Lysosomal cysteine proteases (cathepsins): promising drug targets

The physiological roles of lysosomal cysteine proteases now emerging from research are bringing them increasingly into focus as drug targets. A survey of the available crystal structures and a summary of their specificity determinants are presented here and some directions in inhibitor design are described.

Ping-pong cross-validation in real space: a method for increasing the phasing power of a partial model without risk of model bias

The procedures used to solve the crystal structure of the SecA translocation ATPase are described. MIR phases to a limiting resolution of 4.4 Å were extended to the 2.7 Å resolution limit of the crystals using an iterative density modification and partial model building procedure that employed real-space cross-validation to minimize model bias.

Structural characterization of an MJ1267 ATP-binding cassette crystal with a complex pattern of twinning caused by promiscuous fiber packing

A forensic analysis is presented of a trigonal crystal form with a complex pattern of twinning including merohedral twinning of the dominant lattice. The analysis indicates that twinning is caused by promiscuous parallel and antiparallel packing of helical protein fibers, permitted by the conformational flexibility of the protein combined with the inherent plasticity of the intermolecular interactions between the fibers.

Membrane-protein crystallization in cubo: temperature-dependent phase behaviour of monoolein–detergent mixtures

High-quality three-dimensional crystals of membrane proteins can be produced using lipidic cubic phases. The detailed temperature-dependent phase behaviour of a wide range of monoolein–detergent mixtures is presented to aid the development of streamlined membrane-protein crystallization in cubo.

Structure of a hybrid squash inhibitor in complex with porcine pancreatic elastase at 1.8 Å resolution

The crystal structure of a hybrid squash-type inhibitor in complex with porcine pancreatic elastase is described. The binding loop derived from the turkey ovomucoid inhibitor third domain adopts a `canonical' conformation upon elastase binding.

The structure of the D49 phospholipase A₂ piratoxin III from Bothrops pirajai reveals unprecedented structural displacement of the calcium-binding loop: possible relationship to cooperative substrate binding

The crystal structure of piratoxin III may represent the T-state of this phospholipase A₂ which, unusually, exhibits cooperative substrate binding.

Actin–DBP: the perfect structural fit?

The crystal structure of the actin–DBP complex was determined at 2.4 Å resolution. The implications for the actin-sequestering role of DBP and the model of the actin-filament structure are discussed.

An automatic method to generate force-field parameters for hetero-compounds

A method has been developed that automatically constructs a crystallographic refinement force field (topology and parameter files) for any molecule from a theoretical frequency calculation. The approach has been tested on five proteins containing metal sites or non-standard inhibitors or coenzymes and it is shown that the structures are improved in various aspects.

Use of multiple anomalous dispersion to phase highly merohedrally twinned crystals of interleukin-1β

The crystal structure of a double mutant (F42W/W120F) of interleukin-1β was determined by MAD using data from merohedrally twinned crystals. The detection and overcoming of twinning in interleukin-1β crystals and an analysis of the effect of the twinning on phasing by MAD is presented.

Structure of bovine adenosine deaminase complexed with 6-hy­droxy-1,6-di­hydropurine riboside

The crystal structure of adenosine deaminase (ADA) from bovine intestine complexed with a transition-state analogue, 6-hydroxy-1,6-dihydropurine riboside (HDPR), was solved at 2.5 Å resolution. HDPR tightly interacts with ADA by means of six hydrogen bonds and is entirely enclosed within the active site.

SOMoRe: a multi-dimensional search and optimization approach to molecular replacement

A new molecular-replacement code is introduced that balances local and global optimization through a global six-dimensional search of a low-resolution target function which is followed by multi-start local optimization.

Faster data-collection strategies for structure determination using anomalous dispersion

This paper discusses the results of an analysis of several data sets to determine the minimum amount of data needed to phase with SAD and MAD and the implications for the design of data-collection strategies seeking to minimize the total exposure time and dose absorbed by the crystal.

Refined structure of bovine carboxypeptidase A at 1.25 Å resolution

The crystal structure of bovine carboxypeptidase A has been refined at 1.25 Å resolution and allowed, together with theoretical simulations, an improved description of conformational disorder, solvent networks and protonation states in the resting enzyme.

Crystallization and preliminary X-ray analysis of NADP(H)-dependent alcohol de­hydrogenases from Saccharomyces cerevisiae and Rana perezi

Different crystal forms diffracting to high resolution have been obtained for two NADP(H)-dependent alcohol dehydrogenases: ScADHVI from S. cerevisiae and ADH8 from R. perezi.

Crystallization and preliminary X-ray diffraction analysis of O-acetylserine sulfhydrylase from Aeropyrum pernix K1

An enzyme involved in cysteine biosynthesis in a hyperthermophilic archaeon has been crystallized. The crystal structure to be determined is expected to elucidate versatile substrate specificity in an ancestral-type enzyme.

Crystallization and preliminary X-ray analysis of a thermostable pectate lyase PL 47 from Bacillus sp. TS 47

The thermostable pectate lyase PL 47 from Bacillus sp. TS 47 was crystallized. A complete diffraction data set was collected to 1.8 Å resolution using synchrotron radiation.

Crystallization and preliminary X-ray analysis of recombinant human acid β-glucocerebrosidase, a treatment for Gaucher's disease

Enzyme replacement therapy is clinically effective in reducing the morbidity associated with Gaucher's disease (a lysosomal storage disorder). A 2.7 Å data set has been collected from crystals of the responsible enzyme (β-glucocerebrosidase).

