Protein expression, characterization, crystallization and preliminary X-ray crystallographic analysis of a Fic protein from Clostridium difficile
Fic domains in proteins are found in abundance in nature from the simplest prokaryotes to animals. Interestingly, Fic domains found in two virulence factors of Gram-negative bacteria have recently been demonstrated to catalyse the transfer of the AMP moiety from ATP to small host GTPases. This post-translational modification has attracted considerable interest and a role for adenylylation in pathology and physiology is emerging. This work was aimed at the structural characterization of a newly identified Fic protein of the Gram-positive bacterium Clostridium difficile. A constitutively active inhibitory helix mutant of C. ...
Source: Acta Crystallographica Section F - May 25, 2014 Category: Biochemistry Authors: Welner, D.Dedic, E.van Leeuwen, H.C.Kuijper, E.Bjerrum, M.J.Østergaard, O.Jørgensen, R. Tags: Fic protein Clostridium difficile adenylylation crystallization communications Source Type: research

Crystallization and preliminary X-ray characterization of the 2,4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP
The enzyme 2,4′-dihydroxyacetophenone dioxygenase (or DAD) catalyses the conversion of 2,4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C—C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits each containing nonhaem iron and its sequence suggests that it belongs to the cupin family of dioxygenases. By the use of limited chymotrypsinol...
Source: Acta Crystallographica Section F - May 25, 2014 Category: Biochemistry Authors: Beaven, G.Bowyer, A.Erskine, P.Wood, S.P.McCoy, A.Coates, L.Keegan, R.Lebedev, A.Hopper, D.J.Kaderbhai, M.A.Cooper, J.B. Tags: 2,4 ′ -dihydroxyacetophenone dioxygenase limited proteolysis Alcaligenes sp. 4HAP crystallization communications Source Type: research

Preliminary time-of-flight neutron diffraction studies of Escherichia coli ABC transport receptor phosphate-binding protein at the Protein Crystallography Station
Inorganic phosphate is an essential molecule for all known life. Organisms have developed many mechanisms to ensure an adequate supply, even in low-phosphate conditions. In prokaryotes phosphate transport is instigated by the phosphate-binding protein (PBP), the initial receptor for the ATP-binding cassette (ABC) phosphate transporter. In the crystal structure of the PBP–phosphate complex, the phosphate is completely desolvated and sequestered in a deep cleft and is bound by 13 hydrogen bonds: 12 to protein NH and OH donor groups and one to a carboxylate acceptor group. The carboxylate plays a key recognition role by...
Source: Acta Crystallographica Section F - May 25, 2014 Category: Biochemistry Authors: Sippel, K.H.Bacik, J.Quiocho, F.A.Fisher, S.Z. Tags: phosphate-binding protein Escherichia coli phosphate-binding protein (PBP) crystallization communications Source Type: research

Preliminary joint X-ray and neutron protein crystallographic studies of ecDHFR complexed with folate and NADP+
A crystal of Escherichia coli dihydrofolate reductase (ecDHFR) complexed with folate and NADP+ of 4 × 1.3 × 0.7 mm (3.6 mm3) in size was obtained by sequential application of microseeding and macroseeding. A neutron diffraction data set was collected to 2.0 Å resolution using the IMAGINE diffractometer at the High Flux Isotope Reactor within Oak Ridge National Laboratory. A 1.6 Å resolution X-ray data set was also collected from a smaller crystal at room temperature. The neutron and X-ray data were used together for joint refinement of the ecDHFR–folate–NADP+ ternary-complex stru...
Source: Acta Crystallographica Section F - May 25, 2014 Category: Biochemistry Authors: Wan, Q.Kovalevsky, A.Y.Wilson, M.A.Bennett, B.C.Langan, P.Dealwis, C. Tags: Escherichia coli dihydrofolate reductase protonation state crystallization communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of the peripheral light-harvesting complex LH2 from Marichromatium purpuratum
LH2 from the purple photosynthetic bacterium Marichromatium (formerly known as Chromatium) purpuratum is an integral membrane pigment–protein complex that is involved in harvesting light energy and transferring it to the LH1–RC `core' complex. The purified LH2 complex was crystallized using the sitting-drop vapour-diffusion method at 294 K. The crystals diffracted to a resolution of 6 Å using synchrotron radiation and belonged to the tetragonal space group I4, with unit-cell parameters a = b = 109.36, c = 80.45 Å. The data appeared to be twinned, producing apparent diffraction symmetry I422. T...
Source: Acta Crystallographica Section F - May 24, 2014 Category: Biochemistry Authors: Cranston, L.J.Roszak, A.W.Cogdell, R.J. Tags: Marichromatium purpuratum LH2 peripheral light-harvesting complex crystallization communications Source Type: research

Preliminary crystallographic studies of a Schistosoma mansoni antigen (Sm21.7) dynein light-chain (DLC) domain
Schistosomiasis is an inflammatory chronic disease that represents a major health problem in tropical and subtropical countries. The drug of choice for treatment, praziquantel, is effective in killing adult worms but fails to kill immature forms and prevent reinfection. One prominent antigen candidate for an anti-schistosomiasis vaccine is the protein Sm21.7 (184 amino-acid residues) from Schistosoma mansoni, a tegumental protein capable of reducing the worm burden in a murine immunization model. In the present work, the Sm21.7 gene was cloned and expressed in Escherichia coli and the full-length protein was purified to ho...
Source: Acta Crystallographica Section F - May 24, 2014 Category: Biochemistry Authors: Costa, M.A.F.Rodrigues, F.T.G.Chagas, B.C.A.Rezende, C.M.F.Goes, A.M.Nagem, R.A.P. Tags: Sm21.7 dynein light-chain domain Schistosoma mansoni crystallization communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of (R)-carbonyl reductase from Candida parapsilosis
The NADH-dependent (R)-carbonyl reductase from Candida parapsilosis (RCR) catalyzes the asymmetric reduction of 2-hydroxyacetophenone (HAP) to produce (R)-1-phenyl-1,2-ethanediol [(R)-PED], which is used as a versatile building block for the synthesis of pharmaceuticals and fine chemicals. To gain insight into the catalytic mechanism, the structures of complexes of RCR with ligands, including the coenzyme, are important. Here, the recombinant RCR protein was expressed and purified in Escherichia coli and was crystallized in the presence of NAD+. The crystals, which belonged to the orthorhombic space group P212121, with uni...
Source: Acta Crystallographica Section F - May 24, 2014 Category: Biochemistry Authors: Wang, S.Nie, Y.Yan, X.Ko, T.-P.Huang, C.-H.Chan, H.-C.Guo, R.-T.Xiao, R. Tags: (R)-carbonyl reductase Candida parapsilosis crystallization communications Source Type: research

