Large-scale crystallization and neutron crystallographic analysis of HSP70 in complex with ADP
HSP70 belongs to the heat-shock protein family and binds to unfolded proteins, driven by ATP hydrolysis, in order to prevent aggregation. Previous X-ray crystallographic analyses of HSP70 have shown that HSP70 binds to ADP with internal water molecules. In order to elucidate the role of the water molecules, including their H/D atoms, a neutron diffraction study of the human HSP70 ATPase domain was initiated. Deuterated large crystals of the HSP – ADP complex (1.2 – 1.8   mm3) were successfully grown by large-scale crystallization, and a neutron diffraction experiment at BIODIFF resulted in diffraction to a ma...
Source: Acta Crystallographica Section F - September 22, 2017 Category: Biochemistry Authors: Yokoyama, T. Ostermann, A. Schrader, T.E. Mizuguchi, M. Tags: neutron protein crystallography heat-shock proteins HSP70 large-scale crystallization research communications Source Type: research

Crystal structure of the starch-binding domain of glucoamylase from Aspergillus niger
Glucoamylases are widely used commercially to produce glucose syrup from starch. The starch-binding domain (SBD) of glucoamylase from Aspergillus niger is a small globular protein containing a disulfide bond. The structure of A. niger SBD has been determined by NMR, but the conformation surrounding the disulfide bond was unclear. Therefore, X-ray crystal structural analysis was used to attempt to clarify the conformation of this region. The SBD was purified from an Escherichia coli-based expression system and crystallized at 293   K. The initial phase was determined by the molecular-replacement method, and the asymmetric...
Source: Acta Crystallographica Section F - September 22, 2017 Category: Biochemistry Authors: Suyama, Y. Muraki, N. Kusunoki, M. Miyake, H. Tags: starch-binding domain glucoamylase β -sheet structure disulfide bond Aspergillus niger research communications Source Type: research

Crystal structure of type II NADH:quinone oxidoreductase from Caldalkalibacillus thermarum with an improved resolution of 2.15   Å
Type II NADH:quinone oxidoreductase (NDH-2) is a respiratory enzyme found in the electron-transport chain of many species, with the exception of mammals. It is a 40 – 70   kDa single-subunit monotopic membrane protein that catalyses the oxidation of NADH and the reduction of quinone molecules via the cofactor FAD. NDH-2 is a promising new target for drug development given its essential role in many bacterial species and intracellular parasites. Only two bacterial NDH-2 structures have been reported and these structures are at moderate resolution (2.3 – 2.5   Å ). In this communication, a new crystalli...
Source: Acta Crystallographica Section F - September 22, 2017 Category: Biochemistry Authors: Nakatani, Y. Jiao, W. Arag ã o, D. Shimaki, Y. Petri, J. Parker, E.J. Cook, G.M. Tags: type II NADH:quinone oxidoreductase NDH-2 respiratory enzymes membrane proteins quinone binding research communications Source Type: research

Production, crystallization and structure determination of a mycobacterial glucosylglycerate hydrolase
Glucosylglycerate hydrolase is highly conserved among rapidly growing mycobacteria and has been found to be involved in recovery from nitrogen starvation by promoting the rapid mobilization of the glucosylglycerate that accumulates under these conditions. Here, the production, crystallization and structure determination of glucosylglycerate hydrolase from Mycobacterium hassiacum using two-wavelength anomalous diffraction of selenomethionine-substituted crystals are described. The monoclinic (space group P21) crystals diffracted to ∼ 2.0   Å resolution at a synchrotron-radiation source and contained four molecul...
Source: Acta Crystallographica Section F - September 1, 2017 Category: Biochemistry Authors: Cereija, T.B. Alarico, S. Empadinhas, N. Pereira, P.J.B. Tags: Mycobacterium hassiacum GH63 glucosylglycerate hydrolase nitrogen starvation multiwavelength anomalous diffraction research communications Source Type: research

Ectodomain of plasmodesmata-localized protein 5 in Arabidopsis: expression, purification, crystallization and crystallographic analysis
Plasmodesmata-localized protein 5 (PDLP5) is a cysteine-rich receptor-like protein which is localized on the plasmodesmata of Arabidopsis thaliana. Overexpression of PDLP5 can reduce the permeability of the plasmodesmata and further affect the cell-to-cell movement of viruses and macromolecules in plants. The ectodomain of PDLP5 contains two DUF26 domains; however, the function of these domains is still unknown. Here, the ectodomain of PDLP5 from Arabidopsis was cloned and overexpressed using an insect expression system and was then purified and crystallized. X-ray diffraction data were collected to 1.90   Å resolu...
Source: Acta Crystallographica Section F - August 31, 2017 Category: Biochemistry Authors: Wang, X. Zhu, P. Qu, S. Zhao, J. Singh, P.K. Wang, W. Tags: plasmodesmata N-terminus of PDLP5 DUF26 domain Arabidopsis thaliana research communications Source Type: research

Protein crystallization and initial neutron diffraction studies of the photosystem II subunit PsbO
The PsbO protein of photosystem II stabilizes the active-site manganese cluster and is thought to act as a proton antenna. To enable neutron diffraction studies, crystals of the β -barrel core of PsbO were grown in capillaries. The crystals were optimized by screening additives in a counter-diffusion setup in which the protein and reservoir solutions were separated by a 1% agarose plug. Crystals were cross-linked with glutaraldehyde. Initial neutron diffraction data were collected from a 0.25   mm3 crystal at room temperature using the MaNDi single-crystal diffractometer at the Spallation Neutron Source, Oak Ridge N...
Source: Acta Crystallographica Section F - August 31, 2017 Category: Biochemistry Authors: Bommer, M. Coates, L. Dau, H. Zouni, A. Dobbek, H. Tags: photosystem II PsbO counter-diffusion agarose neutron diffraction capillary cross-linking research communications Source Type: research

