Production, crystallization and X-ray diffraction analysis of a complex between a fragment of the TssM T6SS protein and a camelid nanobody
The type VI secretion system (T6SS) is a machine evolved by Gram-negative bacteria to deliver toxin effectors into target bacterial or eukaryotic cells. The T6SS is functionally and structurally similar to the contractile tail of the Myoviridae family of bacteriophages and can be viewed as a syringe anchored to the bacterial membrane by a transenvelope complex. The membrane complex is composed of three proteins: the TssM and TssL inner membrane components and the TssJ outer membrane lipoprotein. The TssM protein is central as it interacts with both TssL and TssJ, therefore linking the membranes. Using controlled trypsinoly...
Source: Acta Crystallographica Section F - February 19, 2015 Category: Biochemistry Authors: Nguyen, V.S.Spinelli, S.Desmyter, A.Le, T.T.H.Kellenberger, C.Cascales, E.Cambillau, C.Roussel, A. Tags: type VI secretion system TssM nb25 research communications Source Type: research

Crystallization and preliminary X-ray crystallographic analysis of human myotubularin-related protein 1
In this study, human MTMR1 was overexpressed in Escherichia coli, purified and crystallized at 277 K using polyethylene glycol 20 000 as a precipitant. Diffraction data were collected to 2.0 Å resolution using synchrotron radiation. The crystals belonged to space group P1, with unit-cell parameters a = 67.219, b = 96.587, c = 97.581 Å, α = 87.597, β = 86.072, γ = 77.327°. Assuming the presence of four molecules in the asymmetric unit, the calculated Matthews coefficient value was 2.61 Å3 Da−1 and the corresponding solvent content was 52.9%. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - February 19, 2015 Category: Biochemistry Authors: Bong, S.M.Yang, S.W.Choi, J.-W.Kim, S.J.Lee, B.I. Tags: myotubularin MTMR phosphatidylinositol phosphatase research communications Source Type: research

Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of Haemophilus influenzae BamD and BamCD complex
In this study, the recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of H. influenzae BamD and BamCD complex are reported. The genes encoding BamC and BamD were cloned into a pET vector and expressed in E. coli. Affinity, ion-exchange and gel-filtration chromatography were used to obtain high-purity protein for further crystallographic characterization. Using the hanging-drop vapour-diffusion technique, BamD and BamCD protein crystals of suitable size were obtained using protein concentrations of 70 and 50 mg ml−1, respectively. Preliminary X-ray diffraction analysis...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Lei, J.Cai, X.Ma, X.Zhang, L.Li, Y.Dong, X.St Geme III, J.Meng, G. Tags: Haemophilus influenzae BamD BamCD complex research communications Source Type: research

Purification, crystallization and preliminary X-ray diffraction analysis of a soluble variant of the monoglyceride lipase Yju3p from the yeast Saccharomyces cerevisiae
The protein Yju3p is the orthologue of monoglyceride lipases in the yeast Saccharomyces cerevisiae. A soluble variant of this lipase termed s-Yju3p (38.3 kDa) was generated and purified to homogeneity by affinity and size-exclusion chromatography. s-Yju3p was crystallized in a vapour-diffusion setup at 293 K and a complete data set was collected to 2.4 Å resolution. The crystal form was orthorhombic (space group P212121), with unit-cell parameters a = 77.2, b = 108.6, c = 167.7 Å. The asymmetric unit contained four molecules with a solvent content of 46.4%. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Rengachari, S.Aschauer, P.Sturm, C.Oberer, M. Tags: s-Yju3p monoacylglycerol lipase monoglyceride lipase research communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of a putative carbon–carbon bond hydrolase from Mycobacterium abscessus 103
The PhlG protein from Mycobacterium abscessus 103 (mPhlG), which shares 30% sequence identity with phloretin hydrolase from Eubacterium ramulus and 38% sequence identity with 2,4-diacetylphloroglucinol hydrolase from Pseudomonas fluorescens Pf-5, is a putative carbon–carbon bond hydrolase. Here, the expression, purification and crystallization of mPhlG are reported. Crystals were obtained using a precipitant consisting of 100 mM citric acid pH 5.0, 1.0 M lithium chloride, 8%(w/v) polyethylene glycol 6000. The crystals diffracted to 1.87 Å resolution and belonged to space group P21, with unit-cell paramete...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Zhang, Z.Jiang, Y.-L.Wu, Y.He, Y.-X. Tags: PhlG carbon – carbon bond hydrolase Mycobacterium abscessus 103 research communications Source Type: research

