Purification, crystallization and preliminary X-ray crystallographic studies of Rv3899c from Mycobacterium tuberculosis
Rv3899c, a hypothetical protein from Mycobacterium tuberculosis that is conserved within the mycobacteria, is predicted to be secreted and has been found in culture filtrates. Here, Rv3899c has been cloned, expressed in Escherichia coli and purified using standard chromatographic techniques. The hanging-drop vapour-diffusion method with PEG 3350 as a precipitant was used to crystallize the protein. N-terminal sequencing results showed that the amino-acid sequence of the crystallized protein began with GATAG, indicating that it is a fragment containing residues 184–410 of Rv3899c. Rv3899c184–410 crystals exhibit...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Song, Y.Liu, J.Li, D.-F.Li, H.Wang, S.Wang, D.-C.Zhou, J.Bi, L. Tags: Mycobacterium tuberculosis Rv3899c research communications Source Type: research

Purification, crystallization and preliminary X-ray diffraction analysis of SpaD, a backbone-pilin subunit encoded by the fimbrial spaFED operon in Lactobacillus rhamnosus GG
In this study, soluble recombinant SpaD protein was overproduced in Escherichia coli and then purified by Ni2+-chelating affinity and gel-filtration chromatography. After limited proteolysis with α-chymotrypsin, good-quality crystals of SpaD were obtained which diffracted beyond 2.0 Å resolution. These crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 50.11, b = 83.27, c = 149.65 Å. For phasing, sodium iodide-derivatized crystals were prepared using the halide quick-soaking method and diffraction data were collected in-house to a resolution of 2.2 Å. An i...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Chaurasia, P.von Ossowski, I.Palva, A.Krishnan, V. Tags: Lactobacillus rhamnosus GG SpaFED pili fimbria adhesion in-house iodide SAD phasing limited proteolysis research communications Source Type: research

Cleaning protocols for crystallization robots: preventing protease contamination
The protease in the commonly used commercial low-foam enzyme cleaner Zymit cannot be completely blocked by EDTA, a widely used inhibitor of metalloproteases, at concentrations of up to 5 mM. Severe protein degradation was observed in crystallization drops after EDTA-containing wash steps unless residual Zymit protease was removed with NaOH at a concentration of at least 0.1 M. Wash steps with 0.1% SDS were also ineffective in completely removing the remaining Zymit activity. Protocols including wash steps with at least 0.1 M NaOH, as for example specified in the original ZENM protocol, are recommended to completely d...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Naschberger, A.Fürnrohr, B.G.Dunzendorfer-Matt, T.Bonagura, C.A.Wright, D.Scheffzek, K.Rupp, B. Tags: crystallization robotics protease protease inhibitor Zymit cleaning protocol research communications Source Type: research

Crystallization and preliminary X-ray analysis of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1
NAD+-reducing [NiFe] hydrogenases catalyze the oxidoreduction of dihydrogen concomitant with the interconversion of NAD+ and NADH. Here, the isolation, purification and crystallization of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1 are reported. Crystals of the NAD+-reducing [NiFe] hydrogenase were obtained within one week from a solution containing polyethylene glycol using the sitting-drop vapour-diffusion method and micro-seeding. The crystal diffracted to 2.58 Å resolution and belonged to space group C2, with unit-cell parameters a = 131.43, b = 189.71, c = 124.59 Å, &b...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Taketa, M.Nakagawa, H.Habukawa, M.Osuka, H.Kihira, K.Komori, H.Shibata, N.Ishii, M.Igarashi, Y.Nishihara, H.Yoon, K.-S.Ogo, S.Shomura, Y.Higuchi, Y. Tags: NAD -reducing [NiFe] hydrogenase hydrogen metabolism diaphorase respiratory complex I research communications Source Type: research

Structures of the N-acetyltransferase domain of Xylella fastidiosaN-acetyl-l-glutamate synthase/kinase with and without a His tag bound to N-acetyl-l-glutamate
Structures of the catalytic N-acetyltransferase (NAT) domain of the bifunctional N-acetyl-l-glutamate synthase/kinase (NAGS/K) from Xylella fastidiosa bound to N-acetyl-l-glutamate (NAG) with and without an N-terminal His tag have been solved and refined at 1.7 and 1.4 Å resolution, respectively. The NAT domain with an N-terminal His tag crystallized in space group P41212, with unit-cell parameters a = b = 51.72, c = 242.31 Å. Two subunits form a molecular dimer in the asymmetric unit, which contains ∼41% solvent. The NAT domain without an N-terminal His tag crystallized in space group P21, with unit-ce...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Zhao, G.Jin, Z.Allewell, N.M.Tuchman, M.Shi, D. Tags: N-acetylglutamate synthase N-acetylglutamate kinase GCN5 N-acetyltransferase bifunctional enzymes arginine biosynthesis research communications Source Type: research

Crystallization and preliminary X-ray crystallographic analysis of the CARD domain of apoptosis repressor with CARD (ARC)
In this study, the N-terminal CARD domain of ARC was overexpressed, purified and crystallized. X-ray diffraction data were collected to a resolution of 2.1 Å and the crystals were found to belong to space group P61 or P65, with unit-cell parameters a = 98.28, b = 98.28, c = 51.86 Å, α = 90, β = 90, γ = 120°. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Kim, S.H.Park, H.H. Tags: apoptosis ARC CARD research communications Source Type: research

