Synthesis, purification and crystallographic studies of the C-terminal sterol carrier protein type 2 (SCP-2) domain of human hydroxysteroid dehydrogenase-like protein 2
In this study, the C-terminal SCP-2 domain of human HSDL2, including residues Lys318–Arg416, was produced in Escherichia coli, purified and crystallized. X-ray diffraction data were collected to 2.10 Å resolution. The crystal belonged to the trigonal space group P3121 (or P3221), with unit-cell parameters a = b = 70.4, c = 60.6 Å, α = β = 90, γ = 120°. Two protein molecules are present in the asymmetric unit, resulting in a Matthews coefficient of 2.16 Å3 Da−1 and an approximate solvent content of 43%. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Cheng, Z.Li, Y.Sui, C.Sun, X.Xie, Y. Tags: human HSDL2 SCP-2 domian crystallization X-ray diffraction research communications Source Type: research

Structure of human saposin A at lysosomal pH
The saposins are essential cofactors for the normal lysosomal degradation of complex glycosphingolipids by acid hydrolase enzymes; defects in either saposin or hydrolase function lead to severe metabolic diseases. Saposin A (SapA) activates the enzyme β-galactocerebrosidase (GALC), which catalyzes the breakdown of β-d-galactocerebroside, the principal lipid component of myelin. SapA is known to bind lipids and detergents in a pH-dependent manner; this is accompanied by a striking transition from a `closed' to an `open' conformation. However, previous structures were determined at non-lysosomal pH. This work descr...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Hill, C.H.Read, R.J.Deane, J.E. Tags: saposin A lipid-transfer protein sphingolipid activator protein GALC research communications Source Type: research

Expression, purification, crystallization and crystallographic study of the Aspergillus terreus aromatic prenyltransferase AtaPT
Prenylated aromatics are produced by aromatic prenyltransferases during the secondary metabolism of bacteria, fungi and plants. The prenylation of nonprenylated precursors can lead to great chemical diversity and extensive biological properties. Aspergillus terreus aromatic prenyltransferase (AtaPT), which has recently been discovered and characterized, is such an enzyme and is responsible for the prenylation of various aromatic compounds. Here, recombinant AtaPT was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to a resolution of 1.71 Å and the crystal belonged to sp...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Gao, B.Chen, R.Liu, X.Dai, J.Sun, F. Tags: aromatic prenyltransferase Aspergillus terreus prenylation crystallization research communications Source Type: research

Structural characterization of the principal mRNA-export factor Mex67–Mtr2 from Chaetomium thermophilum
Members of the Mex67–Mtr2/NXF–NXT1 family are the principal mediators of the nuclear export of mRNA. Mex67/NXF1 has a modular structure based on four domains (RRM, LRR, NTF2-like and UBA) that are thought to be present across species, although the level of sequence conservation between organisms, especially in lower eukaryotes, is low. Here, the crystal structures of these domains from the thermophilic fungus Chaetomium thermophilum are presented together with small-angle X-ray scattering (SAXS) and in vitro RNA-binding data that indicate that, not withstanding the limited sequence conservation between differen...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Aibara, S.Valkov, E.Lamers, M.H.Dimitrova, L.Hurt, E.Stewart, M. Tags: nuclear transport Mex67 Chaetomium thermophilum RNA binding research communications Source Type: research

Crystallographic analysis of the Arabidopsis thaliana BAG5–calmodulin protein complex
Arabidopsis thaliana BAG5 (AtBAG5) belongs to the plant BAG (Bcl-2-associated athanogene) family that performs diverse functions ranging from growth and development to abiotic stress and senescence. BAG family members can act as nucleotide-exchange factors for heat-shock protein 70 (Hsp70) through binding of their evolutionarily conserved BAG domains to the Hsp70 ATPase domain, and thus may be involved in the regulation of chaperone-mediated protein folding in plants. AtBAG5 is distinguished from other family members by the presence of a unique IQ motif adjacent to the BAG domain; this motif is specific for calmodulin (CaM...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Cui, B.Fang, S.Xing, Y.Shen, Y.Yang, X. Tags: BAG5 calmodulin Arabidopsis thaliana research communications Source Type: research

Fusion-protein-assisted protein crystallization
Fusion proteins can be used directly in protein crystallization to assist crystallization in at least two different ways. In one approach, the `heterologous fusion-protein approach', the fusion partner can provide additional surface area to promote crystal contact formation. In another approach, the `fusion of interacting proteins approach', protein assemblies can be stabilized by covalently linking the interacting partners. The linker connecting the proteins plays different roles in the two applications: in the first approach a rigid linker is required to reduce conformational heterogeneity; in the second, conversely, a f...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Kobe, B.Ve, T.Williams, S.J. Tags: linker heterologous fusion-protein approach fusion of interacting proteins approach membrane-protein crystallization protein interactions recombinant fusion protein research communications Source Type: research

Batch crystallization of rhodopsin for structural dynamics using an X-ray free-electron laser
Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Wu, W.Nogly, P.Rheinberger, J.Kick, L.M.Gati, C.Nelson, G.Deupi, X.Standfuss, J.Schertler, G.Panneels, V. Tags: batch crystallization GPCR serial crystallography FEL dynamics research communications Source Type: research