Crystallization of two operator complexes from the Vibrio cholerae HigBA2 toxin–antitoxin module
The HigA2 antitoxin and the HigBA2 toxin–antitoxin complex from Vibrio cholerae were crystallized in complex with their operator box. Screening of 22 different DNA duplexes led to two crystal forms of HigA2 complexes and one crystal form of a HigBA2 complex. Crystals of HigA2 in complex with a 17 bp DNA duplex belong to space group P3221, with unit-cell parameters a = b = 94.0, c = 123.7 Å, and diffract to 2.3 Å resolution. The second form corresponding to HigA2 in complex with a 19 bp duplex belong to space group P43212 and only diffract to 3.45 Å resolution. Crystals of the HigBA2 toxin&...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Hadži, S.Garcia-Pino, A.Gerdes, K.Lah, J.Loris, R. Tags: persistence toxin – antitoxin module protein DNA complex macromolecular complex research communications Source Type: research

Purification, identification and preliminary crystallographic studies of an allergenic protein from Solanum melongena
Solanum melongena (eggplant), a member of the Solanaceae family, is a widely cultivated vegetable crop and is commonly used as a food throughout the world. Allergic reactions caused by members of this family are well known. However, mechanistic analyses to understand their molecular basis have not been adequately explored. In order to address this issue, the 7S vicilin protein (SM80.1) of size 45 kDa was purified from seeds of S. melongena by ammonium sulfate fractionation and size-exclusion chromatography. Significant homology of SM80.1 to an allergy-related protein from S. lycopersicum was identified through a BLAST se...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Jain, A.Salunke, D.M. Tags: Solanaceae Solanum melongena allergy research communications Source Type: research

Crystallization and preliminary X-ray diffraction studies of the family 54 carbohydrate-binding module from laminarinase (β-1,3-glucanase) Lic16A of Clostridium thermocellum
The crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding module (CBM) from laminarinase Lic16A of the hyperthermophilic anaerobic bacterium Clostridium thermocellum (ctCBM54) are reported. Recombinant ctCBM54 was prepared using an Escherichia coli/pQE30 overexpression system and was crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The crystals belonged to space group P6322, with unit-cell parameters a = b = 130.15, c = 131.05 Å. The three-dimensional structure of ctCBM54 will pro...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Kislitsyn, Y.A.Samygina, V.R.Dvortsov, I.A.Lunina, N.A.Kuranova, I.P.Velikodvorskaya, G.A. Tags: carbohydrate-binding module β -1,3-glucanase Clostridium thermocellum Lic16A research communications Source Type: research

Cloning, refolding, purification and preliminary crystallographic analysis of the sensory domain of the Campylobacter chemoreceptor for multiple ligands (CcmL)
A periplasmic sensory domain of the Campylobacter jejuni chemoreceptor for multiple ligands (CcmL) has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 as a precipitating agent. A complete data set was collected to 1.3 Å resolution using cryocooling conditions and synchrotron radiation. The crystals belonged to space group P21, with unit-cell parameters a = 42.6, b = 138.0, c = 49.0 Å, β = 94.3°. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Machuca, M.A.Liu, Y.C.Beckham, S.A.Roujeinikova, A. Tags: Campylobacter jejuni chemotaxis transducer-like proteins methyl-accepting proteins research communications Source Type: research

Crystallization and preliminary X-ray crystallographic studies of dipeptidyl peptidase 11 from Porphyromonas gingivalis
Dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) preferentially cleaves substrate peptides with Asp and Glu at the P1 position [NH2–P2–P1(Asp/Glu)–P1′–P2′…]. For crystallographic studies, PgDPP11 was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 1.82 Å resolution were collected from an orthorhombic crystal form belonging to space group C2221, with unit-cell parameters a = 99.33, b = 103.60, c = 177.33 Å. Structural analysis by the multi-wavelength anomalous diffra...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Sakamoto, Y.Suzuki, Y.Iizuka, I.Tateoka, C.Roppongi, S.Fujimoto, M.Gouda, H.Nonaka, T.Ogasawara, W.Tanaka, N. Tags: dipeptidyl peptidase DPP11 Porphyromonas gingivalis research communications Source Type: research