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp6
Enpp (ectonucleotide phosphodiesterase/pyrophosphatase) 6 is a membrane-bound glycoprotein that hydrolyzes choline-containing compounds such as lysophosphatidylcholine and glycerophosphorylcholine, and presumably participates in choline metabolism. The catalytic domain of mouse Enpp6 was expressed in HEK293T cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 1.8 Å resolution. The crystal belonged to space group P1, with unit-cell parameters a = 63.7, b = 68.8, c = 69.7 Å, α = 60.6, β = 87.0, γ = 68.1°. Assuming th...
Source: Acta Crystallographica Section F - May 24, 2014 Category: Biochemistry Authors: Morita, J.Kato, K.Mihara, E.Ishitani, R.Takagi, J.Nishimasu, H.Aoki, J.Nureki, O. Tags: Enpp6 choline N-glycosylation crystallization communications Source Type: research

Crystallization and preliminary X-ray crystallographic analysis of a bacterial Asn-transamidosome
In this study, the Asn-transamidosome from Pseudomonas aeruginosa was reconstructed and crystallized by mixing purified GatCAB complex, AspRS and tRNAAsn. The crystal of the Asn-transamidosome belonged to space group P21, with unit-cell parameters a = 93.3, b = 186.0, c = 287.8 Å, β = 93.3°, and diffracted to 3.73 Å resolution. Preliminary X-ray crystallographic analysis showed that the asymmetric unit contained two Asn-transamidosomes, each composed of two GatCABs, one AspRS dimer and two tRNAAsns, indicating that the construction of the current Asn-transamidosome differs from that of Thermus ther...
Source: Acta Crystallographica Section F - May 24, 2014 Category: Biochemistry Authors: Suzuki, T.Yamashita, K.Tanaka, Y.Tanaka, I.Yao, M. Tags: transamidosome aminoacyl-tRNA AspRS GatCAB Asn-tRNAAsn Pseudomonas aeruginosa crystallization communications Source Type: research

Expression, purification, crystallization and preliminary X-ray analysis of the HER3–9E12 Fab complex
9E12 is a fully human immunoglobulin G1/κ monoclonal antibody that is specific for the epidermal growth factor receptor 3 (HER3), the overexpression of which has been detected in many tumour types and is associated with poor survival outcomes. To date, knowledge of the molecular mechanism for targeted antibodies that directly inhibit HER3 signalling is limited. Because knowledge of such therapeutic antibodies would help basic immunological therapeutics, structural insights into the HER3–9E12 Fab complex are important. Recombinant human HER3 and Fab fragments of the 9E12 antibody were cloned, expressed and cryst...
Source: Acta Crystallographica Section F - May 24, 2014 Category: Biochemistry Authors: He, K.Huang, A.Huang, Y.Takeda, H. Tags: HER3 targeted therapy crystallization communications Source Type: research

Crystallization and preliminary X-ray crystallographic studies of dehydroascorbate reductase (DHAR) from Oryza sativa L. japonica
In this study, recombinant OsDHAR protein was purified and crystallized using the sitting-drop vapour-diffusion method at pH 8.0 and 298 K. Plate-shaped crystals were obtained using 0.15 M potassium bromide, 30%(w/v) PEG MME 2000 as a precipitant, and the crystals diffracted to a resolution of 1.9 Å on beamline 5C at the Pohang Accelerator Laboratory. The X-ray diffraction data indicated that the crystal contained one OsDHAR molecule in the asymmetric unit and belonged to space group P21 with unit-cell parameters a = 47.03, b = 48.38, c = 51.83 Å, β = 107.41°. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 24, 2014 Category: Biochemistry Authors: Do, H.Kim, I.-S.Kim, Y.-S.Shin, S.-Y.Kim, J.-J.Mok, J.-E.Park, S.-I.Wi, A.R.Park, H.Kim, H.-W.Yoon, H.-S.Lee, J.H. Tags: ascorbate dehydroascorbate reductase Oryza sativa L. japonica crystallization communications Source Type: research

Structure of transportin SR2, a karyopherin involved in human disease, in complex with Ran
Transportin SR2 (TRN-SR2) is a β-type karyopherin responsible for the nuclear import of specific cargoes, including serine/arginine-rich splicing factors. The protein has been implicated in a variety of human diseases, including HIV infection, primary biliary cirrhosis and limb-girdle muscular dystrophy 1F. Towards understanding its molecular mechanism, a 2.9 Å resolution crystal structure of human TRN-SR2 complexed with the small GTPase Ran has been determined. TRN-SR2 is composed of 20 α-helical HEAT repeats forming a solenoid-like fold. The first nine repeats form a `cradle' for the binding of RanGTP,...
Source: Acta Crystallographica Section F - May 24, 2014 Category: Biochemistry Authors: Tsirkone, V.G.Beutels, K.G.Demeulemeester, J.Debyser, Z.Christ, F.Strelkov, S.V. Tags: nuclear import molecular mechanism transportin SR2 structural communications Source Type: research