Systematic analysis of protein–detergent complexes applying dynamic light scattering to optimize solutions for crystallization trials
This study demonstrates the potential of in situ DLS to optimize solutions of protein–detergent complexes for crystallization applications. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Meyer, A.Dierks, K.Hussein, R.Brillet, K.Brognaro, H.Betzel, C. Tags: dynamic light scattering n-alkyl- β -d-maltopyranosides micelle size protein – detergent complex hydrodynamic radius research communications Source Type: research

Crystallization and preliminary X-ray analysis of the C-terminal fragment of PorM, a subunit of the Porphyromonas gingivalis type IX secretion system
PorM is a membrane protein involved in the assembly of the type IX secretion system (T9SS) from Porphyromonas gingivalis, a major bacterial pathogen responsible for periodontal disease in humans. The periplasmic domain of PorM was overexpressed in Escherichia coli and purified. A fragment of the purified protein was obtained by limited proteolysis. Crystals of this fragment belonged to the tetragonal space group P43212. Native and MAD data sets were recorded to 2.85 and 3.1 Å resolution, respectively, using synchrotron radiation. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Stathopulos, J.Cambillau, C.Cascales, E.Roussel, A.Leone, P. Tags: Porphyromonas gingivalis T9SS research communications Source Type: research

Three-dimensional structure of a variant `Termamyl-like' Geobacillus stearothermophilus α-amylase at 1.9 Å resolution
The enzyme-catalysed degradation of starch is central to many industrial processes, including sugar manufacture and first-generation biofuels. Classical biotechnological platforms involve steam explosion of starch followed by the action of endo-acting glycoside hydrolases termed α-amylases and then exo-acting α-glucosidases (glucoamylases) to yield glucose, which is subsequently processed. A key enzymatic player in this pipeline is the `Termamyl' class of bacterial α-amylases and designed/evolved variants thereof. Here, the three-dimensional structure of one such Termamyl α-amylase variant based upo...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Offen, W.A.Viksoe-Nielsen, A.Borchert, T.V.Wilson, K.S.Davies, G.J. Tags: amylase Geobacillus stearothermophilus Termamyl research communications Source Type: research

Expression, purification and crystallization of a membrane-associated, catalytically active type I signal peptidase from Staphylococcus aureus
Staphylococcus aureus infections are becoming increasingly difficult to treat as they rapidly develop resistance to existing antibiotics. Bacterial type I signal peptidases are membrane-associated, cell-surface serine proteases with a unique catalytic mechanism that differs from that of eukaryotic endoplasmic reticulum signal peptidases. They are thus potential antimicrobial targets. S. aureus has a catalytically active type I signal peptidase, SpsB, that is essential for cell viability. To elucidate its structure, the spsB gene from S. aureus Newman strain was cloned and overexpressed in Escherichia coli. After exploring ...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Ting, Y.T.Batot, G.Baker, E.N.Young, P.G. Tags: SpsB type I signal peptidase Staphylococcus aureus cell secretion maltose-binding fusion protein research communications Source Type: research

Crystallization and preliminary X-ray analysis of the type IV restriction endonuclease ScoMcrA from Streptomyces coelicolor, which cleaves both Dcm-methylated DNA and phosphorothioated DNA
In this study, the ScoMcrA protein was overexpressed, purified and crystallized. The crystals diffracted to 3.35 Å resolution and belonged to space group P212121. The unit-cell parameters were determined to be a = 130.19, b = 139.36, c = 281.01 Å, α = β = γ = 90°. These results will facilitate the detailed structural analysis of ScoMcrA and further elucidation of its biochemical mechanism. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Liu, G.Zhang, Z.Zhao, G.Deng, Z.Wu, G.He, X. Tags: ScoMcrA DNA phosphorothioation Dcm methylation research communications Source Type: research

Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of SF173 from Shigella flexneri
Shigella flexneri is a Gram-negative, anaerobic bacterium in the genus Shigella that can cause diarrhoea in humans. SF173, a hypothetical protein from S. flexneri 5a strain M90T, has been cloned, overexpressed, purified and crystallized as a part of laboratory-scale structural genomics project. The SF173 protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 0.8 M succinic acid pH 7.0 at 293 K. Preliminary X-ray diffraction analysis revealed that the crystal diffracted to 1.47 Å resolution and belonged to space group I432, with unit-cell parameters a = b = c = 110.245 &Arin...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Kim, H.-N.An, J.-G.Lee, Y.-S.Seok, S.-H.Yoo, H.-S.Seo, M.-D. Tags: SF173 Shigella flexneri research communications Source Type: research

Purification, crystallization and preliminary crystallographic analysis of DR0248, an MNT–HEPN fused protein from Deinococcus radiodurans
DR0248 is a protein identified in the Deinococcus radiodurans (DR) genome that is predicted to encompass two domains: an N-terminal minimal nucleotidyl transferase domain (MNT) and a C-terminal higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN). These two domains, usually encoded in two ORFs, have been suggested to play the role of a toxin–antitoxin (TA) system in prokaryotes. Recombinant DR0248 was overexpressed and purified from Escherichia coli and diffraction-quality crystals were obtained in the presence of the detergent molecules dodecyldimethylamine oxide (DDAO) and octaethylene glycol monodode...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Pesce, G.Pellegrino, S.McSweeney, S.Goncalves, A.de Sanctis, D. Tags: Deinococcus radiodurans DR0248 minimal nucleotidyl transferase domain higher eukaryotes and prokaryotes nucleotide-binding domain research communications Source Type: research