Structural analysis of human dual-specificity phosphatase 22 complexed with a phosphotyrosine-like substrate
4-Nitrophenyl phosphate (p-nitrophenyl phosphate, pNPP) is widely used as a small molecule phosphotyrosine-like substrate in activity assays for protein tyrosine phosphatases. It is a colorless substrate that upon hydrolysis is converted to a yellow 4-nitrophenolate ion that can be monitored by absorbance at 405 nm. Therefore, the pNPP assay has been widely adopted as a quick and simple method to assess phosphatase activity and is also commonly used in assays to screen for inhibitors. Here, the first crystal structure is presented of a dual-specificity phosphatase, human dual-specificity phosphatase 22 (DUSP22), in compl...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Lountos, G.T.Cherry, S.Tropea, J.E.Waugh, D.S. Tags: dual-specificity phosphatase 22 4-nitrophenyl phosphate research communications Source Type: research

Crystallization and preliminary X-ray crystallographic analysis of Z-ring-associated protein (ZapD) from Escherichia coli
Bacterial cytokinesis is accomplished by the Z-ring, which is a polymeric structure that includes the tubulin homologue FtsZ at the division site. ZapD, a Z-ring-associated protein, directly binds to FtsZ and stabilizes the polymerization of FtsZ to form a stable Z-ring during cytokinesis. Structural analysis of ZapD from Escherichia coli was performed to investigate the mechanism of ZapD-mediated FtsZ stabilization and polymerization. ZapD was crystallized using a reservoir solution consisting of 1.5 M lithium sulfate, 0.1 M HEPES pH 7.8, 2%(v/v) polyethylene glycol 400. X-ray diffraction data were collected to 2.95...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Son, S.H.Lee, H.H. Tags: bacterial cytokinesis FtsZ ZapD research communications Source Type: research

Crystallization and preliminary crystallographic analysis of the putative sugar-binding protein Msmeg_0515 (AgaE) from Mycobacterium smegmatis
Msmeg_0515, a gene from Mycobacterium smegmatis strain 155 encoding the ligand-binding domain, AgaE, of a putative ABC sugar transporter system, has been cloned into a pET-28a vector system, overexpressed in Escherichia coli and purified. The truncated protein lacking the first 27 residues, which correspond to a N-terminal signal sequence, was crystallized using the sitting-drop vapour-diffusion technique. The crystals of this protein diffracted to 1.48 Å resolution and belonged to space group P212121, with unit-cell parameters a = 64.06, b = 69.26, c = 100.74 Å, α = β = γ = 90° and wit...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Almourfi, F.M.Rodgers, H.F.Sedelnikova, S.E.Baker, P.J. Tags: Msmeg_0515 AgaE Mycobacterium smegmatis research communications Source Type: research

Preliminary X-ray crystallographic analysis of an engineered variant of human chimera-type galectin-3 with a shortened N-terminal domain
How lectins translate sugar-encoded information into cellular effects not only depends on glycan recognition. Other domains of the protein can contribute to the functional profile of a lectin. Human galectin-3 (Gal-3), an adhesion/growth-regulatory galectin, is composed of three different domains and is thus called a chimera-type protein. In addition to the carbohydrate-recognition domain, this lectin encompasses an N-terminal domain consisting of a peptide harbouring two phosphorylation sites and nine non-triple-helical collagen-like repeats. This region plays an as yet structurally undefined role in Gal-3 aggregation and...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Flores-Ibarra, A.Ruiz, F.M.Vértesy, S.André, S.Gabius, H.-J.Romero, A. Tags: collagen galectin-3 glycome lipopolysaccharide research communications Source Type: research

Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN
The adaptor protein LGN interacts via the N-terminal domain comprising eight tetratricopeptide-repeat (TPR) motifs with its partner proteins mInsc, NuMA, Frmpd1 and Frmpd4 in a mutually exclusive manner. Here, the crystal structure of the LGN TPR domain in complex with human Frmpd4 is described at 1.5 Å resolution. In the complex, the LGN-binding region of Frmpd4 (amino-acid residues 990–1011) adopts an extended structure that runs antiparallel to LGN along the concave surface of the superhelix formed by the TPR motifs. Comparison with the previously determined structures of the LGN–Frmpd1, LGN–mI...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Takayanagi, H.Yuzawa, S.Sumimoto, H. Tags: LGN Frmpd4 research communications Source Type: research