Crystallographic analysis of a novel aldo-keto reductase from Thermotoga maritima in complex with NADP+
In this study, Tm1743 was overexpressed in Escherichia coli BL21(DE3) cells with an N-terminal His6 tag and was purified by Ni2+-chelating affinity and size-exclusion chromatography. Purified recombinant enzyme was incubated with its cofactor NADP+ and its substrate ethyl 2-oxo-4-phenylbutyrate (EOPB) for crystallization. Two X-ray diffraction data sets were collected at 2.0 and 1.7 Å resolution from dodecahedral crystals grown from samples containing Tm1743–NADP+–EOPB and Tm1743–NADP+, respectively. Both crystals belonged to space group P3121, with similar unit-cell parameters. However, in the re...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Hou, H.Li, R.Wang, X.Yuan, Z.Liu, X.Chen, Z.Xu, X. Tags: aldo-keto reductases thermostable Thermotoga maritima research communications Source Type: research

Use of dynamic light scattering and small-angle X-ray scattering to characterize new surfactants in solution conditions for membrane-protein crystallization
The structural and interactive properties of two novel hemifluorinated surfactants, F2H9-β-M and F4H5-β-M, the syntheses of which were based on the structure and hydrophobicity of the well known dodecyl-β-maltoside (DD-β-M), are described. The shape of their micellar assemblies was characterized by small-angle X-ray scattering and their intermicellar interactions in crystallizing conditions were measured by dynamic light scattering. Such information is essential for surfactant phase-diagram determination and membrane-protein crystallization. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Dahani, M.Barret, L.-A.Raynal, S.Jungas, C.Pernot, P.Polidori, A.Bonneté, F. Tags: membrane protein surfactant micelle scattering techniques phase diagram crystallization research communications Source Type: research

The MORPHEUS II protein crystallization screen
High-quality macromolecular crystals are a prerequisite for the process of protein structure determination by X-ray diffraction. Unfortunately, the relative yield of diffraction-quality crystals from crystallization experiments is often very low. In this context, innovative crystallization screen formulations are continuously being developed. In the past, MORPHEUS, a screen in which each condition integrates a mix of additives selected from the Protein Data Bank, a cryoprotectant and a buffer system, was developed. Here, MORPHEUS II, a follow-up to the original 96-condition initial screen, is described. Reagents were selec...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Gorrec, F. Tags: macromolecular crystallography protein crystallization crystallization screening crystallization additives heavy-atom derivatization MORPHEUS II research communications Source Type: research

Towards time-resolved serial crystallography in a microfluidic device
Serial methods for crystallography have the potential to enable dynamic structural studies of protein targets that have been resistant to single-crystal strategies. The use of serial data-collection strategies can circumvent challenges associated with radiation damage and repeated reaction initiation. This work utilizes a microfluidic crystallization platform for the serial time-resolved Laue diffraction analysis of macroscopic crystals of photoactive yellow protein (PYP). Reaction initiation was achieved via pulsed laser illumination, and the resultant electron-density difference maps clearly depict the expected pR1/pRE46...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Pawate, A.S.Šrajer, V.Schieferstein, J.Guha, S.Henning, R.Kosheleva, I.Schmidt, M.Ren, Z.Kenis, P.J.A.Perry, S.L. Tags: serial crystallography Laue diffraction time-resolved protein crystallography protein crystallization microfluidics photoactive yellow protein research communications Source Type: research

Do protein crystals nucleate within dense liquid clusters?
Protein-dense liquid clusters are regions of high protein concentration that have been observed in solutions of several proteins. The typical cluster size varies from several tens to several hundreds of nanometres and their volume fraction remains below 10−3 of the solution. According to the two-step mechanism of nucleation, the protein-rich clusters serve as locations for and precursors to the nucleation of protein crystals. While the two-step mechanism explained several unusual features of protein crystal nucleation kinetics, a direct observation of its validity for protein crystals has been lacking. Here, two inde...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Maes, D.Vorontsova, M.A.Potenza, M.A.C.Sanvito, T.Sleutel, M.Giglio, M.Vekilov, P.G. Tags: nucleation two-step mechanism protein-rich clusters crystallization research communications Source Type: research

Trace fluorescent labeling for protein crystallization
Fluorescence can be a powerful tool to aid in the crystallization of proteins. In the trace-labeling approach, the protein is covalently derivatized with a high-quantum-yield visible-wavelength fluorescent probe. The final probe concentration typically labels ≤0.20% of the protein molecules, which has been shown to not affect the crystal nucleation or diffraction quality. The labeled protein is then used in a plate-screening experiment in the usual manner. As the most densely packed state of the protein is the crystalline form, then crystals show as the brightest objects in the well under fluorescent illumination. A stu...
Source: Acta Crystallographica Section F - June 26, 2015 Category: Biochemistry Authors: Pusey, M.Barcena, J.Morris, M.Singhal, A.Yuan, Q.Ng, J. Tags: trace fluorescence labeling crystallization screening intensity research communications Source Type: research