Crystallization and preliminary crystallographic studies of a novel noncatalytic carbohydrate-binding module from the Ruminococcus flavefaciens cellulosome
Microbial degradation of the plant cell wall is a fundamental biological process with considerable industrial importance. Hydrolysis of recalcitrant polysaccharides is orchestrated by a large repertoire of carbohydrate-active enzymes that display a modular architecture in which a catalytic domain is connected via linker sequences to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus potentiating catalysis. The genome of the most abundant ruminal cellulolytic bacterium, Ruminococcus flavefaciens strain FD-1, provides an opportunity to dis...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Venditto, I.Goyal, A.Thompson, A.Ferreira, L.M.A.Fontes, C.M.G.A.Najmudin, S. Tags: Ruminococcus flavefaciens carbohydrate-active enzymes glycoside hydrolase family 9 Cel9A carbohydrate-binding module research communications Source Type: research

Expression and crystallization of a bacterial glycoside hydrolase family 116 β-glucosidase from Thermoanaerobacterium xylanolyticum
The Thermoanaerobacterium xylanolyticum gene product TxGH116, a glycoside hydrolase family 116 protein of 806 amino-acid residues sharing 37% amino-acid sequence identity over 783 residues with human glucosylceramidase 2 (GBA2), was expressed in Escherichia coli. Purification by heating, immobilized metal-affinity and size-exclusion chromatography produced>90% pure TxGH116 protein with an apparent molecular mass of 90 kDa on SDS–PAGE. The purified TxGH116 enzyme hydrolyzed the p-nitrophenyl (pNP) glycosides pNP-β-d-glucoside, pNP-β-d-galactoside and pNP-N-acetyl-β-d-glucopyranoside, as well as cel...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Sansenya, S.Mutoh, R.Charoenwattanasatien, R.Kurisu, G.Ketudat Cairns, J.R. Tags: glycoside hydrolase family 116 β -glucosidase thermophilic research communications Source Type: research

Structures of Drosophila melanogaster Rab2 and Rab3 bound to GMPPNP
This study focuses on Rab2 and Rab3 from Drosophila melanogaster. Whereas Rab2 is involved in vesicle transport between the Golgi and the endoplasmatic reticulum, Rab3 is a key player in exocytosis, and in the synapse it is involved in the assembly of the presynaptic active zone. Here, high-resolution crystal structures of Rab2 and Rab3 in complex with GMPPNP and Mg2+ are presented. In the structure of Rab3 a modified cysteine residue is observed with an enigmatic electron density attached to its thiol function. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Lardong, J.A.Driller, J.H.Depner, H.Weise, C.Petzoldt, A.Wahl, M.C.Sigrist, S.J.Loll, B. Tags: Rab GTPase GMPPNP cysteine modification research communications Source Type: research

Protein purification, crystallization and preliminary X-ray diffraction analysis of l-arabinose isomerase from Lactobacillus fermentum CGMCC2921
l-Arabinose isomerase (AI) catalyzes the isomerization of l-arabinose to l-ribulose, as well as that of d-galactose to d-tagatose. A thermophilic AI derived from Lactobacillus fermentum CGMCC2921 (LFAI) was overexpressed in Escherichia coli BL21 (DE3). This enzyme was purified to over 95% purity by nickel affinity, Mono-Q ion-exchange and size-exclusion chromatography. The LFAI protein was crystallized from either 0.1 M bis-tris pH 6.5, 23% PEG 3350, 0.3 M NaCl (form 1 crystals) or 0.1 M bis-tris pH 6.0, 25% PEG monomethyl ether 5000 (form 2 crystals). Diffraction data from form 1 LFAI crystals were collected to 2.80...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Xu, Z.Li, S.Liang, J.Feng, X.Xu, H. Tags: l-arabinose isomerase d-tagatose synchrotron radiation rare sugars research communications Source Type: research

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of the histone-like HU protein from Spiroplasma melliferum KC3
HU proteins belong to the nucleoid-associated proteins (NAPs) that are involved in vital processes such as DNA compaction and reparation, gene transcription etc. No data are available on the structures of HU proteins from mycoplasmas. To this end, the HU protein from the parasitic mycoplasma Spiroplasma melliferum KC3 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Prismatic crystals of the protein were obtained by the vapour-diffusion technique at 4°C. The crystals diffracted to 1.36 Å resolution (the best resolution ever obtained for a HU protein). The diffraction data were indexed ...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Boyko, K.Gorbacheva, M.Rakitina, T.Korzhenevskiy, D.Vanyushkina, A.Kamashev, D.Lipkin, A.Popov, V. Tags: HU protein three-dimensional structure DNA binding research communications Source Type: research