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the effector-interaction domain of the resistance protein RGA5-A from Oryza sativa L. japonica
RGA5-A, a component of the Pia resistance-protein complex (RGA4/RGA5-A) from Oryza sativa L. japonica, has the ability to interact physically with the effector protein AVR-Pia from Magnaporthe oryzae via its effector-interaction domain RGA5-A_S. The interaction between RGA5-A and AVR-Pia relieves the repression of RGA4, leading to AVR-independent cell death by the freed RGA4. To further understand the details of this interaction, the effector-interaction domain RGA5-A_S was expressed in Escherichia coli and purified to homogeneity. The purified recombinant protein His-RGA5-A_S was successfully crystallized using the sittin...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Huang, D.Zhang, Y.Zhao, Y.Liu, J.Peng, Y.-L. Tags: Oryza sativa L. japonica resistance protein RGA5-A research communications Source Type: research

Structural analysis of ibuprofen binding to human adipocyte fatty-acid binding protein (FABP4)
Inhibition of human adipocyte fatty-acid binding protein (FABP4) has been proposed as a treatment for type 2 diabetes, fatty liver disease and atherosclerosis. However, FABP4 displays a naturally low selectivity towards hydrophobic ligands, leading to the possibility of side effects arising from cross-inhibition of other FABP isoforms. In a search for structural determinants of ligand-binding selectivity, the binding of FABP4 towards a group of small molecules structurally related to the nonsteroidal anti-inflammatory drug ibuprofen was analyzed through X-ray crystallography. Several specific hydrophobic interactions are s...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: González, J.M.Fisher, S.Z. Tags: human fatty-acid binding protein adipocyte aP2 ALBP iLBP FABP4 ibuprofen research communications Source Type: research

The role of flexibility and molecular shape in the crystallization of proteins by surface mutagenesis
Proteins are dynamic systems and interact with their environment. The analysis of crystal contacts in the most accurately determined protein structures (d
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Devedjiev, Y.D. Tags: protein crystallization static disorder conformational entropy surface mutagenesis molecular shape research communications Source Type: research

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of tomato β-galactosidase 4
Plant β-galactosidases play important roles in carbohydrate-reserve mobilization, cell-wall expansion and degradation, and turnover of signalling molecules during ripening. Tomato β-galactosidase 4 (TBG4) not only has β-galactosidase activity but also has exo-β-(1,4)-galactanase activity, and prefers β-(1,4)-galactans longer than pentamers as its substrates; most other β-galactosidases only have the former activity. Recombinant TBG4 protein expressed in the yeast Pichia pastoris was crystallized by the sitting-drop vapour-diffusion method using PEG 10 000 as a precipitant. The crystals belon...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Eda, M.Ishimaru, M.Tada, T. Tags: β -galactosidase Solanum lycopersicum plant cell wall TBG4 research communications Source Type: research

Expression, crystallization and preliminary crystallographic data analysis of VioD, a hydroxylase in the violacein-biosynthesis pathway
Violacein, a natural purple secondary metabolite, is sequentially biosynthesized by five enzymes in the following pathway: VioA–VioB–VioE–VioD–VioC. VioD, a flavin-dependent oxygenase, catalyzes the hydroxylation of the intermediate product prodeoxyviolaceinic acid (PVA) at the 5-position of one indole ring to yield proviolacein. In vitro biochemical data have revealed this process, but the catalytic mechanism still remains largely unclear. Here, the cloning, expression, purification, crystallization and diffraction of VioD are reported. Crystals of VioD diffracted to 1.7 Å resolution and be...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Ran, T.Gao, M.Wei, Q.He, J.Tang, L.Wang, W.Xu, D. Tags: violacein VioD research communications Source Type: research

Structure of the apo anti-influenza CH65 Fab
Influenza viruses remain a persistent challenge to human health owing to their inherent ability to evade the immune response by antigenic drift. However, the discovery of broadly neutralizing antibodies (bnAbs) against divergent viruses has sparked renewed interest in a universal influenza vaccine and novel therapeutic opportunities. Here, a crystal structure at 1.70 Å resolution is presented of the Fab of the human antibody CH65, which has broad neutralizing activity against a range of seasonal H1 isolates. Previous studies proposed that affinity maturation of this antibody lineage pre-organizes the complementarit...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Lee, P.S.Arnell, A.J.Wilson, I.A. Tags: influenza antibody adaptive immunity affinity maturation research communications Source Type: research