Practical macromolecular cryocrystallography
Cryocrystallography is an indispensable technique that is routinely used for single-crystal X-ray diffraction data collection at temperatures near 100 K, where radiation damage is mitigated. Modern procedures and tools to cryoprotect and rapidly cool macromolecular crystals with a significant solvent fraction to below the glass-transition phase of water are reviewed. Reagents and methods to help prevent the stresses that damage crystals when flash-cooling are described. A method of using isopentane to assess whether cryogenic temperatures have been preserved when dismounting screened crystals is also presented. (Source: ...
Source: Acta Crystallographica Section F - June 1, 2015 Category: Biochemistry Authors: Pflugrath, J.W. Tags: cryocrystallography cryoprotectant flash-cooling crystal mounting annealing automounter high pressure research communications Source Type: research

Mode of binding of the antithyroid drug propylthiouracil to mammalian haem peroxidases. Erratum
The results in the article by Singh et al. [(2015), Acta Cryst. F71, 304–310] have been brought into question and the article is retracted. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 27, 2015 Category: Biochemistry Authors: Singh, R.P.Singh, A.Kushwaha, G.S.Singh, A.K.Kaur, P.Sharma, S.Singh, T.P. Tags: lactoperoxidase propylthiouracil distal haem side antithyroid drug retraction addenda and errata Source Type: research

Structure–activity correlations for three pyrido[2,3-d]pyrimidine antifolates binding to human and Pneumocystis carinii dihydrofolate reductase
To further define the interactions that enhance the selectivity of binding and to directly compare the binding of the most potent analogue {N6-methyl-N6-(3,4,5-trifluorophenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine; compound 26} in the series of bicyclic pyrido[2,3-d]pyrimidine analogues of piritrexim (PTX) with native human (h), Pneumocystis carinii (pc) and Pneumocystis jirovecii (pj) dihydrofolate reductase (DHFR) enzymes, the crystal structures of hDHFR complexed with N6-methyl-N6-(4-isopropylphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (compound 22), of hDHFR complexed with compound 26 and of pcDHFR complexed with N6...
Source: Acta Crystallographica Section F - May 27, 2015 Category: Biochemistry Authors: Cody, V.Pace, J.Namjoshi, O.A.Gangjee, A. Tags: dihydrofolate reductase Pneumocystis carinii pathogens antifolate inhibitors conformational analysis research communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of the two distinct types of zebrafish β2-microglobulin
β2-Microglobulin (β2m) noncovalently associates with the heavy chain of major histocompatibility complex class I (MHC I) molecules, which bind foreign antigen peptides to control the cytotoxic T lymphocyte (CTL) immune response. In contrast to mammals, there are distinct types of β2ms derived from two loci in a number of teleost species. In order to clarify the structures of the β2ms, the zebrafish (Danio rerio) β2ms Dare-β2m-I and Dare-β2m-II were expressed in Escherichia coli, purified and crystallized, and diffraction data were collected to 1.6 and 1.9 Å resolution, respectivel...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Chen, Z.Zhang, N.Lu, S.Tariq, M.Wang, J.Xia, C. Tags: zebrafish β 2-microglobulin MHC class I research communications Source Type: research

A preliminary X-ray study of 3-deoxy-d-manno-oct-2-ulosonic acid 8-phosphate phosphatase (YrbI) from Burkholderia pseudomallei
In this study, YrbI from Burkholderia pseudomallei, the causative agent of melioidosis, has been cloned, expressed, purified and crystallized. Synchrotron X-ray data were also collected to 2.25 Å resolution. The crystal belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 63.7, b = 97.5, c = 98.0 Å. A full structural determination is in progress to elucidate the structure–function relationship of this protein. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Park, J.Lee, D.Kim, M.-S.Kim, D.Y.Shin, D.H. Tags: 3-deoxy-d-manno-oct-2-ulosonic acid 8-phosphate phosphatase YrbI Burkholderia pseudomallei research communications Source Type: research

Expression, purification, crystallization and preliminary X-ray analysis of CttA, a putative cellulose-binding protein from Ruminococcus flavefaciens
A number of anaerobic microorganisms produce multi-modular, multi-enzyme complexes termed cellulosomes. These extracellular macromolecular nanomachines are designed for the efficient degradation of plant cell-wall carbohydrates to smaller sugars that are subsequently used as a source of carbon and energy. Cellulolytic strains from the rumens of mammals, such as Ruminococcus flavefaciens, have been shown to have one of the most complex cellulosomal systems known. Cellulosome assembly requires the binding of dockerin modules located in cellulosomal enzymes to cohesin modules located in a macromolecular scaffolding protein. O...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Venditto, I.Bule, P.Thompson, A.Sanchez-Weatherby, J.Sandy, J.Ferreira, L.M.A.Fontes, C.M.G.A.Najmudin, S. Tags: X-dockerin cellulosome CttA Ruminococcus flavefaciens cell surface attachment cellulose-binding protein research communications Source Type: research