The LRR-Roc-COR module of the Chlorobium tepidum Roco protein: crystallization and X-ray crystallographic analysis
Roco proteins are characterized by the presence of a Roc-COR supradomain harbouring GTPase activity, which is often preceded by an LRR domain. The most notorious member of the Roco protein family is the Parkinson's disease-associated LRRK2. The Roco protein from the bacterium Chlorobium tepidum has been used as a model system to investigate the structure and mechanism of this class of enzymes. Here, the crystallization and crystallographic analysis of the LRR-Roc-COR construct of the C. tepidum Roco protein is reported. The LRR-Roc-COR crystals belonged to space group P212121, with unit-cell parameters a = 95.6, b = 129.8,...
Source: Acta Crystallographica Section F - August 21, 2017 Category: Biochemistry Authors: Deyaert, E. Kortholt, A. Vers é es, W. Tags: Roco proteins Chlorobium tepidum LRR-Roc-COR research communications Source Type: research

Characterization and crystal structure of a novel zearalenone hydrolase from Cladophialophora bantiana
Zearalenone (ZEN) is a mycotoxin which causes huge economic losses in the food and animal feed industries. The lactonase ZHD101 from Clonostachys rosea, which catalyzes the hydrolytic degradation of ZEN, is the only known ZEN-detoxifying enzyme. Here, a protein homologous to ZHD101, denoted CbZHD, from Cladophialophora batiana was expressed and characterized. Sequence alignment indicates that CbZHD possesses the same catalytic triad and ZEN-interacting residues as found in ZHD101. CbZHD exhibits optimal enzyme activity at 35 ° C and pH 8, and is sensitive to heat treatment. The crystal structure of apo CbZHD was determ...
Source: Acta Crystallographica Section F - August 21, 2017 Category: Biochemistry Authors: Hui, R. Hu, X. Liu, W. Liu, W. Zheng, Y. Chen, Y. Guo, R.-T. Jin, J. Chen, C.-C. Tags: crystal structure mycotoxins zearalenone Cladophialophora bantiana lactonases research communications Source Type: research

3,6-Anhydro-l-galactonate cycloisomerase from Vibrio sp. strain EJY3: crystallization and X-ray crystallographic analysis
3,6-Anhydro-l-galactonate cycloisomerase (ACI), which is found in the marine bacterium Vibrio sp. strain EJY3, converts 3,6-anhydro-l-galactonate into 2-keto-3-deoxygalactonate. ACI is a key enzyme in the metabolic pathway of 3,6-anhydro-l-galactose (AHG). Study of AHG metabolism is important for the efficient fermentation of agar and biofuel production, because AHG is a sugar that is non-fermentable by commercial microorganisms. The aci gene from Vibrio sp. strain EJY3 was cloned, and the recombinant protein was overexpressed and crystallized in order to determine the structure and understand the function of the protein. ...
Source: Acta Crystallographica Section F - August 21, 2017 Category: Biochemistry Authors: Lee, S. Yun, E.J. Kim, K.H. Kim, H.-Y. Choi, I.-G. Tags: 3,6-anhydro-l-galactonate AHGA 3,6-anhydro-l-galactonate cycloisomerase ACI AHG metabolism agarolytic pathway 3,6-anhydro-l-galactose Vibrio research communications Source Type: research

HicAB toxin – antitoxin complex from Escherichia coli: expression and crystallization
Toxin – antitoxin (TA) systems are widespread in both bacteria and archaea, where they enable cells to adapt to environmental cues. TA systems play crucial roles in various cellular processes, such as programmed cell death, cell growth, persistence and virulence. Here, two distinct forms of the type II toxin – antitoxin complex HicAB were identified and characterized in Escherichia coli K-12, and both were successfully overexpressed and purified. The two proposed forms, HicABL and HicABS, differed in the presence or absence of a seven-amino-acid segment at the N-terminus in the antitoxin HicB. The short form Hi...
Source: Acta Crystallographica Section F - August 14, 2017 Category: Biochemistry Authors: Yang, J. Xu, B. Gao, Z. Zhou, K. Liu, P. Dong, Y. Zhang, J. Liu, Q. Tags: toxin – antitoxin systems HicAB persister cells cell stress Escherichia coli research communications Source Type: research

Crystal structure of the DNA sequence d(CGTGAATTCACG)2 with DAPI
The structure of 4 ′ ,6-diamidine-2-phenylindole (DAPI) bound to the synthetic B-DNA oligonucleotide d(CGTGAATTCACG) has been solved in space group P212121 by single-crystal X-ray diffraction at a resolution of 2.2   Å . The structure is nearly isomorphous to that of the previously reported crystal structure of the oligonucleotide d(CGTGAATTCACG) alone. The adjustments in crystal packing between the native DNA molecule and the DNA – DAPI complex are described. DAPI lies in the narrow minor groove near the centre of the B-DNA fragment, positioned over the A – T base pairs. It is bound to the DNA by...
Source: Acta Crystallographica Section F - August 14, 2017 Category: Biochemistry Authors: Sbirkova-Dimitrova, H.I. Shivachev, B. Tags: DNA single crystal DAPI fluorescent intercalator displacement ligand palindrome research communications Source Type: research

In crystallo activity tests with latent apple tyrosinase and two mutants reveal the importance of the mutated sites for polyphenol oxidase activity
Tyrosinases are type 3 copper enzymes that belong to the polyphenol oxidase (PPO) family and are able to catalyze both the ortho-hydroxylation of monophenols and their subsequent oxidation to o-quinones, which are precursors for the biosynthesis of colouring substances such as melanin. The first plant pro-tyrosinase from Malus domestica (MdPPO1) was recombinantly expressed in its latent form (56.4   kDa) and mutated at four positions around the catalytic pocket which are believed to influence the activity of the enzyme. Mutating the amino acids, which are known as activity controllers, yielded the mutants MdPPO1-Ala239Th...
Source: Acta Crystallographica Section F - July 28, 2017 Category: Biochemistry Authors: Kampatsikas, I. Bijelic, A. Pretzler, M. Rompel, A. Tags: polyphenol oxidase plant tyrosinase latent pro-enzyme type 3 copper enzyme activity controllers research communications Source Type: research