Zinc-substituted pseudoazurin solved by S/Zn-SAD phasing
The copper(II) centre of the blue copper protein pseudoazurin from Alcaligenes faecalis has been substituted by zinc(II) via denaturing the protein, chelation and removal of copper and refolding the apoprotein, followed by the addition of an aqueous solution of ZnCl2. Vapour-diffusion experiments produced colourless hexagonal crystals (space group P65), which when cryocooled had unit-cell parameters a = b = 49.01, c = 98.08 Å. Diffraction data collected at 100 K using a copper sealed tube were phased by the weak anomalous signal of five S atoms and one Zn atom. The structure was fitted manually and refined to 1.6...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Gessmann, R.Papadovasilaki, M.Drougkas, E.Petratos, K. Tags: copper-binding protein metalloprotein protein unfolding and refolding sulfur/zinc SAD zinc substitution research communications Source Type: research

A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes
The lipid cubic phase or in meso method is a robust approach for crystallizing membrane proteins for structure determination. The uptake of the method is such that it is experiencing what can only be described as explosive growth. This timely, comprehensive and up-to-date review introduces the reader to the practice of in meso crystallogenesis, to the associated challenges and to their solutions. A model of how crystallization comes about mechanistically is presented for a more rational approach to crystallization. The possible involvement of the lamellar and inverted hexagonal phases in crystallogenesis and the applicatio...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Caffrey, M. Tags: crystallization lipid cubic phase macromolecular crystallography membrane-protein structure mesophase robot – function water-soluble proteins research communications Source Type: research

Crystals on the cover 2015
(Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - December 23, 2014 Category: Biochemistry Authors: Einspahr, H.Weiss, M.S.Hunter, W.N. Tags: crystallization crystal images editorial Source Type: research

A simple technique to reduce evaporation of crystallization droplets by using plate lids with apertures for adding liquids
A method is described for using plate lids to reduce evaporation in low-volume vapor-diffusion crystallization experiments. The plate lids contain apertures through which the protein and precipitants were added to different crystallization microplates (the reservoir was filled before fitting the lids). Plate lids were designed for each of these commonly used crystallization microplates. This system minimizes the dehydration of crystallization droplets containing just a few nanolitres of protein and precipitant, and results in more reproducible diffraction from the crystals. For each lid design, changes in the weight of the...
Source: Acta Crystallographica Section F - November 28, 2014 Category: Biochemistry Authors: Zipper, L.E.Aristide, X.Bishop, D.P.Joshi, I.Kharzeev, J.Patel, K.B.Santiago, B.M.Joshi, K.Dorsinvil, K.Sweet, R.M.Soares, A.S. Tags: crystallization dehydration vapor diffusion high-throughput screening acoustic droplet ejection in situ X-ray data collection laboratory communications Source Type: research

Crystallization and preliminary crystallographic studies of the hypothetical protein BPSL1038 from Burkholderia pseudomallei
Melioidosis is an infectious disease caused by the pathogenic bacterium Burkholderia pseudomallei. Whole-genome sequencing revealed that the B. pseudomallei genome includes 5855 coding DNA sequences (CDSs), of which ∼25% encode hypothetical proteins. A pathogen-associated hypothetical protein, BPSL1038, was overexpressed in Escherichia coli, purified and crystallized using vapour-diffusion methods. A BPSL1038 protein crystal that grew using sodium formate as precipitant diffracted to 1.55 Å resolution. It belonged to space group C2221, with unit-cell parameters a = 85.36, b = 115.63, c = 46.73 Å. The ca...
Source: Acta Crystallographica Section F - November 28, 2014 Category: Biochemistry Authors: Shaibullah, S.Mohd-Sharif, N.Ho, K.L.Firdaus-Raih, M.Nathan, S.Mohamed, R.Ng, C.L. Tags: Burkholderia pseudomallei BPSL1038 hypothetical protein crystallization communications Source Type: research

Cloning, purification, crystallization and 1.57 Å resolution X-ray data analysis of AmsI, the tyrosine phosphatase controlling amylovoran biosynthesis in the plant pathogen Erwinia amylovora
The Gram-negative bacterium Erwinia amylovora is a destructive pathogen of plants belonging to the Rosaceae family. Amongst its pathogenicity factors, E. amylovora produces the exopolysaccharide amylovoran, which contributes to the occlusion of plant vessels, causing wilting of shoots and eventually resulting in plant death. Amylovoran biosynthesis requires the presence of 12 genes (from amsA to amsL) clustered in the ams region of the E. amylovora genome. They mostly encode glycosyl transferases (AmsG, AmsB, AmsD, AmsE, AmsJ and AmsK), proteins involved in amylovoran translocation and assembly (AmsH, AmsL and AmsC), and a...
Source: Acta Crystallographica Section F - November 28, 2014 Category: Biochemistry Authors: Benini, S.Caputi, L.Cianci, M. Tags: Erwinia exopolysaccharides amylovoran tyrosine phosphatase AmsI crystallization communications Source Type: research