High-resolution crystal structures of two crystal forms of human cyclophilin D in complex with PEG 400 molecules
Cyclophilin D (CypD) is a key mitochondrial target for amyloid-β-induced mitochondrial and synaptic dysfunction and is considered a potential drug target for Alzheimer's disease. The high-resolution crystal structures of primitive orthorhombic (CypD-o) and primitive tetragonal (CypD-t) forms have been determined to 1.45 and 0.85 Å resolution, respectively, and are nearly identical structurally. Although an isomorphous structure of CypD-t has previously been reported, the structure reported here was determined at atomic resolution, while CypD-o represents a new crystal form for this protein. In addition, each c...
Source: Acta Crystallographica Section F - May 24, 2014 Category: Biochemistry Authors: Valasani, K.R.Carlson, E.A.Battaile, K.P.Bisson, A.Wang, C.Lovell, S.Yan, S.S. Tags: cyclophilin D structural communications Source Type: research

Structures and binding specificity of galactose- and mannose-binding lectins from champedak: differences from jackfruit lectins
Galactose-binding and mannose-binding lectins from the champedak fruit, which is native to South-east Asia, exhibit useful potential clinical applications. The specificity of the two lectins for their respective ligands allows the detection of potential cancer biomarkers and monitoring of the glycosylated state of proteins in human serum and/or urine. To fully understand and expand the use of these natural proteins, their complete sequences and crystal structures are presented here, together with details of sugar binding. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 24, 2014 Category: Biochemistry Authors: Gabrielsen, M.Abdul-Rahman, P.S.Othman, S.Hashim, O.H.Cogdell, R.J. Tags: champedak galactose-binding lectin mannose-binding lectin structural communications Source Type: research

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of a ferredoxin/flavodoxin-NADP(H) oxidoreductase (Bc0385) from Bacillus cereus
Ferredoxin/flavodoxin-NADP(H) oxidoreductases (FNRs) are key enzymes involved in catalysing electron transfer between ferredoxins/flavodoxins and NAD(P)H/NAD(P)+. In Bacillus cereus there are three genes that may encode FNRs, and the Bc0385 FNR has been cloned, overexpressed, purified and successfully crystallized in its NADPH/NADP+-free form. Diffraction data have been collected to 2.5 Å resolution from crystals belonging to the orthorhombic space group P21212, with unit-cell parameters a = 57.2, b = 164.3, c = 95.0 Å, containing two FNR molecules in the asymmetric unit. The structure of the Bc0385 FNR has...
Source: Acta Crystallographica Section F - May 10, 2014 Category: Biochemistry Authors: Skråmo, S.Hersleth, H.-P.Hammerstad, M.Andersson, K.K.Røhr, Å.K. Tags: ferredoxin/flavodoxin-NADP(H) oxidoreductase flavoprotein Bacillus cereus electron transfer crystallization communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of Hypocrea jecorina Cel7A in two new crystal forms
Cel7A (previously known as cellobiohydrolase I) from Hypocrea jecorina was crystallized in two crystalline forms, neither of which have been previously reported. Both forms co-crystallize under the same crystallization conditions. The first crystal form belonged to space group C2, with unit-cell parameters a = 152.5, b = 44.9, c = 57.6 Å, β = 101.2°, and diffracted X-rays to 1.5 Å resolution. The second crystal form belonged to space group P6322, with unit-cell parameters a = b ≃ 155, c ≃ 138 Å, and diffracted X-rays to 2.5 Å resolution. The crystals were obtained using full-len...
Source: Acta Crystallographica Section F - May 10, 2014 Category: Biochemistry Authors: Bodenheimer, A.M.Cuneo, M.J.Swartz, P.D.He, J.O'Neill, H.M.Myles, D.A.A.Evans, B.R.Meilleur, F. Tags: Cel7A Hypocrea jecorina crystallization communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of proximal thread matrix protein 1 (PTMP1) from Mytilus galloprovincialis
In order to deal with the dynamic ocean environment, blue mussels adhere to various surfaces via their collagenous byssal threads. PTMP1 (proximal thread matrix protein 1) is one identified matrix protein residing in the proximal thread and is capable of collagen binding. Its sequence comprises two von Willebrand factor type A-like repeats. In order to characterize the structure and domain architecture of PTMP1, recombinant protein was crystallized by vapour diffusion. The obtained crystals diffracted to 1.95 Å resolution and belonged to space group P21, with unit-cell parameters a = 62.0, b = 62.3, c = 122.6 &Ar...
Source: Acta Crystallographica Section F - May 10, 2014 Category: Biochemistry Authors: Suhre, M.H.Scheibel, T.Steegborn, C.Gertz, M. Tags: Mytilus galloprovincialis mussel byssus byssal thread matrix protein proximal thread matrix protein 1 crystallization communications Source Type: research

Characterization, crystallization and preliminary X-ray crystallographic analysis of the Uba5 fragment necessary for high-efficiency activation of Ufm1
In this study, Uba5 adenylation domains with different C-terminal region lengths were cloned, expressed and purified. The results of an in vitro truncation assay suggest that Uba5 residues 57–363 comprise the minimal fragment required for the high-efficiency activation of Ufm1. Crystallization of Uba5 residues 57–363 was performed at 277 K using PEG 3350 as the precipitant, and crystals optimized by microseeding diffracted to 2.95 Å resolution, with unit-cell parameters a = b = 97.66, c = 144.83 Å, α = β = 90, γ = 120°. There is one molecule in the asymmetric unit; the Mat...
Source: Acta Crystallographica Section F - May 10, 2014 Category: Biochemistry Authors: Xie, S. Tags: Uba5 adenylation domain crystallization communications Source Type: research

Crystallization and preliminary X-ray crystallographic studies of the ArsI C–As lyase from Thermomonospora curvata
Arsenic is a ubiquitous and carcinogenic environmental element that enters the biosphere primarily from geochemical sources, but also through anthropogenic activities. Microorganisms play an important role in the arsenic biogeochemical cycle by biotransformation of inorganic arsenic into organic arsenicals and vice versa. ArsI is a microbial nonheme ferrous-dependent dioxygenase that transforms toxic methylarsonous acid to the less toxic inorganic arsenite by C–As bond cleavage. An ArsI ortholog from the thermophilic bacterium Thermomonospora curvata was expressed, purified and crystallized. The crystals diffracted t...
Source: Acta Crystallographica Section F - May 10, 2014 Category: Biochemistry Authors: Nadar, S.V.Yoshinaga, M.Kandavelu, P.Sankaran, B.Rosen, B.P. Tags: ArsI Thermomonospora curvata arsenic crystallization communications Source Type: research