Expression and crystallographic studies of the D1D2 domains of C4.4A, a homologous protein to the urokinase receptor
C4.4A is a glycosylphosphatidylinositol-anchored membrane protein comprised of two LU domains (Ly6/uPAR-like domains) and an extensively O-glycosylated C-terminal Ser/Thr/Pro-rich region. C4.4A is a novel biomarker for squamous epithelial differentiation. Its expression is dysregulated under various pathological conditions and it is a robust biomarker for poor prognosis in various malignant conditions such as pulmonary adenocarcinoma. To facilitate crystallization, the two LU domains were excised from intact C4.4A by limited proteolysis, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals di...
Source: Acta Crystallographica Section F - July 27, 2017 Category: Biochemistry Authors: Chen, S. Lin, L. Yuan, C. G å rdsvoll, H. Kriegbaum, M.C. Ploug, M. Huang, M. Tags: C4.4A LU domains glycosylation prognostic cancer biomarkers research communications Source Type: research

Filament-like DREP4 CIDE domain: characterization and preliminary X-ray crystallographic studies
In this study, it was found that DREP4 CIDE domains form filament-like structures in solution. The length of the highly ordered filament-like structure is dependent on the salt concentration. By adjusting the salt concentration the DREP4 CIDE domain could be crystallized, and X-ray diffraction data were collected to a resolution of 1.9   Å . The crystals were found to belong to the orthorhombic space group P212121, with unit-cell parameters a = 53.08, b = 76.58, c = 174.59   Å . (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - July 26, 2017 Category: Biochemistry Authors: Park, H.H. Tags: apoptosis DNA fragmentation factor CIDE domain DREP4 research communications Source Type: research

The quorum-quenching lactonase from Alicyclobacter acidoterrestris: purification, kinetic characterization, crystallization and crystallographic analysis
Lactonases comprise a class of enzymes that hydrolyze lactones, including acyl-homoserine lactones (AHLs); the latter are used as chemical signaling molecules by numerous Gram-negative bacteria. Lactonases have therefore been demonstrated to quench AHL-based bacterial communication. In particular, lactonases are capable of inhibiting bacterial behaviors that depend on these chemicals, such as the formation of biofilms or the expression of virulence factors. A novel representative from the metallo- β -lactamase superfamily, named AaL, was isolated from the thermoacidophilic bacterium Alicyclobacter acidoterrestris. Kin...
Source: Acta Crystallographica Section F - July 26, 2017 Category: Biochemistry Authors: Bergonzi, C. Schwab, M. Chabriere, E. Elias, M. Tags: quorum sensing quorum quenching lactonases thermophiles Alicyclobacter acidoterrestris research communications Source Type: research

High-resolution structure of a Kazal-type serine protease inhibitor from the dengue vector Aedes aegypti
Blood-feeding exoparasites are rich sources of protease inhibitors, and the mosquito Aedes aegypti, which is a vector of Dengue virus, Yellow fever virus, Chikungunya virus and Zika virus, is no exception. AaTI is a single-domain, noncanonical Kazal-type serine proteinase inhibitor from A. aegypti that recognizes both digestive trypsin-like serine proteinases and the central protease in blood clotting, thrombin, albeit with an affinity that is three orders of magnitude lower. Here, the 1.4   Å resolution crystal structure of AaTI is reported from extremely tightly packed crystals ( ∼ 22% solvent content), revea...
Source: Acta Crystallographica Section F - July 26, 2017 Category: Biochemistry Authors: Torquato, R.J.S. Lu, S. Martins, N.H. Tanaka, A.S. Pereira, P.J.B. Tags: anticoagulants thrombin trypsin salivary glands protein – protein interactions Kazal inhibitor Aedes aegypti Dengue fever Yellow fever Zika virus research communications Source Type: research

Crystal structure of the putative cytoplasmic protein STM0279 (Hcp2) from Salmonella typhimurium
In this study, the crystal structure and the oligomeric state in solution of Hcp2 from S. typhimurium (StHcp2) were investigated. The crystal structure refined to 3.0   Å resolution showed that the protein is composed of a β -barrel domain with extended loops and can form hexameric rings as observed in known Hcp homologues. Mutation of the extended loop was found to partly destabilize the hexameric conformation into monomers or cause the production of inclusion bodies, suggesting it has an important role in hexameric ring formation. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - July 26, 2017 Category: Biochemistry Authors: Lin, Q.-P. Gao, Z.-Q. Geng, Z. Zhang, H. Dong, Y.-H. Tags: type VI secretion system crystal structure haemolysin co-regulated protein hexameric rings T6SS assembly research communications Source Type: research

Production, biophysical characterization and crystallization of Pseudomonas putida GraA and its complexes with GraT and the graTA operator
The graTA operon from Pseudomonas putida encodes a toxin – antitoxin module with an unusually moderate toxin. Here, the production, SAXS analysis and crystallization of the antitoxin GraA, the GraTA complex and the complex of GraA with a 33   bp operator fragment are reported. GraA forms a homodimer in solution and crystallizes in space group P21, with unit-cell parameters a = 66.9, b   = 48.9, c = 62.7   Å , β = 92.6 ° . The crystals are likely to contain two GraA dimers in the asymmetric unit and diffract to 1.9   Å resolution. The GraTA complex forms a heterotetramer in solution. Cr...
Source: Acta Crystallographica Section F - July 26, 2017 Category: Biochemistry Authors: Talavera, A. Tamman, H. Ainelo, A. Had ž i, S. Garcia-Pino, A. H õ rak, R. Konijnenberg, A. Loris, R. Tags: persistence toxin – antitoxin module protein DNA complex macromolecular complex GraT GraA Pseudomonas putida ribosome biogenesis research communications Source Type: research