Expression, purification and crystallization of two endonuclease III enzymes from Deinococcus radiodurans
Endonuclease III is a bifunctional DNA glycosylase that removes a wide range of oxidized bases in DNA. Deinococcus radiodurans is an extreme radiation-resistant and desiccation-resistant bacterium and possesses three genes encoding endonuclease III enzymes in its genome: DR2438 (EndoIII-1), DR0289 (EndoIII-2) and DR0982 (EndoIII-3). Here, EndoIII-1 and an N-terminally truncated form of EndoIII-3 (EndoIII-3Δ76) have been expressed, purified and crystallized, and preliminary X-ray crystallographic analyses have been performed to 2.15 and 1.31 Å resolution, respectively. The EndoIII-1 crystals belonged to the mo...
Source: Acta Crystallographica Section F - November 28, 2014 Category: Biochemistry Authors: Sarre, A.Ökvist, M.Klar, T.Moe, E.Timmins, J. Tags: Deinococcus radiodurans endonuclease III EndoIII-1 EndoIII-3 crystallization communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of Trap1 complexed with Hsp90 inhibitors
Hsp90 is a molecular chaperone responsible for the assembly and regulation of many cellular client proteins. In particular, Trap1, a mitochondrial Hsp90 homologue, plays a pivotal role in maintaining mitochondrial integrity, protecting against apoptosis in cancer cells. The N (N-terminal)-M (middle) domain of human Trap1 was crystallized in complex with Hsp90 inhibitors (PU-H71 and BIIB-021) by the hanging-drop vapour-diffusion method at pH 6.5 and 293 K using 15% PEG 8K as a precipitant. Diffraction data were collected from crystals of the Trap1–PU-H71 (2.7 Å) and Trap1–BIIB-021 (3.1 Å) compl...
Source: Acta Crystallographica Section F - November 28, 2014 Category: Biochemistry Authors: Jeong, H.Kang, B.H.Lee, C. Tags: Trap1 PU-H71 BIIB-021 crystallization communications Source Type: research

Preliminary crystallographic analysis of Xyn52B2, a GH52 β-d-xylosidase from Geobacillus stearothermophilus T6
Geobacillus stearothermophilus T6 is a thermophilic bacterium that possesses an extensive hemicellulolytic system, including over 40 specific genes that are dedicated to this purpose. For the utilization of xylan, the bacterium uses an extracellular xylanase which degrades xylan to decorated xylo-oligomers that are imported into the cell. These oligomers are hydrolyzed by side-chain-cleaving enzymes such as arabinofuranosidases, acetylesterases and a glucuronidase, and finally by an intracellular xylanase and a number of β-xylosidases. One of these β-xylosidases is Xyn52B2, a GH52 enzyme that has already proved t...
Source: Acta Crystallographica Section F - November 28, 2014 Category: Biochemistry Authors: Dann, R.Lansky, S.Lavid, N.Zehavi, A.Belakhov, V.Baasov, T.Dvir, H.Manjasetty, B.Belrhali, H.Shoham, Y.Shoham, G. Tags: Geobacillus stearothermophilus glycoside hydrolase GH52 xylosidase xylose xylobiose xylan utilization enzymatic glycosynthesis glycosynthase selenomethionine SAD data collection crystallization communications Source Type: research

Membrane proteins, detergents and crystals: what is the state of the art?
At the time when the first membrane-protein crystal structure was determined, crystallization of these molecules was widely perceived as extremely arduous. Today, that perception has changed drastically, and the process is regarded as routine (or nearly so). On the occasion of the International Year of Crystallography 2014, this review presents a snapshot of the current state of the art, with an emphasis on the role of detergents in this process. A survey of membrane-protein crystal structures published since 2012 reveals that the direct crystallization of protein–detergent complexes remains the dominant methodology;...
Source: Acta Crystallographica Section F - November 28, 2014 Category: Biochemistry Authors: Loll, P.J. Tags: membrane proteins crystallization detergents crystallization communications Source Type: research

The times they are a-changin' – news from Acta Crystallographica Section F
(Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - November 28, 2014 Category: Biochemistry Authors: Hunter, W.N.Weiss, M.S. Tags: editorial crystallization research communications Source Type: research

Preparation, purification, crystallization and preliminary crystallographic analysis of dual-domain β-propeller phytase from Bacillus sp. HJB17
β-Propeller phytases (BPPs) are abundant in nature. Recently, dual-domain BPPs have been found in which the typical BPP domain is responsible for phytate hydrolysis. The dual-domain BPP (PhyH) from Bacillus sp. HJB17 was obtained with an incomplete N-terminal BPP domain (PhyH-DI; residues 41–318) and a typical BPP domain (PhyH-DII; residues 319–644) at the C-terminus. PhyH-DI was found to act synergistically (with a 1.2–2.5-fold increase in phosphate release) with PhyH-DII, other BPPs (PhyP and 168PhyA) and a histidine acid phosphatase. The structure of PhyH was therefore studied with the aim of expl...
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Lu, F.Guo, G.Li, Q.Feng, D.Liu, Y.Huang, H.Yang, P.Gao, W.Yao, B. Tags: β -propeller phytases PhyHT Bacillus sp. HJB17 crystallization communications Source Type: research

Optimization of the crystallizability of a single-chain antibody fragment
Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagnostic potential. Knowledge of their three-dimensional structure is important for understanding their antigen-binding mode as well as for protein-engineering approaches such as antibody humanization. A major obstacle to the crystallization of single-chain variable antibody fragments is their relatively poor homogeneity caused by spontaneous oligomerization. A new approach to optimization of the crystallizability of single-chain variable antibody fragments is demonstrated using a representative single-chain variable fragment deri...
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Škerlová, J.Král, V.Fábry, M.Sedláček, J.Veverka, V.Řezáčová, P. Tags: single-chain antibody fragment Thermofluor assay differential scanning fluorimetry crystallizability optimization oligomerization crystallization laboratory communications Source Type: research