Crystallization and preliminary crystallographic analysis of defective pollen wall (DPW) protein from Oryza sativa
The defective pollen wall (dpw) gene of Oryza sativa encodes a fatty acid reductase (DPW) which plays important roles in primary fatty alcohol synthesis. DPW catalyzes the synthesis of 1-hexadecanol. The enzyme shows a higher specificity for palmitoyl-ACP than for palmitoyl-CoA as the substrate, and can only use NADPH as the cofactor. To gain an understanding of the molecular mechanism underlying the reaction catalyzed by DPW, the gene encoding DPW without the N-terminal 80 amino acids (DPWΔ80) was cloned into pET-28a vector and was overexpressed in Escherichia coli. DPWΔ80 was purified to homogeneity and scree...
Source: Acta Crystallographica Section F - May 10, 2014 Category: Biochemistry Authors: Wang, W.Ma, Y.Suo, Y.Yan, L.Zhang, D.Miao, C. Tags: DPW Oryza sativa fatty acid reductase crystallization communications Source Type: research

Overproduction, purification, crystallization and preliminary X-ray characterization of the family 46 carbohydrate-binding module (CBM46) of endo-β-1,4-glucanase B (CelB) from Bacillus halodurans
Plant cell-wall polysaccharides offer an abundant energy source utilized by many microorganisms, thus playing a central role in carbon recycling. Aerobic microorganisms secrete carbohydrate-active enzymes (CAZymes) that catabolize this composite structure, comprising cellulose, hemicellulose and lignin, into simple compounds such as glucose. Carbohydrate-binding modules (CBMs) enhance the efficacy of associated CAZYmes. They are organized into families based on primary-sequence homology. CBM family 46 contains more than 40 different members, but has yet to be fully characterized. Here, a recombinant derivative of the C-ter...
Source: Acta Crystallographica Section F - May 10, 2014 Category: Biochemistry Authors: Venditto, I.Santos, H.Ferreira, L.M.A.Sakka, K.Fontes, C.M.G.A.Najmudin, S. Tags: endo- β -1,4-glucanase carbohydrate-binding module family 46 Bacillus halodurans crystallization communications Source Type: research

Expression, purification and preliminary crystallographic analysis of Mycobacterium tuberculosis CysQ, a phosphatase involved in sulfur metabolism
CysQ is part of the sulfur-activation pathway that dephosphorylates 3′-phosphoadenosine 5′-monophosphate (PAP) to regenerate adenosine 5′-monophosphate (AMP) and free phosphate. PAP is the product of sulfate-transfer reactions from sulfotransferases that use the universal sulfate donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS). In some organisms PAP is also the product of PAPS reductases that reduce sulfate from PAPS to sulfite. CysQ from Mycobacterium tuberculosis, which plays an important role in the biosynthesis of sulfated glycoconjugates, was successfully purified and crystallized in 2...
Source: Acta Crystallographica Section F - May 10, 2014 Category: Biochemistry Authors: Erickson, A.Sarsam, R.Fisher, A.J. Tags: CysQ monophosphatase Mycobacterium tuberculosis Rv2131c ′ ,5 -bisphosphate nucleotidase -phosphoadenosine 5 -phosphate PAP crystallization communications Source Type: research

Crystallization and preliminary X-ray crystallographic analysis of carboxyl-terminal region 4 of SigR from Streptomyces coelicolor A3(2)
Full-length SigR from Streptomyces coelicolor A3(2) was overexpressed in Escherichia coli, purified and submitted to crystallization trials using either polyethylene glycol 3350 or 4000 as a precipitant. X-ray diffraction data were collected to 2.60 Å resolution under cryoconditions using synchrotron X-rays. The crystal packs in space group P43212, with unit-cell parameters a = b = 42.14, c = 102.02 Å. According to the Matthews coefficient, the crystal asymmetric unit cannot contain the full-length protein. Molecular replacement with the known structures of region 2 and region 4 as independent search models...
Source: Acta Crystallographica Section F - May 10, 2014 Category: Biochemistry Authors: Kim, K.Y.Kim, S.Park, J.K.Song, H.Park, S. Tags: ECF sigma factor SigR Streptomyces coelicolor A3(2) crystallization communications Source Type: research

Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the bovine CD8αα–BoLA-2*02201 complex
In order to clarify the structural characteristics of the bovine MHC class I molecule (BoLA-I) complexed with CD8αα (CD8αα–BoLA-I), bovine CD8αα, BoLA-I (BoLA-2*02201) and β2m were expressed and purified, and were then assembled with a peptide derived from Foot-and-mouth disease virus (FMDV-VP1YY9) and crystallized. The crystal diffracted to 1.7 Å resolution and belonged to space group P21, with unit-cell parameters a = 53.9, b = 103.8, c = 61.8 Å, α = γ = 90, β = 96°. The asymmetric unit contained one complex, with a Matthews coefficient o...
Source: Acta Crystallographica Section F - May 10, 2014 Category: Biochemistry Authors: Wang, Z.Chen, R.Tariq, M.Jiang, B.Chen, Z.Xia, C. Tags: bovine MHC class I molecule BoLA-I CD8 α crystallization communications Source Type: research