Methylation, crystallization and SAD phasing of the Csu pilus CsuC – CsuE chaperone – adhesin subunit pre-assembly complex from Acinetobacter baumannii
Acinetobacter baumannii is one of the most difficult Gram-negative bacteria to control and treat. This pathogen forms biofilms on hospital surfaces and medical devices using Csu pili assembled via the archaic chaperone – usher pathway. To uncover the mechanism of bacterial attachment to abiotic surfaces, it was aimed to determine the crystal structure of the pilus tip adhesin CsuE. The CsuC – CsuE chaperone – subunit pre-assembly complex was purified from the periplasm of Escherichia coli overexpressing CsuC and CsuE. Despite the high purity of the complex, no crystals could be obtained. This challenge wa...
Source: Acta Crystallographica Section F - July 26, 2017 Category: Biochemistry Authors: Pakharukova, N. Tuittila, M. Paavilainen, S. Zavialov, A. Tags: chaperone – usher pathway archaic pili biofilm Acinetobacter baumannii adhesion CsuC CsuE research communications Source Type: research

1.12   Å resolution crystal structure of the catalytic domain of the plasmid-mediated colistin resistance determinant MCR-2
MCR-2 confers resistance to colistin, a `last-line' antibiotic against extensively resistant Gram-negative pathogens. It is a plasmid-encoded phosphoethanolamine transferase that is closely related to MCR-1. To understand the diversity in the MCR family, the 1.12   Å resolution crystal structure of the catalytic domain of MCR-2 was determined. Variable amino acids are located distant from both the di-zinc active site and the membrane-proximal face. The exceptionally high resolution will provide an accurate starting model for further mechanistic studies. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - July 26, 2017 Category: Biochemistry Authors: Coates, K. Walsh, T.R. Spencer, J. Hinchliffe, P. Tags: MCR-1 antibiotic resistance colistin MCR-2 polymixin research communications Source Type: research

The putative polyketide cyclase MSMEG_0129 from Mycobacterium smegmatis: purification, crystallization and X-ray crystallographic analysis
Mycobacterium tuberculosis Rv0164 has previously been identified as a human T-cell antigen that induces significant production of IFN- γ in human peripheral blood mononuclear cells. M. smegmatis MSMEG_0129 shares 59% sequence identity with Rv0164. Based on sequence alignment, both proteins are predicted to be members of the cyclase/dehydrase family, which is part of a large group of enzymes referred to as type II polyketide synthases (PKSs). In biosynthetic pathways mediated by type II PKSs, cyclases catalyze the conversion of linear poly- β -ketones to cyclized intermediates. To date, no mycobacterial type II P...
Source: Acta Crystallographica Section F - June 30, 2017 Category: Biochemistry Authors: Zheng, S. Zhou, Y. Fleming, J. Zhou, Y. Liu, W. Bi, L. Tags: Mycobacterium tuberculosis Rv0164 MSMEG_0129 type II polyketide synthase crystallization research communications Source Type: research

Crystal structure of the N-terminal domain of VqsR from Pseudomonas aeruginosa at 2.1   Å resolution
VqsR is a quorum-sensing (QS) transcriptional regulator which controls QS systems (las, rhl and pqs) by directly downregulating the expression of qscR in Pseudomonas aeruginosa. As a member of the LuxR family of proteins, VqsR shares the common motif of a helix – turn – helix (HTH)-type DNA-binding domain at the C-terminus, while the function of its N-terminal domain remains obscure. Here, the crystal structure of the N-terminal domain of VqsR (VqsR-N; residues 1 – 193) was determined at a resolution of 2.1   Å . The structure is folded into a regular α – β – α sandwic...
Source: Acta Crystallographica Section F - June 28, 2017 Category: Biochemistry Authors: He, Q. Wang, K. Su, T. Wang, F. Gu, L. Xu, S. Tags: VqsR LuxR quorum sensing Pseudomonas aeruginosa research communications Source Type: research

The multidrug-resistance transporter MdfA from Escherichia coli: crystallization and X-ray diffraction analysis
The active efflux of antibiotics by multidrug-resistance (MDR) transporters is a major pathway of drug resistance and complicates the clinical treatment of bacterial infections. MdfA is a member of the major facilitator superfamily (MFS) from Escherichia coli and provides resistance to a wide variety of dissimilar toxic compounds, including neutral, cationic and zwitterionic substances. The 12-transmembrane-helix MdfA was expressed as a GFP-octahistidine fusion protein with a TEV protease cleavage site. Following tag removal, MdfA was purified using two chromatographic steps, complexed with a Fab fragment and further purif...
Source: Acta Crystallographica Section F - June 20, 2017 Category: Biochemistry Authors: Nagarathinam, K. Jaenecke, F. Nakada-Nakura, Y. Hotta, Y. Liu, K. Iwata, S. Stubbs, M.T. Nomura, N. Tanabe, M. Tags: MFS transporter multidrug resistance membrane protein crystallization antibody fragment lipidic cubic phase research communications Source Type: research

Structure of bovine cytochrome c oxidase crystallized at a neutral pH using a fluorinated detergent
Cytochrome c oxidase (CcO) couples proton pumping to O2 reduction. Its enzymatic activity depends sensitively on pH over a wide range. However, owing to difficulty in crystallizing this protein, X-ray structure analyses of bovine CcO aimed at understanding its reaction mechanism have been conducted using crystals prepared at pH 5.7, which is significantly lower than that in the cell. Here, oxidized CcO at pH 7.3 was crystallized using a fluorinated octyl-maltoside derivative, and the structure was determined at 1.77   Å resolution. No structural differences between crystals obtained at the neutral pH and the acidic...
Source: Acta Crystallographica Section F - June 20, 2017 Category: Biochemistry Authors: Luo, F. Shinzawa-Itoh, K. Hagimoto, K. Shimada, A. Shimada, S. Yamashita, E. Yoshikawa, S. Tsukihara, T. Tags: cytochrome c oxidase membrane-protein complex X-ray structure neutral pH research communications Source Type: research