Crystallization and preliminary crystallographic analysis of the major acid phosphatase from Legionella pneumophila
The major acid phosphatase from Legionella pneumophila (LpMAP) belongs to the histidine acid phosphatase superfamily. It contains the characteristic histidine acid phosphatase (HAP) sequence motif RHGXRXP responsible for the hydrolysis of a phosphoryl group from phosphate monoesters under acidic conditions. Here, the crystallization and preliminary X-ray analysis of crystals of LpMAP in the apo form and in complex with l-(+)-tartrate are described. By using the hanging-drop vapour-diffusion method, apo LpMAP and LpMAP–tartrate were crystallized in space group P21, with unit-cell parameters a = 91.50, b = 56.48, ...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Zhou, D.Pan, Y.Chen, X.Zhang, N.Ge, H. Tags: Legionella pneumophila major acid phosphatase histidine acid phosphatase research communications Source Type: research

Protein production, crystallization and preliminary crystallographic analysis of the four N-terminal immunoglobulin domains of Down syndrome cell adhesion molecule 1
In this study, eight different isforms of Dscam1 Ig1–4 have been cloned, overexpressed, purified to homogeneity and crystallized. X-ray data were collected to 1.9–4.0 Å resolution. These structures will provide the opportunity to perform extensive structural comparisons of different Dscam1 isoforms and provide insight into its specificity. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Cheng, L.Li, S.-A.Yu, Y.Chen, Q. Tags: Dscam1 RNA splicing homophilic dimer horseshoe configuration crystallization research communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of the Csu pili CsuC–CsuA/B chaperone–major subunit pre-assembly complex from Acinetobacter baumannii
The attachment of many Gram-negative pathogens to biotic and abiotic surfaces is mediated by fimbrial adhesins, which are assembled via the classical, alternative and archaic chaperone–usher (CU) pathways. The archaic CU fimbrial adhesins have the widest phylogenetic distribution, yet very little is known about their structure and mechanism of assembly. To elucidate the biogenesis of archaic CU systems, structural analysis of the Csu fimbriae, which are used by Acinetobacter baumannii to form stable biofilms and cause nosocomial infection, was focused on. The major fimbriae subunit CsuA/B complexed with the CsuC chap...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Pakharukova, N.Tuittila, M.Paavilainen, S.Zavialov, A. Tags: chaperone – usher pathway archaic fimbriae biofilm Acinetobacter baumannii assembly adhesion research communications Source Type: research

Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of NAD synthetase from methicillin-resistant Staphylococcus aureus
Staphylococcus aureus is an important human and animal pathogen that causes a wide range of infections. The prevalence of multidrug-resistant S. aureus strains in both hospital and community settings makes it imperative to characterize new drug targets to combat S. aureus infections. In this context, enzymes involved in NAD metabolism and synthesis are significant drug targets as NAD is a central player in several cellular processes. NAD synthetase catalyzes the last step in the biosynthesis of nicotinamide adenine dinucleotide, making it a crucial intermediate enzyme linked to the biosynthesis of several amino acids, puri...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Arbade, G.K.Srivastava, S.K. Tags: NAD synthetase Staphylococcus aureus research communications Source Type: research

Purification, crystallization and preliminary X-ray diffraction analysis of 3-ketoacyl-CoA thiolase A1887 from Ralstonia eutropha H16
The gene product of A1887 from Ralstonia eutropha (ReH16_A1887) has been annotated as a 3-ketoacyl-CoA thiolase, an enzyme that catalyzes the fourth step of β-oxidation degradative pathways by converting 3-ketoacyl-CoA to acyl-CoA. ReH16_A1887 was overexpressed and purified to homogeneity by affinity and size-exclusion chromatography. The degradative thiolase activity of the purified ReH16_A1887 was measured and enzyme-kinetic parameters for the protein were obtained, with Km, Vmax and kcat values of 158 µM, 32 mM min−1 and 5 × 106 s−1, respectively. The ReH16_A1887 protein was crystall...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Kim, J.Kim, K.-J. Tags: 3-ketoacyl-CoA thiolase Ralstonia eutropha β -oxidation degradative pathway research communications Source Type: research

Structure of the catalytic domain of Mre11 from Chaetomium thermophilum
Together with the Rad50 ATPase, the Mre11 nuclease forms an evolutionarily conserved protein complex that plays a central role in the repair of DNA double-strand breaks (DSBs). Mre11–Rad50 detects and processes DNA ends, and has functions in the tethering as well as the signalling of DSBs. The Mre11 dimer can bind one or two DNA ends or hairpins, and processes DNA endonucleolytically as well as exonucleolytically in the 3′-to-5′ direction. Here, the crystal structure of the Mre11 catalytic domain dimer from Chaetomium thermophilum (CtMre11CD) is reported. CtMre11CD crystals diffracted to 2.8 Å res...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Seifert, F.U.Lammens, K.Hopfner, K.-P. Tags: Mre11 nuclease MRN complex research communications Source Type: research

Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora
Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P212121 and P1211 and diffracted to ∼1.65 Å resolution. Co...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Molitor, C.Mauracher, S.G.Rompel, A. Tags: aurone synthase latent proenzyme polyphenol oxidase liquid – liquid phase separation polyoxometalate research communications Source Type: research

