X-ray crystallographic analysis of a bacterial Asn-transamidosome

A bacterial Asn-transamidosome composed of two GatCABs, one AspRS dimer and two tRNA^Asns from P. aeruginosa was crystallized and the crystal diffracted to 3.73 Å resolution.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp6

The extracellular catalytic domain of mouse Enpp6 was expressed in mammalian cells, purified and crystallized. An X-ray diffraction data set was collected to 1.8 Å resolution.

Crystallization and preliminary X-ray diffraction analysis of (R)-carbonyl reductase from Candida parapsilosis

(R)-Carbonyl reductase from C. parapsilosis was crystallized in the presence of its cofactor NAD⁺. Preliminary X-ray diffration analysis was performed.

Preliminary crystallographic studies of a Schistosoma mansoni antigen (Sm21.7) dynein light-chain (DLC) domain

Sm21.7, an S. mansoni tegumental protein that reduces the worm burden in a murine immunization model, was cloned, expressed and purified. Its C-terminal domain was crystallized and a native crystal diffracted to 2.05 Å resolution. An NaI derivative used for SIRAS phasing diffracted to 1.95 Å resolution.

Crystallization and preliminary X-ray diffraction analysis of the peripheral light-harvesting complex LH2 from Marichromatium purpuratum

The LH2 complex from the purple photosynthetic bacterium M. purpuratum was purified and was crystallized using the sitting-drop vapour-diffusion method. X-ray diffraction data collection from the crystals revealed that the complex is an octamer.

Preliminary joint X-ray and neutron protein crystallographic studies of ecDHFR complexed with folate and NADP⁺

A 2.0 Å resolution neutron data set and a 1.6 Å resolution X-ray data set were collected for joint X-ray/neutron refinement of the ecDHFR–folate–NADP⁺ complex in order to study the reaction mechanism of dihydrofolate reductase.

Preliminary time-of-flight neutron diffraction studies of Escherichia coli ABC transport receptor phosphate-binding protein at the Protein Crystallography Station

This paper describes the growth of large crystals of and the collection of neutron time-of-flight data from an H/D-exchanged crystal of the bacterial phosphate-binding protein PBP.

Crystallization and preliminary X-ray characterization of the 2,4′-di­hydroxyaceto­phenone dioxygenase from Alcaligenes sp. 4HAP

The enzyme 2,4′-dihydroxyacetophenone dioxygenase (or DAD) catalyses the conversion of 2,4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. By the use of limited proteolysis, the DAD enzyme from Alcaligenes sp. 4HAP has been crystallized in a form that diffracts synchrotron radiation to a resolution of 2.2 Å.

Protein expression, characterization, crystallization and preliminary X-ray crystallographic analysis of a Fic protein from Clostridium difficile

A Fic protein from the pathogenic bacterium C. difficile R20291 has been cloned, expressed and purified. The purified protein was characterized by mass spectrometry and circular-dichroism spectroscopy, and was crystallized. In addition, a diffraction data set was collected and processed to 1.68 Å resolution.

Crystallization and preliminary X-ray crystallographic analysis of polyphenol oxidase from Juglans regia (jrPPO1)

The crystallization and preliminary X-ray crystallographic analysis of a plant PPO exhibiting monophenolase activity from J. regia (jrPPO1) in its active form (Asp¹⁰¹–Arg⁴⁴⁵) are reported.