Assembly of Francisella novicida Cpf1 endonuclease in complex with guide RNA and target DNA
Bacteria and archaea use the CRISPR – Cas system as an adaptive response against infection by foreign nucleic acids. Owing to its remarkable flexibility, this mechanism has been harnessed and adopted as a powerful tool for genome editing. The CRISPR – Cas system includes two classes that are subdivided into six types and 19 subtypes according to conservation of the cas gene and loci organization. Recently, a new protein with endonuclease activity belonging to class 2 type V has been identified. This endonuclease, termed Cpf1, in complex with a single CRISPR RNA (crRNA) is able to recognize and cleave a target D...
Source: Acta Crystallographica Section F - June 20, 2017 Category: Biochemistry Authors: Alc ó n, P. Montoya, G. Stella, S. Tags: crystallization protein – DNA interaction RNA interaction data collection genome editing CRISPR Cas research communications Source Type: research

Full-length nisin immunity protein NisI from Lactococcus lactis in a lipid-free form: crystallization and X-ray analysis
NisI is a lantibiotic-binding lipoprotein that is specific for nisin. Nisin-producing microorganisms use NisI as an immunity protein for self-protection against nisin. Here, the purification, crystallization and preliminary X-ray diffraction of full-length NisI from Lactobacillus lactis in a lipid-free form (NisI22-C) are reported. Importantly, reductive methylation of the lysine residues in NisI22-C was essential for initial crystallization. Only methylated NisI22-C crystallized. The optimized crystals of methylated NisI22-C were grown in 30 – 40   mM ammonium sulfate, 0.1   M sodium acetate pH 4.6, 16 – 1...
Source: Acta Crystallographica Section F - June 16, 2017 Category: Biochemistry Authors: Jeong, J.H. Ha, S.C. Tags: nisin lantibiotics immunity protein reductive methylation research communications Source Type: research

Crystal structure of a family 6 cellobiohydrolase from the basidiomycete Phanerochaete chrysosporium
Cellobiohydrolases belonging to glycoside hydrolase family 6 (CBH II, Cel6A) play key roles in the hydrolysis of crystalline cellulose. CBH II from the white-rot fungus Phanerochaete chrysosporium (PcCel6A) consists of a catalytic domain (CD) and a carbohydrate-binding module connected by a linker peptide, like other known fungal cellobiohydrolases. In the present study, the CD of PcCel6A was crystallized without ligands, and p-nitrophenyl β -d-cellotrioside (pNPG3) was soaked into the crystals. The determined structures of the ligand-free and pNPG3-soaked crystals revealed that binding of cellobiose at substrate subs...
Source: Acta Crystallographica Section F - June 16, 2017 Category: Biochemistry Authors: Tachioka, M. Nakamura, A. Ishida, T. Igarashi, K. Samejima, M. Tags: cellulases Phanerochaete chrysosporium cellobiohydrolase biomass utilization carbohydrate-active enzymes research communications Source Type: research

DNA-binding domain of myelin-gene regulatory factor: purification, crystallization and X-ray analysis
In this study, the MRF DBD was cloned, purified and crystallized in order to understand the molecular mechanism that regulates the transcription of myelin genes. Selenomethionine was subsequently introduced into the crystals to obtain the phases for the MRF DBD structure. The native and selenomethionine-labelled crystals exhibited diffraction to 2.50 and 2.51   Å resolution, respectively. The crystals belonged to space group P321 and the selenomethionine-labelled crystals had unit-cell parameters a = 104.0, b = 104.0, c = 46.7   Å , α = 90, β = 90, γ = 120 ° . The calculated Matthews co...
Source: Acta Crystallographica Section F - June 16, 2017 Category: Biochemistry Authors: Wu, W. Zhen, X. Shi, N. Tags: myelin-gene regulation MRF DNA-binding domain trimeric transcription factor X-ray crystallography research communications Source Type: research

Crystal structure of a β -aminopeptidase from an Australian Burkholderia sp.
β -Aminopeptidases are a unique group of enzymes that have the unusual capability to hydrolyze N-terminal β -amino acids from synthetic β -peptides. β -Peptides can form secondary structures mimicking α -peptide-like structures that are resistant to degradation by most known proteases and peptidases. These characteristics of β -peptides give them great potential as peptidomimetics. Here, the X-ray crystal structure of BcA5-BapA, a β -aminopeptidase from a Gram-negative Burkholderia sp. that was isolated from activated sludge from a wastewater-treatment plant in Australia, is reported. Th...
Source: Acta Crystallographica Section F - June 16, 2017 Category: Biochemistry Authors: John-White, M. Dumsday, G.J. Johanesen, P. Lyras, D. Drinkwater, N. McGowan, S. Tags: BcA5-BapA β -aminopeptidases crystallization Burkholderia sp. -amino acids Ntn hydrolases research communications Source Type: research

Recombinant ACHT1 from Arabidopsis thaliana: crystallization and X-ray crystallographic analysis
Thioredoxins (Trxs) play important roles in chloroplasts by linking photosynthetic light reactions to a series of plastid functions. They execute their function by regulating the oxidation and reduction of disulfide bonds. ACHT1 (atypical cysteine/histidine-rich Trx1) is a thylakoid-associated thioredoxin-type protein found in the Arabidopsis thaliana chloroplast. Recombinant ACHT1 protein was overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion method. The crystal diffracted to 1.7   Å resolution and a complete X-ray data set was collected. Preliminary crystallographic analysis sugg...
Source: Acta Crystallographica Section F - June 16, 2017 Category: Biochemistry Authors: Pan, W. Wang, J. Yang, Y. Liu, L. Zhang, M. Tags: thioredoxins photosynthetic light reactions Arabidopsis thaliana disulfide bonds chloroplasts ACHT1 research communications Source Type: research

The structure of a complex of the lactonohydrolase zearalenone hydrolase with the hydrolysis product of zearalenone at 1.60   Å resolution
Zearalenone hydrolase (ZHD) is an α / β -hydrolase that detoxifies and degrades the lactone zearalenone (ZEN), a naturally occurring oestrogenic mycotoxin that contaminates crops. Several apoenzyme and enzyme – substrate complex structures have been reported in the resolution range 2.4 – 2.6   Å . However, the properties and mechanism of this enzyme are not yet fully understood. Here, a 1.60   Å resolution structure of a ZHD – product complex is reported which was determined from a C-terminally His6-tagged ZHD crystal soaked with 2   mM ZEN for 30   min. It shows that after ...
Source: Acta Crystallographica Section F - June 16, 2017 Category: Biochemistry Authors: Qi, Q. Yang, W.-J. Zhou, H.-J. Ming, D.-M. Sun, K.-L. Xu, T.-Y. Hu, X.-J. Lv, H. Tags: lactonohydrolase Clonostachys rosea zearalenone catalysis mechansim research communications Source Type: research