Structure of the MarR family protein Rv0880 from Mycobacterium tuberculosis
Rv0880 from the pathogen Mycobacterium tuberculosis is classified as a MarR family protein in the Pfam database. It consists of 143 amino acids and has an isoelectric point of 10.9. Crystals of Rv0880 belonged to space group P1, with unit-cell parameters a = 54.97, b = 69.60, c = 70.32 Å, α = 103.71, β = 111.06, γ = 105.83°. The structure of the MarR family transcription regulator Rv0880 was solved at a resolution of 2.0 Å with an Rcryst and Rfree of 21.2 and 24.9%, respectively. The dimeric structure resembles that of other MarR proteins, with each subunit comprising a winged helix&nda...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Gao, Y.-R.Feng, N.Chen, T.Li, D.-F.Bi, L.-J. Tags: MarR Mycobacterium tuberculosis transcription factor MarR family protein research communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of the CRISPR–Cas RNA-silencing Cmr complex
Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNA (crRNA) and CRISPR-associated (Cas) proteins constitute a prokaryotic adaptive immune system (CRISPR–Cas system) that targets and degrades invading genetic elements. The type III-B CRISPR–Cas Cmr complex, composed of the six Cas proteins (Cmr1–Cmr6) and a crRNA, captures and cleaves RNA complementary to the crRNA guide sequence. Here, a Cmr1-deficient functional Cmr (CmrΔ1) complex composed of Pyrococcus furiosus Cmr2–Cmr3, Archaeoglobus fulgidus Cmr4–Cmr5–Cmr6 and the 39-mer P. furiosus 7.01-crRNA was pr...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Osawa, T.Inanaga, H.Numata, T. Tags: Cmr complex CRISPR-Cas crRNA research communications Source Type: research

Purification, crystallization and X-ray crystallographic studies of a Bacillus cereus MepR-like transcription factor, BC0657
In this study, a Bacillus cereus MepR-like transcription regulator, BC0657, was crystallized. The BC0657 crystals diffracted to 2.05 Å resolution and belonged to either space group P6222 or P6422, with unit-cell parameters a = 110.57, b = 110.57, c = 67.29 Å. There was one molecule per asymmetric unit. Future comparative structural studies on BC0657 would extend knowledge of ligand-induced transcriptional regulatory mechanisms in the MarR family and would make a significant contribution to the design of antibiotic drugs against multidrug-resistant bacteria. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Cho, M.U.Kim, M.I.Hong, M. Tags: MepR transcription factor Bacillus cereus research communications Source Type: research

X-ray structure of cyanide-bound bovine heart cytochrome c oxidase in the fully oxidized state at 2.0 Å resolution
The X-ray structure of cyanide-bound bovine heart cytochrome c oxidase in the fully oxidized state was determined at 2.0 Å resolution. The structure reveals that the peroxide that bridges the two metals in the fully oxidized state is replaced by a cyanide ion bound in a nearly symmetric end-on fashion without significantly changing the protein conformation outside the two metal sites. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Yano, N.Muramoto, K.Mochizuki, M.Shinzawa-Itoh, K.Yamashita, E.Yoshikawa, S.Tsukihara, T. Tags: membrane protein cytochrome c oxidase research communications Source Type: research

Structure of Chlamydomonas reinhardtii THB1, a group 1 truncated hemoglobin with a rare histidine–lysine heme ligation
THB1 is one of several group 1 truncated hemoglobins (TrHb1s) encoded in the genome of the unicellular green alga Chlamydomonas reinhardtii. THB1 expression is under the control of NIT2, the master regulator of nitrate assimilation, which also controls the expression of the only nitrate reductase in the cell, NIT1. In vitro and physiological evidence suggests that THB1 converts the nitric oxide generated by NIT1 into nitrate. To aid in the elucidation of the function and mechanism of THB1, the structure of the protein was solved in the ferric state. THB1 resembles other TrHb1s, but also exhibits distinct features associate...
Source: Acta Crystallographica Section F - May 20, 2015 Category: Biochemistry Authors: Rice, S.L.Boucher, L.E.Schlessman, J.L.Preimesberger, M.R.Bosch, J.Lecomte, J.T.J. Tags: 2/2 hemoglobin distal ligand nitric oxide dioxygenase nitrogen metabolism lysine coordination research communications Source Type: research

Crystallization of interleukin-18 for structure-based inhibitor design
In this study, surface-entropy reduction (SER) and rational protein design were employed to facilitate the crystallization of hIL-18. The results provide an excellent platform for structure-based drug design. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 20, 2015 Category: Biochemistry Authors: Krumm, B.Meng, X.Xiang, Y.Deng, J. Tags: cytokines interleukin-18 immune defense surface-entropy reduction research communications Source Type: research

The structure of a contact-dependent growth-inhibition (CDI) immunity protein from Neisseria meningitidis MC58
Contact-dependent growth inhibition (CDI) is an important mechanism of intercellular competition between neighboring Gram-negative bacteria. CDI systems encode large surface-exposed CdiA effector proteins that carry a variety of C-terminal toxin domains (CdiA-CTs). All CDI+ bacteria also produce CdiI immunity proteins that specifically bind to the cognate CdiA-CT and neutralize its toxin activity to prevent auto-inhibition. Here, the X-ray crystal structure of a CdiI immunity protein from Neisseria meningitidis MC58 is presented at 1.45 Å resolution. The CdiI protein has structural homology to the Whirly family of ...
Source: Acta Crystallographica Section F - May 20, 2015 Category: Biochemistry Authors: Tan, K.Johnson, P.M.Stols, L.Boubion, B.Eschenfeldt, W.Babnigg, G.Hayes, C.S.Joachimiak, A.Goulding, C.W. Tags: contact-dependent growth inhibition CdiA-CT toxin domain CdiI immunity protein – immunity protein complex Neisseria meningitidis docking studies research communications Source Type: research