Crystallization and preliminary X-ray crystallographic analysis of a putative acetylxylan esterase from Talaromyces cellulolyticus
Acetylxylan esterase (AXE) catalyzes the hydrolytic cleavage of the ester bond between acetic acid and hemicellulose in plant cell walls. A putative AXE gene exhibiting high homology to carbohydrate esterase family 3 was found in the genome database of the fungus Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus). A truncated form of the protein, the catalytic domain of the enzyme, was prepared and crystallized. The best crystal was obtained at 293 K using 0.17 M ammonium sulfate, 28% PEG 4000, 5%(v/v) glycerol, 0.5%(w/v) n-octyl-β-d-glucoside. X-ray diffraction data were collected to 1.50 ...
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Watanabe, M.Ishikawa, K. Tags: biomass fungus hemicellulose cellulase crystallization communications Source Type: research

Crystallization and preliminary X-ray crystallographic analysis of a putative feruloyl esterase from Talaromyces cellulolyticus
Feruloyl esterase (FAE; EC 3.1.1.73) catalyzes the cleavage of the ester bond between ferulic acid and polysaccharides in plant cell walls, and thus holds significant potential for the industrial utilization of biomass saccharification. A feruloyl esterase was identified from the genome database of Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus). The gene consists of the catalytic domain and a carbohydrate-binding module connected through a serine/threonine-rich linker region. The recombinant enzyme was prepared, purified and crystallized at 293 K using 0.1 M imidazole pH 8.0, 0.2 M calcium a...
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Watanabe, M.Ishikawa, K. Tags: biomass fungus carbohydrate-binding module cellulase crystallization communications Source Type: research

Crystallization and preliminary crystallographic analysis of human aquaporin 1 at a resolution of 3.28 Å
Aquaporin water channels (AQPs) are found in almost every organism from humans to bacteria. In humans, 13 classes of AQPs control water and glycerol homeostasis. Knockout studies have suggested that modulating the activity of AQPs could be beneficial for the treatment of several pathologies. In particular, aquaporin 1 is a key factor in cell migration and angiogenesis, and constitutes a possible target for anticancer compounds and also for the treatment of glaucoma. Here, a preliminary crystallographic analysis at 3.28 Å resolution of crystals of human aquaporin 1 (hAQP1) obtained from protein expressed in Sf9 inse...
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Ruiz Carrillo, D.To Yiu Ying, J.Darwis, D.Soon, C.H.Cornvik, T.Torres, J.Lescar, J. Tags: aquaporin 1 water channel baculovirus crystal packing crystallization communications Source Type: research

Expression, purification and crystallization of a novel carbohydrate-binding module from the Ruminococcus flavefaciens cellulosome
Anaerobic bacteria organize carbohydrate-active enzymes into a multi-component complex, the cellulosome, which degrades cellulose and hemicellulose highly efficiently. Genome sequencing of Ruminococcus flavefaciens FD-1 offers extensive information on the range and diversity of the enzymatic and structural components of the cellulosome. The R. flavefaciens FD-1 genome encodes over 200 dockerin-containing proteins, most of which are of unknown function. One of these modular proteins comprises a glycoside hydrolase family 5 catalytic module (GH5) linked to an unclassified carbohydrate-binding module (CBM-Rf1) and a dockerin....
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Venditto, I.Centeno, M.S.J.Ferreira, L.M.A.Fontes, C.M.G.A.Najmudin, S. Tags: Ruminococcus flavefaciens cellulosome glycoside hydrolase family 5 catalytic module novel carbohydrate-binding module crystallization communications Source Type: research

Crystallization and preliminary X-ray crystallographic analysis of ribosome assembly factors: the Rpf2–Rrs1 complex
In this study, Rpf2 and Rrs1 from Aspergillus nidulans were co-overexpressed in Escherichia coli, purified and crystallized. Subsequent analysis revealed that these crystals contained the central core region of the complex consisting of both N-terminal domains. X-ray diffraction data were collected to 2.35 Å resolution. Preliminary analysis revealed that the crystals belonged to space group P212121, with unit-cell parameters a = 54.1, b = 123.3, c = 133.8 Å. There are two complexes in the asymmetric unit. Structure determination using selenomethionine-labelled protein is in progress. (Source: Acta...
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Asano, N.Nakamura, A.Komoda, K.Kato, K.Tanaka, I.Yao, M. Tags: ribosome assembly factors Rpf2 Rrs1 Aspergillus nidulans crystallization communications Source Type: research

Crystallization and preliminary X-ray crystallographic study of human Hikeshi, a new nuclear transport receptor for Hsp70
This study is the first to yield structural insight into this highly unusual fourth import receptor after importins, NTF2 and TAP. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Song, J.Lee, S.J. Tags: Hikeshi heat-shock proteins nuclear transport receptor FG-nucleoporins crystallization communications Source Type: research