Crystallization and preliminary crystallographic studies of PotA, a membrane-associated ATPase of the spermidine-preferential uptake system in Thermotoga maritima
A membrane-associated ATPase, PotA, is a component of the spermidine-preferential uptake system in prokaryotes that plays an important role in normal cell growth by regulating the cellular polyamine concentration. No three-dimensional structures of membrane-associated ATPases in polyamine-uptake systems have been determined to date. Here, the crystallization and preliminary X-ray diffraction analysis of PotA from Thermotoga maritima are reported. Diffraction data were collected and processed to 2.7 Å resolution from both native and selenomethionine-labelled crystals. Preliminary crystallographic analysis revealed t...
Source: Acta Crystallographica Section F - May 10, 2014 Category: Biochemistry Authors: Sugiyama, S.Kashiwagi, K.Kakinouchi, K.Tomitori, H.Kanai, K.Murata, M.Adachi, H.Matsumura, H.Takano, K.Murakami, S.Inoue, T.Mori, Y.Igarashi, K. Tags: PotA polyamine-transport system ABC transporter Thermotoga maritima crystallization communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of AntE, a crotonyl-CoA carboxylase/reductase from Streptomyces sp. NRRL 2288
AntE from Streptomyces sp. NRRL 2288 is a crotonyl-CoA carboxylase/reductase that catalyzes the reductive carboxylation of various α,β-unsaturated acyl-CoAs to provide the building block at the C7 position for antimycin A biosynthesis. Recombinant AntE expressed in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group I222 or I212121, with unit-cell parameters a = 76.4, b = 96.7, c = 129.6 Å, α = β = γ = 90.0°. A diffraction data set was collected at the KEK Photon Factory to 2.29 Å resolution. (Source: Acta Crysta...
Source: Acta Crystallographica Section F - May 10, 2014 Category: Biochemistry Authors: Zhang, L.Chen, J.Mori, T.Yan, Y.Liu, W.Abe, I. Tags: AntE crotonyl-CoA carboxylase/reductase polyketide Streptomyces sp. NRRL 2288 crystallization communications Source Type: research

Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones
CsyB from Aspergillus oryzae is a novel type III polyketide synthase that catalyzes the formation of csypyrone B1 [4-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)butyric acid] from fatty acyl-CoA, malonyl-CoA and acetoacetyl-CoA. Recombinant CsyB expressed in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space P21, with unit-cell parameters a = 70.0, b = 104.8, c = 73.5 Å, β = 114.4°. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 10, 2014 Category: Biochemistry Authors: Yang, D.Mori, T.Matsui, T.Hashimoto, M.Morita, H.Fujii, I.Abe, I. Tags: Aspergillus oryzae chalcone synthase csypyrone B1 CsyB polyketide synthase crystallization communications Source Type: research

GlmU (N-acetylglucosamine-1-phosphate uridyltransferase) bound to three magnesium ions and ATP at the active site
N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme exclusive to prokaryotes, belongs to the family of sugar nucleotidyltransferases (SNTs). The enzyme binds GlcNAc-1-P and UTP, and catalyzes a uridyltransfer reaction to synthesize UDP-GlcNAc, an important precursor for cell-wall biosynthesis. As many SNTs are known to utilize a broad range of substrates, substrate specificity in GlmU was probed using biochemical and structural studies. The enzymatic assays reported here demonstrate that GlmU is specific for its natural substrates UTP and GlcNAc-1-P. The crystal structure of GlmU bound to ATP an...
Source: Acta Crystallographica Section F - May 10, 2014 Category: Biochemistry Authors: Vithani, N.Bais, V.Prakash, B. Tags: ATP binding enzyme inhibitor substrate specificity sugar nucleotidyltransferase structural communications Source Type: research

Structure of a His170Tyr mutant of thermostable pNPPase from Geobacillus stearothermophilus
This study potentially offers a molecular basis for both investigation of the catalytic mechanism and thermostable protein engineering. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 9, 2014 Category: Biochemistry Authors: Shen, T.Guo, Z.Ji, C. Tags: thermostability TpNPPase directed evolution random mutagenesis structural communications Source Type: research

Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD
Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosynthesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegma...
Source: Acta Crystallographica Section F - April 25, 2014 Category: Biochemistry Authors: Vit, A.Misson, L.Blankenfeldt, W.Seebeck, F.P. Tags: biosynthesis ergothioneine mycobacteria selenium SAD crystallization communications Source Type: research

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal NHL domain of human TRIM2
The tripartite motif-containing protein 2 (TRIM2) functions as an E3 ubiquitin ligase. Loss of function of TRIM2 has been shown to result in early-onset axonal neuropathy. As a member of the TRIM–NHL family of proteins, TRIM2 has a conserved modular architecture that includes N-terminal RING finger and B-box domains, a middle coiled-coil domain and a C-terminal NHL domain. To characterize the functional role of its NHL domain from the perspective of structural biology, a truncation of human TRIM2 (residues 465–744) was expressed, purified and crystallized. Rod-shaped crystals were obtained that diffracted X-ray...
Source: Acta Crystallographica Section F - April 25, 2014 Category: Biochemistry Authors: Guan, X.Li, J.Lü, X.Dong, Y.Chen, W.Li, X. Tags: TRIM2 NHL domain crystallization communications Source Type: research

Expression, purification and crystallization of MnSOD from Arabidopsis thaliana
Manganese superoxide dismutase (MnSOD) is an essential primary antioxidant enzyme. MnSOD plays an important role in plant tolerance to abiotic stress and is a target candidate for increasing stress tolerance in crop plants. Although the structure and kinetic parameters of MnSODs from several organisms have been determined, this information is still lacking for plant MnSODs. Here, recombinant MnSOD from Arabidopsis thaliana (AtMnSOD) was expressed, purified and crystallized. A nearly complete data set could only be obtained when a total rotation range of 180° was imposed during data collection, despite the seemingly tet...
Source: Acta Crystallographica Section F - April 25, 2014 Category: Biochemistry Authors: Marques, A.T.Santos, S.P.Rosa, M.G.Rodrigues, M.A.A.Abreu, I.A.Frazão, C.Romão, C.V. Tags: oxidative stress SOD metalloproteins plants ROS crystallization communications Source Type: research