Expression, crystallization and X-ray diffraction analysis of a complex between B7-H6, a tumor cell ligand for the natural cytotoxicity receptor NKp30, and an inhibitory antibody
Natural killer (NK) cells are essential components of the innate immune response to tumors and viral infections. In humans, the activating natural cytotoxicity receptor NKp30 plays a major role in NK cell-mediated tumor cell lysis. NKp30 recognizes the cell-surface protein B7-H6, which is expressed on tumor, but not healthy, cells. A mouse monoclonal antibody (17B1.3) against human B7-H6 has been developed (Kd = 0.2 µM) to investigate NKp30-mediated NK cell activation and to target tumors expressing B7-H6. Surprisingly, 17B1.3 blocks NK cell activation without interfering with the binding of B7-H6 to NKp30. Underst...
Source: Acta Crystallographica Section F - May 20, 2015 Category: Biochemistry Authors: Xu, X.Li, Y.Gauthier, L.Chen, Q.Vivier, E.Mariuzza, R.A. Tags: NK cell NKp30 B7-H6 antibody research communications Source Type: research

Functional characterization of heat-shock protein 90 from Oryza sativa and crystal structure of its N-terminal domain
In this study, biochemical characterization of an Hsp90 protein from rice (Oryza sativa; OsHsp90) has been performed and the crystal structure of its N-terminal domain (OsHsp90-NTD) was determined. The binding of OsHsp90 to its substrate ATP and the inhibitor 17-AAG was studied by fluorescence spectroscopy. The protein also exhibited a weak ATPase activity. The crystal structure of OsHsp90-NTD was solved in complex with the nonhydrolyzable ATP analogue AMPPCP at 3.1 Å resolution. The domain was crystallized by cross-seeding with crystals of the N-terminal domain of Hsp90 from Dictyostelium discoideum, which shares ...
Source: Acta Crystallographica Section F - May 20, 2015 Category: Biochemistry Authors: Raman, S.Suguna, K. Tags: Hsp90 Oryza sativa 17-AAG ATPase activity cross-seeding research communications Source Type: research

High-resolution crystal structure of the leucine-rich repeat domain of the human tumour suppressor PP32A (ANP32A)
Acidic leucine-rich nuclear phosphoprotein 32A (PP32A) is a tumour suppressor whose expression is altered in many cancers. It is an apoptotic enhancer that stimulates apoptosome-mediated caspase activation and also forms part of a complex involved in caspase-independent apoptosis (the SET complex). Crystals of a fragment of human PP32A corresponding to the leucine-rich repeat domain, a widespread motif suitable for protein–protein interactions, have been obtained. The structure has been refined to 1.56 Å resolution. This domain was previously solved at 2.4 and 2.69 Å resolution (PDB entries 2je0 and 2...
Source: Acta Crystallographica Section F - May 20, 2015 Category: Biochemistry Authors: Zamora-Caballero, S.Šiaučiunaite-Gaubard, L.Bravo, J. Tags: PP32A LRR domain structure tumour suppressor SET complex research communications Source Type: research

Expression, purification and crystallization of a family 55 β-1,3-glucanase from Chaetomium thermophilum
A β-1,3-glucanase from the thermophilic fungus Chaetomium thermophilum was overexpressed in Pichia pastoris, purified and crystallized in the presence of 1.8 M sodium/potassium phosphate pH 6.8 as a precipitant. Data to 2.0 Å resolution were collected in-house at 293 K from a single crystal. The crystal was found to belong to space group P21, with unit-cell parameters a = 64.1, b = 85.8, c = 68.5 Å, β = 93.1° and one molecule in the asymmetric unit. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 20, 2015 Category: Biochemistry Authors: Papageorgiou, A.C.Li, D. Tags: cellulose glucanase in situ diffraction seeding thermophilic fungus research communications Source Type: research

Purification, crystallization and preliminary X-ray diffraction analysis of the Escherichia coli common pilus chaperone EcpB
Pili are key cell-surface components that allow the attachment of bacteria to both biological and abiotic solid surfaces, whilst also mediating interactions between themselves. In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone–usher (CU) pathway that plays a major role in both early biofilm formation and host-cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under de...
Source: Acta Crystallographica Section F - May 20, 2015 Category: Biochemistry Authors: Garnett, J.A.Diallo, M.Matthews, S.J. Tags: pili Ecp Escherichia coli research communications Source Type: research