Purification, crystallization and preliminary X-ray crystallographic studies of KstR2 (ketosteroid regulatory protein) from Mycobacterium tuberculosis
KstR2 (Rv3557c) is one of two TetR-family transcriptional repressors of cholesterol metabolism in Mycobacterium tuberculosis. The ability to degrade cholesterol fully is important for pathogenesis, and therefore this repressor was expressed, purified and crystallized. Crystals of KstR2 diffracted to better than 1.9 Å resolution and belonged to space group C2, with unit-cell parameters a = 72.3, b = 90.3, c = 49.7 Å, α = γ = 90, β = 128.2°. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Dawes, S.S.Kendall, S.L.Baker, E.N.Lott, J.S. Tags: cholesterol TetR family Mycobacterium tuberculosis steroid crystallization communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of UspE from Escherichia coli
Universal stress proteins (Usps) are among the most highly induced genes when bacteria are subjected to several stress conditions such as heat shock, nutrient starvation or the presence of oxidants or other stress agents. Escherichia coli has five small Usps and one tandem-type Usp. UspE (or YdaA) is the tandem-type Usp and consists of two Usp domains arranged in tandem. To date, the structure of UspE remains to be elucidated. To contribute to the molecular understanding of the function of the tandem-type UspE, UspE from E. coli was overexpressed and the recombinant protein was purified using Ni–NTA affinity, Q anion...
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Xu, Y.Quan, C.-S.Jin, X.Jin, X.Zhao, J.Li, X.Zheng, W.Jin, L.Liu, D.Fan, S.Ha, N.-C. Tags: tandem-type UspE universal stress proteins Usp domain crystallization communications Source Type: research

Crystallization and preliminary X-ray characterization of the full-length bacteriophytochrome from the plant pathogen Xanthomonas campestris pv. campestris
Phytochromes give rise to the largest photosensor family known to date. However, they are underrepresented in the Protein Data Bank. Plant, cyanobacterial, fungal and bacterial phytochromes share a canonical architecture consisting of an N-terminal photosensory module (PAS2–GAF–PHY domains) and a C-terminal variable output module. The bacterium Xanthomonas campestris pv. campestris, a worldwide agricultural pathogen, codes for a single bacteriophytochrome (XccBphP) that has this canonical architecture, bearing a C-terminal PAS9 domain as the output module. Full-length XccBphP was cloned, expressed and purified ...
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Klinke, S.Otero, L.H.Rinaldi, J.Sosa, S.Guimarães, B.G.Shepard, W.E.Goldbaum, F.A.Bonomi, H.R. Tags: light-signalling pathway photosensor photoreceptor biliverdin Xanthomonas campestris pv. campestris bacteriophytochrome crystallization communications Source Type: research

Expression, purification and X-ray crystallographic analysis of the Helicobacter pylori blood group antigen-binding adhesin BabA
Helicobacter pylori is a human pathogen that colonizes about 50% of the world's population, causing chronic gastritis, duodenal ulcers and even gastric cancer. A steady emergence of multiple antibiotic resistant strains poses an important public health threat and there is an urgent requirement for alternative therapeutics. The blood group antigen-binding adhesin BabA mediates the intimate attachment to the host mucosa and forms a major candidate for novel vaccine and drug development. Here, the recombinant expression and crystallization of a soluble BabA truncation (BabA25–460) corresponding to the predicted extracel...
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Subedi, S.Moonens, K.Romão, E.Lo, A.Vandenbussche, G.Bugaytsova, J.Muyldermans, S.Borén, T.Remaut, H. Tags: Helicobacter pylori BabA adhesin nanobody crystallization communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of a trimodular endo-β-1,4-glucanase (Cel5B) from Bacillus halodurans
Cellulases catalyze the hydrolysis of cellulose, the major constituent of plant biomass and the most abundant organic polymer on earth. Cellulases are modular enzymes containing catalytic domains connected, via linker sequences, to noncatalytic carbohydrate-binding modules (CBMs). A putative modular endo-β-1,4-glucanase (BhCel5B) is encoded at locus BH0603 in the genome of Bacillus halodurans. It is composed of an N-terminal glycoside hydrolase family 5 catalytic module (GH5) followed by an immunoglobulin-like module and a C-terminal family 46 CBM (BhCBM46). Here, the crystallization and preliminary X-ray diffraction ...
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Venditto, I.Santos, H.Sandy, J.Sanchez-Weatherby, J.Ferreira, L.M.A.Sakka, K.Fontes, C.M.G.A.Najmudin, S. Tags: endo- β -1,4-glucanase Cel5B family 5 glycoside hydrolase (GH5) family 46 carbohydrate-binding module (CBM46) plant cell-wall degradation Bacillus halodurans crystallization communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of the Sel1-like repeats of SEL1L
In this study, the purification, crystallization and preliminary X-ray diffraction analysis of recombinant mouse SEL1L (residues 348–533) are reported. The crystals were obtained by the hanging-drop vapour-diffusion method at pH 8.5 and 277 K using 30% 2-propanol as a precipitant. Optimized crystals diffracted to 3.3 Å resolution at a synchrotron-radiation source. Preliminary X-ray diffraction analysis revealed that the crystals belonged to space group P21 and contained four molecules per asymmetric unit, with a solvent content of 44%. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Jeong, H.Lee, H.Lee, C. Tags: SEL1L Sel1-like repeats crystallization communications Source Type: research