Crystal structure of recombinant phosphoribosylpyrophosphate synthetase 2 from Thermus thermophilus HB27 complexed with ADP and sulfate ions
Phosphoribosylpyrophosphate synthetase (PRPPS) from the thermophilic bacterial strain Thermus thermophilus HB27 catalyzes the synthesis of phosphoribosylpyrophosphate from ribose 5-phosphate and ATP, and belongs to the class I PRPPSs. The three-dimensional structure of the recombinant enzyme was solved at 2.2   Å resolution using crystals grown in microgravity from protein solution containing ATP, magnesium and sulfate ions. An ADP molecule was located in the active site of each subunit of the hexameric enzyme molecule and sulfate ions were located in both the active and allosteric sites. It was found that the cata...
Source: Acta Crystallographica Section F - May 31, 2017 Category: Biochemistry Authors: Timofeev, V.I. Sinitsyna, E.V. Kostromina, M.A. Muravieva, T.I. Makarov, D.A. Mikheeva, O.O. Kuranova, I.P. Esipov, R.S. Tags: transferases phosphoribosylpyrophosphate synthetase PRPP synthetase Thermus thermophilus HB27 microgravity research communications Source Type: research

Crystal structure of the multiple antibiotic resistance regulator MarR from Clostridium difficile
Regulators of multiple antibiotic resistance (MarRs) are key players against toxins in prokaryotes. MarR homologues have been identified in many bacterial and archaeal species which pose daunting antibiotic resistance issues that threaten public health. The continuous prevalence of Clostridium difficile infection (CDI) throughout the world is associated with the abuse of antibiotics, and antibiotic treatments of CDI have limited effect. In the genome of C. difficile strain 630, the marR gene (ID 4913953) encodes a MarR protein. Here, MarR from C. difficile (MarRC.difficile) was subcloned and crystallized for the first time...
Source: Acta Crystallographica Section F - May 31, 2017 Category: Biochemistry Authors: Peng, J.W. Yuan, H. Tan, X.S. Tags: Clostridium difficile MarR crystal structure transcription factors DNA binding antibiotic resistance research communications Source Type: research

Crystal structure of N-acetylmannosamine kinase from Fusobacterium nucleatum
Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5 ′ -triphosphate to the C6 position of N-acetylmanno...
Source: Acta Crystallographica Section F - May 31, 2017 Category: Biochemistry Authors: Caing-Carlsson, R. Goyal, P. Sharma, A. Ghosh, S. Setty, T.G. North, R.A. Friemann, R. Ramaswamy, S. Tags: sialic acid catabolism N-acetylmannosamine kinase Fusobacterium nucleatum research communications Source Type: research

The crystal structure of human DEAH-box RNA helicase 15 reveals a domain organization of the mammalian DEAH/RHA family
DEAH-box RNA helicase 15 (DHX15) plays important roles in RNA metabolism, including in splicing and in ribosome biogenesis. In addition, mammalian DHX15 also mediates the innate immune sensing of viral RNA. However, structural information on this protein is not available, although the structure of the fungal orthologue of this protein, Prp43, has been elucidated. Here, the crystal structure of the ADP-bound form of human DHX15 is reported at a resolution of 2.0   Å . This is the first structure to be revealed of a member of the mammalian DEAH-box RNA helicase (DEAH/RHA) family in a nearly complete form, including t...
Source: Acta Crystallographica Section F - May 25, 2017 Category: Biochemistry Authors: Murakami, K. Nakano, K. Shimizu, T. Ohto, U. Tags: RNA helicase 15 DEAH-box DEAH/RHA family innate immunity DHX15 research communications Source Type: research

Phosphate-binding protein from Polaromonas JS666: purification, characterization, crystallization and sulfur SAD phasing
Phosphate-binding proteins (PBPs) are key proteins that belong to the bacterial ABC-type phosphate transporters. PBPs are periplasmic (or membrane-anchored) proteins that capture phosphate anions from the environment and release them to the transmembrane transporter. Recent work has suggested that PBPs have evolved for high affinity as well as high selectivity. In particular, a short, unique hydrogen bond between the phosphate anion and an aspartate residue has been shown to be critical for selectivity, yet is not strictly conserved in PBPs. Here, the PBP from Polaromonas JS666 is focused on. Interestingly, this PBP is pre...
Source: Acta Crystallographica Section F - May 25, 2017 Category: Biochemistry Authors: Pegos, V.R. Hey, L. LaMirande, J. Pfeffer, R. Lipsh, R. Amitay, M. Gonzalez, D. Elias, M. Tags: phosphate-binding protein phosphate ABC transporter molecular specificity Polaromonas JS666 research communications Source Type: research

Crystal structure of the active form of native human thymidylate synthase in the absence of bound substrates
Human thymidylate synthase (hTS) provides the sole de novo intracellular source of thymidine 5 ′ -monophosphate (dTMP). hTS is required for DNA replication prior to cell division, making it an attractive target for anticancer chemotherapy and drug discovery. hTS binds 2 ′ -deoxyuridine 5 ′ -monophosphate (dUMP) and the folate co-substrate N5,N10-methylenetetrahydrofolate (meTHF) in a pocket near the catalytic residue Cys195. The catalytic loop, which is composed of amino-acid residues 181 – 197, can adopt two distinct conformations related by a 180 ° rotation. In the active conformation Cys195 i...
Source: Acta Crystallographica Section F - May 25, 2017 Category: Biochemistry Authors: Deschamps, P. R é ty, S. Bareille, J. Leulliot, N. Tags: thymidylate synthase active form low-salt conditions DNA replication research communications Source Type: research