Overexpression, purification, crystallization and preliminary X-ray diffraction of the nisin resistance protein from Streptococcus agalactiae
Nisin is a 34-amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics. Nisin displays a high bactericidal activity against various Gram-positive bacteria, including some human-pathogenic strains. However, there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome. The encoded protein, NSR, cleaves off the last six amino acids of nisin, thereby reducing its bactericidal efficacy. An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH1. The protein w...
Source: Acta Crystallographica Section F - May 20, 2015 Category: Biochemistry Authors: Khosa, S.Hoeppner, A.Kleinschrodt, D.Smits, S.H.J. Tags: lantibiotic nisin resistance immunity lipoprotein Lactococcus lactis research communications Source Type: research

Crystallographic study of a novel DNA-binding domain of human HLTF involved in the template-switching pathway to avoid the replication arrest caused by DNA damage
In this study, the HIRAN domain of human HLTF was successfully crystallized. The crystals belonged to space group P41212 or P43212, with unit-cell parameters a = b = 130.0, c = 150.1 Å. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 20, 2015 Category: Biochemistry Authors: Ikegaya, Y.Hara, K.Hishiki, A.Yokoyama, H.Hashimoto, H. Tags: HIRAN domain HLTF template switching DNA-binding domain research communications Source Type: research

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of CofB, the minor pilin subunit of CFA/III from human enterotoxigenic Escherichia coli
In this study, constructs of wild-type CofB with an N-terminal truncation and the corresponding SeMet derivative were cloned, expressed, purified and crystallized. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 103.97, c = 364.57 Å for the wild-type construct and a = b = 103.47, c = 362.08 Å for the SeMet-derivatized form. Although the diffraction quality of these crystals was initially very poor, dehydration of the crystals substantially improved the resolution limit from ∼4.0 to ∼2.0 Å. The initial phase was solved by the single-wavelength anom...
Source: Acta Crystallographica Section F - May 20, 2015 Category: Biochemistry Authors: Kawahara, K.Oki, H.Fukakusa, S.Maruno, T.Kobayashi, Y.Motooka, D.Taniguchi, T.Honda, T.Iida, T.Nakamura, S.Ohkubo, T. Tags: ETEC type IVb pili minor pilin CFA/III CofB research communications Source Type: research

Multiple crystal forms of N,N′-diacetylchitobiose deacetylase from Pyrococcus furiosus
Native N,N′-diacetylchitobiose deacetylase from Pyrococcus furiosus (Pf-Dac) and its selenomethionine derivative (Se-Pf-Dac) were crystallized and analyzed in the presence and absence of cadmium ion. The four crystal structures fell into three different crystal-packing groups, with the cadmium-free Pf-Dac and Se-Pf-Dac belonging to the same space group, with homologous unit-cell parameters. The crystal structures in the presence of cadmium contained distorted octahedral cadmium complexes coordinated by three chlorides, two O atoms and an S or Se atom from the N-terminal methionine or selenomethionine, respectively. T...
Source: Acta Crystallographica Section F - May 20, 2015 Category: Biochemistry Authors: Nakamura, T.Niiyama, M.Hashimoto, W.Ida, K.Abe, M.Morita, J.Uegaki, K. Tags: N,N ′ -diacetylchitobiose deacetylase crystal-packing interaction selenomethionine cadmium complex research communications Source Type: research

Structure of human dual-specificity phosphatase 7, a potential cancer drug target
Human dual-specificity phosphatase 7 (DUSP7/Pyst2) is a 320-residue protein that belongs to the mitogen-activated protein kinase phosphatase (MKP) subfamily of dual-specificity phosphatases. Although its precise biological function is still not fully understood, previous reports have demonstrated that DUSP7 is overexpressed in myeloid leukemia and other malignancies. Therefore, there is interest in developing DUSP7 inhibitors as potential therapeutic agents, especially for cancer. Here, the purification, crystallization and structure determination of the catalytic domain of DUSP7 (Ser141–Ser289/C232S) at 1.67 &Arin...
Source: Acta Crystallographica Section F - May 20, 2015 Category: Biochemistry Authors: Lountos, G.T.Austin, B.P.Tropea, J.E.Waugh, D.S. Tags: dual-specificity phosphatase DUSP DUSP7 MAP kinase phosphatase PTP research communications Source Type: research

The structure of hookworm platelet inhibitor (HPI), a CAP superfamily member from Ancylostoma caninum
Secreted protein components of hookworm species include a number of representatives of the cysteine-rich/antigen 5/pathogenesis-related 1 (CAP) protein family known as Ancylostoma-secreted proteins (ASPs). Some of these have been considered as candidate antigens for the development of vaccines against hookworms. The functions of most CAP superfamily members are poorly understood, but one form, the hookworm platelet inhibitor (HPI), has been isolated as a putative antagonist of the platelet integrins αIIbβ3 and α2β1. Here, the crystal structure of HPI is described and its structural features are examin...
Source: Acta Crystallographica Section F - May 20, 2015 Category: Biochemistry Authors: Ma, D.Francischetti, I.M.B.Ribeiro, J.M.C.Andersen, J.F. Tags: antigen V hemostasis parasitism research communications Source Type: research