Expression, crystallization and preliminary crystallographic study of the functional mutant (N60K) of nonstructural protein 9 from Human coronavirus HKU1
Human coronavirus HKU1 (HCoV-HKU1), which mainly causes acute self-limited respiratory-tract infections, belongs to group A of the Betacoronavirus genus. Coronavirus genomes encode 16 nonstructural proteins (nsp1–16), which assemble into a large replication–transcription complex mediating virus propagation. Nonstructural protein 9, which binds to the single-stranded DNA/RNA, has been shown to be indispensible for viral replication. Interestingly, a functional mutant (N60K) of nsp9 was identified to compensate for a 6 nt insertion mutation of the 3′-untranslated region (UTR), which is critical for viral ...
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Chen, X.Tan, Y.Wang, F.Wang, J.Zhao, Q.Li, S.Fu, S.Chen, C.Yang, H. Tags: Human coronavirus HKU1 nonstructural protein 9 N60K mutant crystallization communications Source Type: research

Crystallization and preliminary X-ray study of biosynthetic alanine racemase from Pseudomonas aeruginosa PAO1
Biosynthetic alanine racemase (AlrPA) from Pseudomonas aeruginosa PAO1 carrying a His6 tag was expressed in Escherichia coli BL21 (DE3) cells and purified by Ni2+-chelating affinity and anion-exchange chromatography for X-ray crystallographic analysis. Crystals were grown by the hanging-drop vapour-diffusion method at 289 K in a solution consisting of 4%(v/v) Tacsimate pH 5.0, 14%(w/v) polyethylene glycol 3350 with a protein concentration of 8 mg ml−1. The crystal diffracted to 2.76 Å resolution and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 74.12, b = 76.97, c = 154...
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Zhou, H.Li, Z.Zhang, G.Xu, S.Tang, Z.Zhu, X.Dong, H.Ju, J. Tags: biosynthetic alanine racemase Pseudomonas aeruginosa crystallization communications Source Type: research

Crystallization and preliminary crystallographic study of Feline infectious peritonitis virus main protease in complex with an inhibitor
In this study, the main protease of FIPV in complex with a Michael acceptor-type inhibitor was crystallized. The complex crystals diffracted to 2.5 Å resolution and belonged to space group I422, with unit-cell parameters a = 112.3, b = 112.3, c = 102.1 Å. There is one molecule per asymmetric unit. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Wang, J.Wang, F.Tan, Y.Chen, X.Zhao, Q.Fu, S.Li, S.Chen, C.Yang, H. Tags: Feline infectious peritonitis virus main protease N3 inhibitor crystallization communications Source Type: research

Crystallization and preliminary crystallographic study of Porcine epidemic diarrhea virus main protease in complex with an inhibitor
In this study, the main protease of Porcine epidemic diarrhea virus in complex with a Michael acceptor was crystallized. The complex crystals diffracted to 2.5 Å resolution and belonged to space group R3, with unit-cell parameters a = 175.3, b = 175.3, c = 58.7 Å. Two molecules were identified per asymmetric unit. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Tan, Y.Wang, F.Chen, X.Wang, J.Zhao, Q.Li, S.Wang, Z.Fu, S.Chen, C.Yang, H. Tags: Porcine epidemic diarrhea virus main protease N3 inhibitor crystallization communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of the phosphate-binding protein PhoX from Xanthomonas citri
Xanthomonas axonopodis pv. citri (X. citri) is an important bacterium that causes citrus canker disease in plants in Brazil and around the world, leading to significant economic losses. Determination of the physiology and mechanisms of pathogenesis of this bacterium is an important step in the development of strategies for its containment. Phosphate is an essential ion in all microrganisms owing its importance during the synthesis of macromolecules and in gene and protein regulation. Interestingly, X. citri has been identified to present two periplasmic binding proteins that have not been further characterized: PstS, from ...
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Pegos, V.R.Medrano, F.J.Balan, A. Tags: Xanthomonas axonopodis pv. citri PhoX periplasmic binding proteins crystallization communications Source Type: research

The structure of the cyanobactin domain of unknown function from PatG in the patellamide gene cluster
Patellamides are members of the cyanobactin family of ribosomally synthesized and post-translationally modified cyclic peptide natural products, many of which, including some patellamides, are biologically active. A detailed mechanistic understanding of the biosynthetic pathway would enable the construction of a biotechnological `toolkit' to make novel analogues of patellamides that are not found in nature. All but two of the protein domains involved in patellamide biosynthesis have been characterized. The two domains of unknown function (DUFs) are homologous to each other and are found at the C-termini of the multi-domain...
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Mann, G.Koehnke, J.Bent, A.F.Graham, R.Houssen, W.Jaspars, M.Schwarz-Linek, U.Naismith, J.H. Tags: cyanobactins patellamides PatG RiPPs structural communications Source Type: research

Structures of heterodimeric POZ domains of Miz1/BCL6 and Miz1/NAC1
The POZ domain is an evolutionarily conserved protein–protein interaction domain that is found in approximately 40 mammalian transcription factors. POZ domains mediate both homodimerization and the heteromeric interactions of different POZ-domain transcription factors with each other. Miz1 is a POZ-domain transcription factor that regulates cell-cycle arrest and DNA-damage responses. The activities of Miz1 are altered by its interaction with the POZ-domain transcriptional repressors BCL6 and NAC1, and these interactions have been implicated in tumourigenesis in B-cell lymphomas and in ovarian serous carcinomas that o...
Source: Acta Crystallographica Section F - November 14, 2014 Category: Biochemistry Authors: Stead, M.A.Wright, S.C. Tags: BTB domain forced heterodimer tethered heterodimer transcriptional repressor Miz1/BCL6 Miz1/NAC1 structural communications Source Type: research