The purification, crystallization and preliminary X-ray diffraction analysis of two isoforms of meso-diaminopimelate decarboxylase from Arabidopsis thaliana
Diaminopimelate decarboxylase catalyses the last step in the diaminopimelate-biosynthetic pathway leading to S-lysine: the decarboxylation of meso-diaminopimelate to form S-lysine. Lysine biosynthesis occurs only in microorganisms and plants, and lysine is essential for the growth and development of animals. Thus, the diaminopimelate pathway represents an attractive target for antimicrobial and herbicide treatments and has received considerable attention from both a mechanistic and a structural viewpoint. Diaminopimelate decarboxylase has only been characterized in prokaryotic species. This communication describes the firs...
Source: Acta Crystallographica Section F - April 25, 2014 Category: Biochemistry Authors: Oliver, M.R.Crowther, J.M.Leeman, M.M.Kessans, S.A.North, R.A.Donovan, K.A.Griffin, M.D.W.Suzuki, H.Hudson, A.O.Kasanmascheff, M.Dobson, R.C.J. Tags: diaminopimelate decarboxylase antibiotics antibiotic resistance drug discovery lysine biosynthesis S-lysine herbicides crystallization communications Source Type: research

Purification, crystallization and phase determination of the DR1998 haem b catalase from Deinococcus radiodurans
This study reports the purification and crystallization of the DR1998 catalase from D. radiodurans. The crystals diffracted to 2.6 Å resolution and belonged to space group C2221, with unit-cell parameters a = 97.33, b = 311.88, c = 145.63 Å, suggesting that they contain four molecules per asymmetric unit. The initial phases were determined by molecular replacement and the obtained solution shows the typical catalase quaternary structure. A preliminary model of the protein structure has been built and refinement is currently in progress. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - April 25, 2014 Category: Biochemistry Authors: Borges, P.T.Miranda, C.S.Santos, S.P.Carita, J.N.Frazão, C.Romão, C.V. Tags: metalloproteins reactive oxygen species bacteria crystallization communications Source Type: research

Purification, crystallization and preliminary X-ray characterization of the third ScaB cohesin in complex with an ScaA X-dockerin from Acetivibrio cellulolyticus
Interactions between cohesin and dockerin modules are critical for the formation of the cellulosome, which is responsible for the efficient degradation of plant cell-wall carbohydrates by anaerobes. Type I dockerin modules found in modular enzymatic components interact with type I cohesins in primary scaffoldins, enabling the assembly of the multi-enzyme complex. In contrast, type II dockerins located in primary scaffoldins bind to type II cohesins in adaptor scaffoldins or anchoring scaffoldins located at the bacterial envelope, contributing to the cell-surface attachment of the entire complex. Acetivibrio cellulolyticus ...
Source: Acta Crystallographica Section F - April 25, 2014 Category: Biochemistry Authors: Cameron, K.Alves, V.D.Bule, P.Ferreira, L.M.A.Fontes, C.M.G.A.Najmudin, S. Tags: cellulosome cohesins dockerins Acetivibrio cellulolyticus crystallization communications Source Type: research

Structure of tyrosine aminotransferase from Leishmania infantum
The trypanosomatid parasite Leishmania infantum is the causative agent of visceral leishmaniasis (VL), which is usually fatal unless treated. VL has an incidence of 0.5 million cases every year and is an important opportunistic co-infection in HIV/AIDS. Tyrosine aminotransferase (TAT) has an important role in the metabolism of trypanosomatids, catalyzing the first step in the degradation pathway of aromatic amino acids, which are ultimately converted into their corresponding l-2-oxoacids. Unlike the enzyme in Trypanosoma cruzi and mammals, L. infantum TAT (LiTAT) is not able to transaminate ketoglutarate. Here, the structu...
Source: Acta Crystallographica Section F - April 25, 2014 Category: Biochemistry Authors: Moreno, M.A.Abramov, A.Abendroth, J.Alonso, A.Zhang, S.Alcolea, P.J.Edwards, T.Lorimer, D.Myler, P.J.Larraga, V. Tags: tyrosine aminotransferase Leishmania infantum structural communications Source Type: research

Crystallization and preliminary crystallographic analysis of human muscle phosphofructokinase, the main regulator of glycolysis
Whereas the three-dimensional structure and the structural basis of the allosteric regulation of prokaryotic 6-phosphofructokinases (Pfks) have been studied in great detail, knowledge of the molecular basis of the allosteric behaviour of the far more complex mammalian Pfks is still very limited. The human muscle isozyme was expressed heterologously in yeast cells and purified using a five-step purification protocol. Protein crystals suitable for diffraction experiments were obtained by the vapour-diffusion method. The crystals belonged to space group P6222 and diffracted to 6.0 Å resolution. The 3.2 Å resol...
Source: Acta Crystallographica Section F - April 25, 2014 Category: Biochemistry Authors: Kloos, M.Brüser, A.Kirchberger, J.Schöneberg, T.Sträter, N. Tags: human muscle phosphofructokinase allosteric regulation structural communications Source Type: research

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylmannosamine-6-phosphate 2-epimerase from methicillin-resistant Staphylococcus aureus
Sialic acids are one of the most important carbohydrate classes in biology. Some bacterial pathogens can scavenge sialic acids from their surrounding environment and degrade them as a source of carbon, nitrogen and energy. This sequestration and subsequent catabolism of sialic acid require a cluster of genes known as the `Nan-Nag' cluster. The enzymes coded by these genes are important for pathogen colonization and persistence. Importantly, the Nan-Nag genes have proven to be essential for Staphylococcus aureus growth on sialic acids, suggesting that the pathway is a viable antibiotic drug target. The enzyme N-acetylmannos...
Source: Acta Crystallographica Section F - April 17, 2014 Category: Biochemistry Authors: North, R.A.Kessans, S.A.Griffin, M.D.W.Watson, A.J.A.Fairbanks, A.J.Dobson, R.C.J. Tags: N-acetylmannosamine-6-phosphate 2-epimerase NanE sialic acid catabolism Staphylococcus aureus MRSA crystallization communications Source Type: research