Expression, purification and preliminary crystallographic analysis of a haem-utilizing protein, HutX, from Vibrio cholerae
Vibrio cholerae, the causative agent of cholera, has developed a variety of mechanisms to obtain the limited-availability iron from human hosts. One important method for iron acquisition is through haem-uptake systems. Although the transport of haem has been widely studied, the fate of haem once it enters the cytoplasm remains an open question. Here, preliminary X-ray crystallographic analysis was performed on HutX, a member of the conserved haem-utilization operon from V. cholerae strain N16961. The crystals of HutX were found to belong to the orthorhombic space group C2221, with unit-cell parameters a = 50.1, b = 169.0, ...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Su, T.Chi, K.Wang, K.Guo, L.Huang, Y. Tags: haem uptake iron acquisition Vibrio cholerae research communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of Paenibacillus barcinonensis xylanase 10C containing the CBM22-1–CBM22-2 tandem
This study represents the first structure containing two contiguous CBM22 modules, which will contribute to a better understanding of the role that this multiplicity plays in fine-tuning substrate affinity. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Sainz-Polo, M.Á.González, B.Pastor, F.I.J.Sanz-Aparicio, J. Tags: carbohydrate-binding domain CBM22 Paenibacillus bacterial xylanase xylan-binding domain research communications Source Type: research

Preliminary X-ray analysis of the binding domain of the soybean vacuolar sorting receptor complexed with a sorting determinant of a seed storage protein
β-Conglycinin is a major seed storage protein in soybeans, which are an important source of protein. The major subunits (α, α′ and β) of β-conglycinin are sorted to protein-storage vacuoles in seed cells. Vacuolar sorting receptor (VSR) is an integral membrane protein that recognizes the sorting determinant of vacuolar proteins, including β-conglycinin, and regulates their sorting process. Vacuolar sorting determinants of the α′ and β subunits of β-conglycinin exist in their C-terminal peptides. Here, the preliminary X-ray diffraction analysis of the binding domai...
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Maruyama, N.Goshi, T.Sugiyama, S.Niiyama, M.Adachi, H.Takano, K.Murakami, S.Inoue, T.Mori, Y.Matsumura, H.Mikami, B. Tags: soybean seed storage protein β -conglycinin vacuolar sorting receptor research communications Source Type: research

Latest methods of fluorescence-based protein crystal identification
Successful protein crystallization screening experiments are dependent upon the experimenter being able to identify positive outcomes. The introduction of fluorescence techniques has brought a powerful and versatile tool to the aid of the crystal grower. Trace fluorescent labeling, in which a fluorescent probe is covalently bound to a subpopulation ( (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - January 28, 2015 Category: Biochemistry Authors: Meyer, A.Betzel, C.Pusey, M. Tags: protein crystal identification fluorescence research communications Source Type: research

The three-dimensional structure of the cellobiohydrolase Cel7A from Aspergillus fumigatus at 1.5 Å resolution
The enzymatic degradation of plant cell-wall cellulose is central to many industrial processes, including second-generation biofuel production. Key players in this deconstruction are the fungal cellobiohydrolases (CBHs), notably those from family GH7 of the carbohydrate-active enzymes (CAZY) database, which are generally known as CBHI enzymes. Here, three-dimensional structures are reported of the Aspergillus fumigatus CBHI Cel7A solved in uncomplexed and disaccharide-bound forms at resolutions of 1.8 and 1.5 Å, respectively. The product complex with a disaccharide in the +1 and +2 subsites adds to the growing thre...
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Moroz, O.V.Maranta, M.Shaghasi, T.Harris, P.V.Wilson, K.S.Davies, G.J. Tags: cellulase biofuels carbohydrate-active enzyme thermal stability research communications Source Type: research

Cloning, expression, refolding, purification and preliminary crystallographic analysis of the sensory domain of the Campylobacter chemoreceptor for aspartate A (CcaA)
In this study, the sensory domain of the C. jejuni chemoreceptor for aspartate A (CcaA) has been expressed in Escherichia coli and purified from inclusion bodies. The urea-denatured protein was refolded and then crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as a precipitating agent. A complete data set has been collected to 1.4 Å resolution using cryocooling conditions and synchrotron radiation. The crystals belonged to space group P1, with unit-cell parameters a = 39.3, b = 43.3, c = 50.9 Å, α = 92.5, β = 111.4, γ = 114.7°. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - December 24, 2014 Category: Biochemistry Authors: Machuca, M.A.Liu, Y.C.Roujeinikova, A. Tags: Campylobacter jejuni chemotaxis transducer-like proteins methyl-accepting proteins research communications Source Type: research

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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