Crystal structure of the Thermoplasma acidophilum protein Ta1207
The crystal structure of the Ta1207 protein from Thermoplasma acidophilum is reported. Ta1207 was identified in a screen for high-molecular-weight protein complexes in T. acidophilum. In solution, Ta1207 forms homopentamers of 188   kDa. The crystal structure of recombinant Ta1207 solved by Se-MAD at 2.4   Å resolution revealed a complex with fivefold symmetry. In the crystal lattice, calcium ions induce the formation of a nanocage from two pentamers. The Ta1207 protomers comprise two domains with the same novel α / β topology. A deep pocket with a binding site for a negatively charged group suggests t...
Source: Acta Crystallographica Section F - May 25, 2017 Category: Biochemistry Authors: Pathare, G.R. Nagy, I. Hubert, Á . Thomas, D.R. Bracher, A. Tags: pentamers archaeon thermostability nanotechnology Thermoplasma acidophilum Ta1207 research communications Source Type: research

Crystallization and X-ray diffraction analysis of SpaE, a basal pilus protein from the gut-adapted Lactobacillus rhamnosus GG
SpaE is the predicted basal pilin subunit in the sortase-dependent SpaFED pilus from the gut-adapted and commensal Lactobacillus rhamnosus GG. Thus far, structural characterization of the cell-wall-anchoring basal pilins has remained difficult and has been limited to only a few examples from pathogenic genera and species. To gain a further structural understanding of the molecular mechanisms that are involved in the anchoring and assembly of sortase-dependent pili in less harmful bacteria, L. rhamnosus GG SpaE for crystallization was produced by recombinant expression in Escherichia coli. Although several attempts to cryst...
Source: Acta Crystallographica Section F - May 25, 2017 Category: Biochemistry Authors: Mishra, A.K. Megta, A.K. Palva, A. von Ossowski, I. Krishnan, V. Tags: adhesion probiotics basal pilin host – microbe interaction sortase-dependent pili Lactobacillus rhamnosus GG SpaE research communications Source Type: research

Crystal structures of human Fabs targeting the Bexsero meningococcal vaccine antigen NHBA
This study also provides indirect evidence for the intrinsically disordered nature of two N-terminal regions of NHBA. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 25, 2017 Category: Biochemistry Authors: Maritan, M. Cozzi, R. Lo Surdo, P. Veggi, D. Bottomley, M.J. Malito, E. Tags: fragment antigen binding Fabs monoclonal antibodies mAbs NHBA Neisseria vaccine Bexsero research communications Source Type: research

Crystal structure of Rv1220c, a SAM-dependent O-methyltransferase from Mycobacterium tuberculosis
Rv1220c from Mycobacterium tuberculosis is annotated as an O-methyltransferase (MtbOMT). Currently, no structural information is available for this protein. Here, the crystal structure of MtbOMT refined to 2.0   Å resolution is described. The structure reveals the presence of a methyltransferase fold and shows clear electron density for one molecule of S-adenosylmethionine (SAM), which was apparently bound by the protein during its production in Escherichia coli. Although the overall structure of MtbOMT resembles the structures of O-methyltransferases from Cornybacterium glutamicum, Coxiella burnetti and Alfa alfa,...
Source: Acta Crystallographica Section F - May 11, 2017 Category: Biochemistry Authors: Yan, Q. Shaw, N. Qian, L. Jiang, D. Tags: S-adenosyl-l-methionine His – Asp pair crystal structure dihydroxycinnamic acid Mycobacterium tuberculosis Rv1220c research communications Source Type: research

Crystallization and biochemical characterization of an archaeal lectin from Methanococcus voltae A3
A lectin from Methanococcus voltae A3 has been cloned, expressed, purified and characterized. The lectin appears to be specific for complex sugars. The protein crystallized in a tetragonal space group, with around 16 subunits in the asymmetric unit. Sequence comparisons indicate the lectin to have a β -prism I fold, with poor homology to lectins of known three-dimensional structure. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - April 28, 2017 Category: Biochemistry Authors: Sivaji, N. Abhinav, K.V. Vijayan, M. Tags: archaeal lectins β -prism I fold X-ray studies Methanococcus voltae research communications Source Type: research

Crystal structure of CD27 in complex with a neutralizing noncompeting antibody
CD27 is a T-cell and B-cell co-stimulatory glycoprotein of the tumor necrosis factor (TNF) receptor superfamily that is dependent on the availability of the TNF-like ligand CD70. Therapeutic approaches to treating autoimmune diseases and cancers with antagonistic and agonistic anti-CD27 monoclonal antibodies (mAbs), respectively, have recently been developed. Mouse anti-human CD27 mAb 2177 shows potency in neutralizing CD70-induced signaling; however, it does not block the binding of soluble CD70. To provide insight into the mechanism of action of the mAb, the crystal structure of the CD27 extracellular domain in complex w...
Source: Acta Crystallographica Section F - April 26, 2017 Category: Biochemistry Authors: Teplyakov, A. Obmolova, G. Malia, T.J. Gilliland, G.L. Tags: CD27 cysteine-rich domain antibodies crystal structure epitopes tumor necrosis factor research communications Source Type: research

Camelid nanobodies used as crystallization chaperones for different constructs of PorM, a component of the type IX secretion system from Porphyromonas gingivalis
PorM is a membrane protein that is involved in the assembly of the type IX secretion system (T9SS) in Porphyromonas gingivalis, a major bacterial pathogen that is responsible for periodontal disease in humans. In the context of structural studies of PorM to better understand T9SS assembly, four camelid nanobodies were selected, produced and purified, and their specific interaction with the N-terminal or C-terminal part of the periplasmic domain of PorM was investigated. Diffracting crystals were also obtained, and the structures of the four nanobodies were solved by molecular replacement. Furthermore, two nanobodies were u...
Source: Acta Crystallographica Section F - April 26, 2017 Category: Biochemistry Authors: Duhoo, Y. Roche, J. Trinh, T.T.N. Desmyter, A. Gaubert, A. Kellenberger, C. Cambillau, C. Roussel, A. Leone, P. Tags: camelid nanobodies type IX secretion system crystallization chaperones PorM Porphyromonas gingivalis research communications Source Type: research