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylmannosamine kinase from methicillin-resistant Staphylococcus aureus
N-Acetylmannosamine kinase (EC 2.7.1.60) is involved in the catabolism of sialic acid for many bacterial pathogens implicated in human disease such as Escherichia coli, Staphylococcus aureus, Vibrio cholerae and V. vulnificus. Interestingly, some human commensals and bacterial pathogens can scavenge sialic acids from their surrounding environment and degrade them as a source of carbon, nitrogen and energy. This process requires a cluster of genes known as the `Nan-Nag cluster', which have proven to be essential for S. aureus growth on sialic acids, suggesting that the pathway is a viable antimicrobial drug target. The enzy...
Source: Acta Crystallographica Section F - April 17, 2014 Category: Biochemistry Authors: North, R.A.Seizova, S.Stampfli, A.Kessans, S.A.Suzuki, H.Griffin, M.D.W.Kvansakul, M.Dobson, R.C.J. Tags: N-acetylmannosamine kinase NanK sialic acid catabolism Staphylococcus aureus MRSA crystallization communications Source Type: research

Expression, crystallization and preliminary X-ray crystallographic analysis of DNA-directed RNA polymerase subunit L from Thermococcus onnurineus NA1
RNA polymerase (RNAP) plays a crucial role in gene expression in all organisms. It is a multiprotein complex that produces primary transcript RNA. Generally, the basal transcription apparatus in archaea is simpler than the eukaryotic RNA polymerase II counterpart. To understand the structure and function of archaeal RNAP, the TON-0309 gene encoding DNA-directed RNA polymerase subunit L (ToRNAP_L) from Thermococcus onnurineus NA1 was cloned and the protein was overexpressed in Escherichia coli, purified and crystallized. The purified protein was crystallized using the hanging-drop vapour-diffusion method and the crystal dif...
Source: Acta Crystallographica Section F - April 17, 2014 Category: Biochemistry Authors: Ho, T.-H.Hong, M.-K.Ngo, H.-P.-T.Kang, L.-W. Tags: DNA-directed RNA polymerase subunit L Thermococcus onnurineus NA1 crystallization communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of a novel β-l-arabinofuranosidase (HypBA1) from Bifidobacterium longum
The β-l-arabinofuranosidase (HypBA1) from Bifidobacterium longum JCM 1217 hydrolyzes the β-1,2-linked arabinofuranose disaccharide to release l-arabinoses. HypBA1 was classified into glycoside hydrolase family 127 (GH127) by the CAZy website (http://www.cazy.org/). The enzyme was expressed in Escherichia coli and the purified recombinant protein was crystallized. Crystals belonging to the primitive hexagonal space group P3x21, with unit-cell parameters a = b = 75.9, c = 254.0 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.78 Å resolution. A BLASTP search (http://b...
Source: Acta Crystallographica Section F - April 17, 2014 Category: Biochemistry Authors: Zhu, Z.He, M.Huang, C.-H.Ko, T.-P.Zeng, Y.-F.Huang, Y.-N.Jia, S.Lu, F.Liu, J.-R.Guo, R.-T. Tags: hydroxyproline-rich glycoproteins glycoside hydrolase HypBA1 Bifidobacterium longum crystallization communications Source Type: research

Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycan
Amidation of peptidoglycan is an essential feature in Staphylococcus aureus that is necessary for resistance to β-lactams and lysozyme. GatD, a 27 kDa type I glutamine amidotransferase-like protein, together with MurT ligase, catalyses the amidation reaction of the glutamic acid residues of the peptidoglycan of S. aureus. The native and the selenomethionine-derivative proteins were crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol, sodium acetate and calcium acetate. The crystals obtained diffracted beyond 1.85 and 2.25 Å, respectively, and belonged to space group P212121. ...
Source: Acta Crystallographica Section F - April 17, 2014 Category: Biochemistry Authors: Vieira, D.Figueiredo, T.A.Verma, A.Sobral, R.G.Ludovice, A.M.de Lencastre, H.Trincao, J. Tags: GatD glutamine amidotransferase-like protein Staphylococcus aureus crystallization communications Source Type: research

Expression, purification and preliminary crystallographic analysis of the N-terminal domain of Trypanosoma brucei BILBO1
Trypanosoma brucei is a unicellular parasite that causes sleeping sickness in sub-Saharan Africa. It has a unique flagellar pocket (FP) at the base of the single flagellum. The FP is the sole site for endocytosis and exocytosis activity and plays crucial roles in the defence of the cell against the host immune response. In the neck region of the FP is an electron-dense material termed the flagellar pocket collar (FPC). T. brucei BILBO1 (TbBILBO1) was the first cytoskeletal protein to be characterized in the FPC. This protein is highly conserved among trypanosomatids and is essential for FP biogenesis. Structural informatio...
Source: Acta Crystallographica Section F - April 17, 2014 Category: Biochemistry Authors: Vidilaseris, K.Dong, G. Tags: BILBO1 flagellar pocket Trypanosoma brucei crystallization communications Source Type: research

Expression, crystallization and preliminary crystallographic data analysis of PigI, a putative l-prolyl-AMP ligase from the prodigiosin synthetic pathway in Serratia
Prodigiosin, a member of the prodiginines, is a tripyrrole red pigment synthesized by Serratia and some other microbes. A bifurcated biosynthesis pathway of prodigiosin has been proposed in Serratia in which MBC (4-methoxy-2,2′-bipyrrole-5-carbaldehyde) and MAP (2-methyl-3-N-amyl-pyrrole) are synthesized separately and then condensed by PigC to form prodigiosin. The first step for the synthesis of MBC is the activation of l-proline by PigI, but its catalytic mechanism has remained elusive. To elucidate its mechanism, recombinant PigI was purified and crystallized. Crystals obtained by the sitting-drop method belonged...
Source: Acta Crystallographica Section F - April 17, 2014 Category: Biochemistry Authors: Han, N.Ran, T.Lou, X.Gao, Y.He, J.Tang, L.Xu, D.Wang, W. Tags: Serratia PigI crystallization communications Source Type: research