Crystallization and preliminary X-ray diffraction analysis of the di-haem cytochrome c peroxidase from Pseudomonas stutzeri

Cytochrome c peroxidase from P. stutzeri has been crystallized using sodium citrate and PEG 8000 as precipitants. The crystals belong to space group P2₁, with unit-cell parameters a = 69.29, b = 143.31, c = 76.83 Å, β = 100.78°.

Crystallization and preliminary X-ray diffraction studies of α-cyclodextrin glucanotransferase isolated from Bacillus macerans

The α-cyclodextrin glucanotransferase from B. macerans was purified homogeneously and crystallized. The crystals were suitable for X-ray analysis and diffracted to at least 2 Å.

Preliminary X-ray characterization of the ribonuclease P (C5 protein) from Escherichia coli: expression, crystallization and cryoconditions

The gene for E. coli ribonuclease P protein (also known as C5 protein) and its mutant C5-C113A have been expressed as GST-fusion proteins in E. coli at a high level. After cleavage of the fusion protein, highly purified functional C5 protein is obtained that can be crystallized and characterized by X-ray diffraction.

Crystallization and preliminary X-ray diffraction studies of an α-methylacyl-CoA racemase from Mycobacterium tuberculosis

α-Methylacyl-CoA racemase from M. tuberculosis has been expressed in E. coli, purified and crystallized. Native crystals belonging to space group P6₁22 or P6₅22 diffract to 2.8 Å.

Expression, purification and preliminary X-ray characterization of N-acetyl-γ-glutamyl-phosphate reductase from Thermus thermophilus HB8

N-Acetyl-γ-glutamyl-phosphate reductase from T. thermophilus HB8 has been expressed in E. coli, purified and crystallized. The crystals belong to space group P6₂22 or P6₄22 and diffraction data have been collected to 2.2 Å resolution.

Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the F1 antigen Caf1M–Caf1 chaperone–subunit pre-assembly complex from Yersinia pestis

Crystals of the Y. pestis capsular antigen pre-assembly complex Caf1M–Caf1 have been obtained. Diffraction data from native, SeMet and Pt-labelled crystals were used to obtain a 2 Å electron-density map.

Crystallization and preliminary X-ray crystallographic analysis of Sclerotium rolfsii lectin

The S. rolfsii lectin was crystallized and X-ray diffraction data were collected to 1.1 Å resolution.

Definition of domain boundaries and crystallization of the SMN Tudor domain

The boundaries of the Tudor domain of the spinal muscular neuron protein SMN have been defined using NMR and crystals that diffract to 1.8 Å resolution are reported.

Purification, crystallization and preliminary X-ray analysis of a μ-like calpain

A hybrid calpain containing 85% of μ-calpain sequence, and very close in properties to μ-calpain, has been crystallized. The structure may help in understanding the difference in Ca²⁺ requirement between μ- and m-calpain.

Crystallization and preliminary X-ray analysis of TBP-interacting protein from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1

The 26 kDa TBP-interacting protein from the hyperthermophilic archaeon T. kodakaraensis KOD1 (Tk-TIP26) was crystallized and the crystals diffracted to 2.2 Å. Selenomethionine-substituted Tk-TIP26 was also crystallized and MAD data was collected. A Bijvoet difference Patterson map showed strong peaks sufficient to determine the positions of the Se atoms.

Expression, crystallization and activities of the two family 11 aldo–keto reductases from Bacillus subtilis

Two aldo-keto reductases from B. subtilis have been crystallized and their NADPH-dependent oxidoreductase activities have been kinetically characterized.

X-ray crystallographic characterization and phasing of a fucose-specific lectin from Aleuria aurantia

A fucose-specific lectin from A. aurantia was crystallized. 2.2 Å resolution data sets were collected and phased by the MAD method using an Hg derivative.

Crystallization of hamster di­hydroorotase: involvement of a di­sulfide-linked tetrameric form

The dihydroorotase domain from hamster was crystallized by vapour diffusion. The crystals were found to exclusively consist of a disulfide-linked tetrameric form of the enzyme.

Improved three-dimensional growth of manganese superoxide dismutase crystals on the International Space Station

Preliminary results on the improved crystallization of manganese superoxide dismutase in the microgravity environment of the International Space Station are reported. Crystal volume was 80-times larger than the best earth crystal and diffraction to 1.26 Å resolution was observed.

Structure of the Yersinia enterocolitica molecular-chaperone protein SycE

A crystal structure of the Y. enterocolitica molecular-chaperone protein SycE shows binding of its carboxy-terminal peptide to a hydrophobic patch on a neighboring molecule.

In-house phase determination of the lima bean trypsin inhibitor: a low-resolution sulfur-SAD case

The unknown structure of the lima bean trypsin inhibitor was solved from the anomalous signal of the native sulfur using 3 Å in-house Cu Kα data and refined against 2 Å synchrotron data.

The advantages of using a modified microbatch method for rapid screening of protein crystallization conditions

A modified microbatch technique is described which can be used to rapidly screen crystallization conditions. The characteristics of crystallization using silicone or paraffin oil are described.

De novo purification scheme and crystallization conditions yield high-resolution structures of chitinase A and its complex with the inhibitor allosamidin

Homogeneous sample of chitinase A from Serratia marcescens yielded crystals that provided a structure to 1.55 Å resolution. Co-crystallization of the enzyme with its potent inhibitor allosamidin provided the structure of the complex to 1.9 Å resolution

Pushing the boundaries of molecular replacement with maximum likelihood. Erratum

An erratum to the paper by Read (2001) Acta Cryst. D57, 1373–1382.