Transcription factor Rv0081 from Mycobacterium tuberculosis: purification, crystallization and initial crystallographic analysis
Because of its high infectivity and pathogenicity, Mycobacterium tuberculosis is a serious threat to human health. While the transcription-regulatory system of M.   tuberculosis remains incompletely understood, Rv0081, an essential regulatory hub, is known to mediate the initial response to hypoxia in the long-term survival of M. tuberculosis. Here, the production, crystallization and initial X-ray crystallographic analysis of Rv0081 are reported. The crystals of Rv0081 belonged to space group P62, with unit-cell parameters a = 67.48, b   =   67.48, c   =   40.84   Å , γ = 120 ° . Th...
Source: Acta Crystallographica Section F - April 26, 2017 Category: Biochemistry Authors: Dong, S. Ding, Z. Wang, Y. Yang, Y. Mao, Y. Wang, Y. Tags: Rv0081 transcription factor Mycobacterium tuberculosis research communications Source Type: research

Crystal structure of the RNA 2 ′ ,3 ′ -cyclic phosphodiesterase from Deinococcus radiodurans
2 ′ ,3 ′ -Cyclic phosphodiesterase (CPDase) homologues have been found in all domains of life and are involved in diverse RNA and nucleotide metabolisms. The CPDase from Deinococcus radiodurans was crystallized and the crystals diffracted to 1.6   Å resolution, which is the highest resolution currently known for a CPDase structure. Structural comparisons revealed that the enzyme is in an open conformation in the absence of substrate. Nevertheless, the active site is well formed, and the representative motifs interact with sulfate ion, which suggests a conserved catalytic mechanism. (Source: Acta Crystal...
Source: Acta Crystallographica Section F - April 26, 2017 Category: Biochemistry Authors: Han, W. Cheng, J. Zhou, C. Hua, Y. Zhao, Y. Tags: RNA repair CPDase RNA 2 ′ ,3 -cyclic phosphodiesterase Deinococcus radiodurans research communications Source Type: research

Structural insights into the mechanism of the drastic changes in enzymatic activity of the cytochrome P450 vitamin D3 hydroxylase (CYP107BR1) caused by a mutation distant from the active site
Cytochromes P450 (P450s) are haem-containing enzymes that catalyze medically and industrially important oxidative reactions, and many P450s have been subjected to directed evolution and site-directed mutagenesis to improve their activity and substrate specificity. Nonetheless, in most cases the mechanism that leads to drastic changes in specific activity after the introduction of an amino-acid substitution distant from the active-site pocket is unclear. Here, two crystal structures of inactive mutants of the P450 vitamin D3 hydroxylase (Vdh), Vdh-F106V and Vdh-L348M, which were obtained in the course of protein-engineering...
Source: Acta Crystallographica Section F - April 26, 2017 Category: Biochemistry Authors: Yasutake, Y. Kameda, T. Tamura, T. Tags: crystal structure cytochrome P450 CYP vitamin D3 hydroxylase conformational changes research communications Source Type: research

Crystal structure of d(CCGGGGTACCCCGG)2 at 1.4   Å resolution
The X-ray crystal structure of the DNA tetradecamer sequence d(CCGGGGTACCCCGG)2 is reported at 1.4   Å resolution in the tetragonal space group P41212. The sequence was designed to fold as a four-way junction. However, it forms an A-type double helix in the presence of barium chloride. The metal ion could not be identified in the electron-density map. The crystallographic asymmetric unit consists of one A-type double helix with 12 base pairs per turn, in contrast to 11 base pairs per turn for canonical A-DNA. A large number of solvent molecules have been identified in both the grooves of the duplex and around the b...
Source: Acta Crystallographica Section F - April 26, 2017 Category: Biochemistry Authors: Karthik, S. Thirugnanasambandam, A. Mandal, P.K. Gautham, N. Tags: d(CCGGGGTACCCCGG)2 A-DNA duplex X-ray crystallography tetragonal space group research communications Source Type: research

Increasing the soluble expression and crystallization of the Escherichia coli quorum-sensing protein LsrK
LsrK is one of the key components of the luxS-regulated (lsr) operon in Escherichia coli and plays an important role during the quorum-sensing (QS) process mediated by autoinducer-2 (AI-2). The AI-2 molecule is imported into the cell by the LsrACB transporter and is subsequently phosphorylated (to AI-2-P) by LsrK. AI-2-P binds to the repressor protein of the lsr operon (LsrR) and triggers various cellular responses related to QS by dissociating LsrR from the DNA. Although a large amount of purified LsrK is required for structural studies, recombinant GST-LsrK was mostly expressed in an insoluble form. To enhance the solubl...
Source: Acta Crystallographica Section F - April 26, 2017 Category: Biochemistry Authors: Ha, J.-H. Eo, Y. Ahn, H.-C. Ryu, K.-S. Tags: chaperones heat shock LsrK complex LsrK crystal soluble expression of LsrK osmotic shock quorum sensing research communications Source Type: research

Structure of NADP+-bound 7 β -hydroxysteroid dehydrogenase reveals two cofactor-binding modes
In mammals, bile acids/salts and their glycine and taurine conjugates are effectively recycled through enterohepatic circulation. 7 β -Hydroxysteroid dehydrogenases (7 β -HSDHs; EC 1.1.1.201), including that from the intestinal microbe Collinsella aerofaciens, catalyse the NADPH-dependent reversible oxidation of secondary bile-acid products to avoid potential toxicity. Here, the first structure of NADP+ bound to dimeric 7 β -HSDH is presented. In one active site, NADP+ adopts a conventional binding mode similar to that displayed in related enzyme structures. However, in the other active site a unique binding...
Source: Acta Crystallographica Section F - April 26, 2017 Category: Biochemistry Authors: Wang, R. Wu, J. Jin, D.K. Chen, Y. Lv, Z. Chen, Q. Miao, Q. Huo, X. Wang, F. Tags: crystal structure NADP -bound 7 β -HSDH rational protein engineering research communications Source Type: research