A structural characterization of the isoniazid Mycobacterium tuberculosis drug target, Rv2971, in its unliganded form
Aldo-keto reductases (AKR) are a large superfamily of NADPH-dependent oxidoreductases and play a role in detoxification of toxic metabolites. Rv2971, an AKR in Mycobacterium tuberculosis, has recently been identified as a target of isoniazid, a key first-line drug against tuberculosis. Here, the cloning, expression, purification, crystallization and structural characterization of Rv2971 are described. To gain insight into its function, the crystal structure of Rv2971 was successfully determined to 1.60 Å resolution in its unliganded form. The structure exhibits a TIM-barrel fold typical of AKRs, revealing structura...
Source: Acta Crystallographica Section F - April 17, 2014 Category: Biochemistry Authors: Shahine, A.Prasetyoputri, A.Rossjohn, J.Beddoe, T. Tags: Rv2971 AKR5H1 Mycobacterium tuberculosis isoniazid structural communications Source Type: research

Structure determination of a sugar-binding protein from the phytopathogenic bacterium Xanthomonas citri
The uptake of maltose and related sugars in Gram-negative bacteria is mediated by an ABC transporter encompassing a periplasmic component (the maltose-binding protein or MalE), a pore-forming membrane protein (MalF and MalG) and a membrane-associated ATPase (MalK). In the present study, the structure determination of the apo form of the putative maltose/trehalose-binding protein (Xac-MalE) from the citrus pathogen Xanthomonas citri in space group P6522 is described. The crystals contained two protein molecules in the asymmetric unit and diffracted to 2.8 Å resolution. Xac-MalE conserves the structural and functiona...
Source: Acta Crystallographica Section F - April 17, 2014 Category: Biochemistry Authors: Medrano, F.J.de Souza, C.S.Romero, A.Balan, A. Tags: malE Xanthomonas citri ABC transporters structural communications Source Type: research

SPINE-compatible `carboloops': a new microshaped vitreous carbon sample mount for X-ray and neutron crystallography
A novel vitreous carbon mount for macromolecular crystallography, suitable for neutron and X-ray crystallographic studies, has been developed. The technology described here is compatible both with X-ray and neutron cryo-crystallography. The mounts have low density and low background scattering for both neutrons and X-rays. They are prepared by laser cutting, allowing high standards of production quality, the ability to custom-design the mount to specific crystal sizes and large-scale production. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - April 15, 2014 Category: Biochemistry Authors: Romoli, F.Mossou, E.Cuypers, M.van der Linden, P.Carpentier, P.Mason, S.A.Forsyth, V.T.McSweeney, S. Tags: neutron crystallography X-ray crystallography sample mounting laser cutting laboratory communications Source Type: research

Expression, purification and crystallization of the SKICH domain of human TAX1BP1
TAX1BP1 is a highly conserved, pleiotropic protein that plays many essential functions in human cells, including negative regulation of inflammatory and antimicrobial responses mediated by NF-κB and IRF3 signaling, inhibition of apoptosis, transcriptional coactivation and autophagy etc. TAX1BP1 contains a SKICH domain at the N-terminus, three coiled-coil domains in the middle and two ubiquitin-binding zinc-finger motifs at the C-terminus. The SKICH domain and the linker sequence between the SKICH domain and the coiled-coil region mediate interaction with ubiquitin-like proteins of the LC3/GABARAP family, which are au...
Source: Acta Crystallographica Section F - April 15, 2014 Category: Biochemistry Authors: Yang, Y.Wang, G.Huang, X.Du, Z. Tags: TAX1BP1 A20 SKICH domain innate immune signaling NF- κ IRF3 autophagy receptor crystallization communications Source Type: research

Crystallization and preliminary X-ray crystallographic analysis of a novel α-l-arabinofuranosidase (CtGH43) from Clostridium thermocellum ATCC 27405
The truncated carbohydrate-active enzyme belonging to family 43 glycoside hydrolase from Clostridium thermocellum (CtGH43) is an α-l-arabinofuranosidase that in combination with endoxylanase leads to complete breakdown of l-arabinosyl-substituted xylans. The recombinant enzyme CtGH43 from C. thermocellum was overexpressed in Escherichia coli and purified by immobilized metal-ion affinity chromatography. The recombinant CtGH43 has a molecular mass of 35.86 kDa. Preliminary structural characterization was carried out on CtGH43 crystallized from different conditions, which gave either cube-shaped or brick-shaped cryst...
Source: Acta Crystallographica Section F - April 15, 2014 Category: Biochemistry Authors: Goyal, A.Ahmed, S.Fontes, C.M.G.A.Najmudin, S. Tags: glycoside hydrolase CtGH43 α -l-arabinofuranosidase C. thermocellum crystallization communications Source Type: research

Purification, crystallization and preliminary X-ray data collection of the N-terminal domain of the 26S proteasome regulatory subunit p27 and its complex with the ATPase domain of Rpt5 from Mus musculus
The protein 26S proteasome regulatory subunit p27 is one of the four chaperones that help in the assembly of the 19S regulatory particle (RP) of the 26S proteasome. In the present work, the N-terminus of p27 (residues 1–128) from Mus musculus was cloned, expressed, purified and crystallized alone and in complex with the C-terminal ATPase domain of Rpt5 (residues 173–442). The crystals of p27(1–128) diffracted to 1.7 Å resolution and belonged to space group P212121, with unit-cell parameters a = 26.79, b = 30.39, c = 145.06 Å. Resolution-dependent Matthews coefficient probability analysis s...
Source: Acta Crystallographica Section F - April 15, 2014 Category: Biochemistry Authors: Diao, W.Yang, X.Zhou, H. Tags: 26S proteasome regulatory subunit p27 chaperones ATPase domain of Rpt5 Mus musculus crystallization communications Source Type: research