Iron superoxide dismutases in eukaryotic pathogens: new insights from Apicomplexa and Trypanosoma structures
This report presents the structures of three iron-dependent superoxide dismutases (FeSODs) from Trypanosoma cruzi, Leishmania major and Babesia bovis. Comparison with existing structures from Plasmodium and other trypanosome isoforms shows a very conserved overall fold with subtle differences. In particular, structural data suggest that B. bovis FeSOD may display similar resistance to peroxynitrite-mediated inactivation via an intramolecular electron-transfer pathway as previously described in T. cruzi FeSOD isoform B, thus providing valuable information for structure-based drug design. Furthermore, lysine-acetylation resu...
Source: Acta Crystallographica Section F - May 7, 2015 Category: Biochemistry Authors: Phan, I.Q.H.Davies, D.R.Moretti, N.S.Shanmugam, D.Cestari, I.Anupama, A.Fairman, J.W.Edwards, T.E.Stuart, K.Schenkman, S.Myler, P.J. Tags: iron superoxide dismutase Trypanosoma Apicomplexa research communications Source Type: research

Macromolecular crystallography and what it can contribute to antiparasite drug discovery
(Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - April 24, 2015 Category: Biochemistry Authors: Hunter, W.N.Weiss, M.S. Tags: molecular parasitology special issues editorial Source Type: research

Structures of prostaglandin F synthase from the protozoa Leishmania major and Trypanosoma cruzi with NADP
The crystal structures of prostaglandin F synthase (PGF) from both Leishmania major and Trypanosoma cruzi with and without their cofactor NADP have been determined to resolutions of 2.6 Å for T. cruzi PGF, 1.25 Å for T. cruzi PGF with NADP, 1.6 Å for L. major PGF and 1.8 Å for L. major PGF with NADP. These structures were determined by molecular replacement to a final R factor of less than 18.6% (Rfree of less than 22.9%). PGF in the infectious protozoa L. major and T. cruzi is a potential therapeutic target. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - April 21, 2015 Category: Biochemistry Authors: Moen, S.O.Fairman, J.W.Barnes, S.R.Sullivan, A.Nakazawa-Hewitt, S.Van Voorhis, W.C.Staker, B.L.Lorimer, D.D.Myler, P.J.Edwards, T.E. Tags: prostaglandin infectious diseases leishmaniasis Chagas disease SSGCID research communications Source Type: research

Structure of Plasmodium falciparum orotate phosphoribosyltransferase with autologous inhibitory protein–protein interactions
The most severe form of malaria is caused by the obligate parasite Plasmodium falciparum. Orotate phosphoribosyltransferase (OPRTase) is the fifth enzyme in the de novo pyrimidine-synthesis pathway in the parasite, which lacks salvage pathways. Among all of the malaria de novo pyrimidine-biosynthesis enzymes, the structure of P. falciparum OPRTase (PfOPRTase) was the only one unavailable until now. PfOPRTase that could be crystallized was obtained after some low-complexity sequences were removed. Four catalytic dimers were seen in the asymmetic unit (a total of eight polypeptides). In addition to revealing unique amino aci...
Source: Acta Crystallographica Section F - April 21, 2015 Category: Biochemistry Authors: Kumar, S.Krishnamoorthy, K.Mudeppa, D.G.Rathod, P.K. Tags: pyrimidine de novo salvage PRPP OMP prodrugs peptide inhibition research communications Source Type: research

Structure of an ADP-ribosylation factor, ARF1, from Entamoeba histolytica bound to Mg2+–GDP
Entamoeba histolytica is the etiological agent of amebiasis, a diarrheal disease which causes amoebic liver abscesses and amoebic colitis. Approximately 50 million people are infected worldwide with E. histolytica. With only 10% of infected people developing symptomatic amebiasis, there are still an estimated 100 000 deaths each year. Because of the emergence of resistant strains of the parasite, it is necessary to find a treatment which would be a proper response to this challenge. ADP-ribosylation factor (ARF) is a member of the ARF family of GTP-binding proteins. These proteins are ubiquitous in eukaryotic cells; they...
Source: Acta Crystallographica Section F - April 21, 2015 Category: Biochemistry Authors: Serbzhinskiy, D.A.Clifton, M.C.Sankaran, B.Staker, B.L.Edwards, T.E.Myler, P.J. Tags: GDP-ribosylation factor small GTPase ARFA1 structural genomics Seattle Structural Genomics Center for Infectious Disease SSGCID signaling protein research communications Source Type: research

Identification and structure solution of fragment hits against kinetoplastid N-myristoyltransferase
Trypanosoma brucei N-myristoyltransferase (TbNMT) is an attractive therapeutic target for the treatment of human African trypanosomiasis. Pyrazole sulfonamide (DDD85646), a potent inhibitor of TbNMT, has been identified in previous studies; however, poor central nervous system exposure restricts its use to the haemolymphatic form (stage 1) of the disease. In order to identify new chemical matter, a fragment screen was carried out by ligand-observed NMR spectroscopy, identifying hits that occupy the DDD85646 binding site. Crystal structures of hits from this assay have been obtained in complex with the closely related NMT f...
Source: Acta Crystallographica Section F - April 21, 2015 Category: Biochemistry Authors: Robinson, D.A.Wyatt, P.G. Tags: N-myristoyltransferase Trypanosoma brucei African trypanosomiasis research communications Source Type: research