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1.  Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active site 
The structure of L. donovani pteridine reductase has been targeted to assist in a program of structure-based inhibitor research. Crystals that diffracted to 2.5 Å resolution were obtained and the structure has been solved. Unfortunately, the active site is disordered and this crystal form is unsuitable for use in characterizing enzyme–ligand interactions.
Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species, protozoa that are responsible for a range of serious diseases found in tropical and subtropical parts of the world. As part of a structure-based approach to inhibitor development, specifically targeting Leishmania species, well ordered crystals of L. donovani PTR1 were sought to support the characterization of complexes formed with inhibitors. An efficient system for recombinant protein production was prepared and the enzyme was purified and crystallized in an orthorhombic form with ammonium sulfate as the precipitant. Diffraction data were measured to 2.5 Å resolution and the structure was solved by molecular replacement. However, a sulfate occupies a phosphate-binding site used by NADPH and occludes cofactor binding. The nicotinamide moiety is a critical component of the active site and without it this part of the structure is disordered. The crystal form obtained under these conditions is therefore unsuitable for the characterization of inhibitor complexes.
doi:10.1107/S174430911004724X
PMCID: PMC3079966  PMID: 21206018
antifolates; pteridine reductase; Leishmania; pterins; Trypanosoma
2.  Purification, crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding region of the Streptococcus gordonii adhesin GspB 
The carbohydrate-binding region of GspB from S. gordonii strain M99 was crystallized in space group P212121 and data were collected to 1.3 Å resolution.
The carbohydrate-binding region of the bacterial adhesin GspB from Strepto­coccus gordonii strain M99 (GspBBR) was expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. Separate sparse-matrix screening of GspBBR buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts and additives supported crystallization of GspBBR in each buffer. While both sets of conditions supported crystal growth in space group P212121, the crystals had distinct unit-cell parameters of a = 33.3, b = 86.7, c = 117.9 Å for crystal form 1 and a = 34.6, b = 98.3, c = 99.0 Å for crystal form 2. Additive screening improved the crystals grown in both conditions such that diffraction extended to beyond 2 Å resolution. A complete data set has been collected to 1.3 Å resolution with an overall R merge value of 0.04 and an R merge value of 0.33 in the highest resolution shell.
doi:10.1107/S1744309110036535
PMCID: PMC3001660  PMID: 21045307
GspB; glycoproteins; Streptococcus gordonii; sialic acid; adhesins; endocarditis; lectins
3.  Purification, crystallization, and preliminary x-ray diffraction analysis of the carbohydrate-binding region from the Streptococcus gordonii adhesin GspB 
The carbohydrate binding region of the bacterial adhesin GspB from Streptococcus gordonii strain M99 (GspBBR) was expressed in Escherichia coli and purified using affinity and size exclusion chromatography. Separate sparse-matrix screening of GspBBR buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts, and additives supported crystallization of GspBBR in each buffer. While both sets of conditions supported crystal growth in space group P212121, these had distinct unit cell dimensions of a=33.3 Å, b=86.6 Å, c=117.9 Å for crystal form one and a=34.6 Å, b=98.3 Å, c=99.0 Å for crystal form two. Additive screening improved the crystals grown in both conditions such that diffraction extended beyond 2 Å resolution. A complete data set has been collected to 1.3 Å resolution with an overall Rsym value of 0.04 and an Rsym value of 0.33 in the highest resolution shell.
doi:10.1107/S1744309110036535
PMCID: PMC3001660  PMID: 21045307
GspB; glycoprotein; Streptococcus gordonii; sialic acid; adhesin; endocarditis; lectin
4.  Purification, crystallization and preliminary X-ray diffraction of the G3BP1 NTF2-like domain 
The human G3BP1 NTF2-like domain was crystallized. Diffraction data were collected to 3.6 Å resolution.
The nuclear transport factor 2-like (NTF2-like) domain of human G3BP1 was subcloned, overexpressed in Escherichia coli and purified. Crystals were obtained using the hanging-drop vapour-diffusion method. Diffraction data were collected to 3.6 Å resolution using synchrotron radiation. The crystals belonged to the hexagonal space group P6322, with unit-cell parameters a = b = 89.84, c = 70.02 Å. The crystals contained one molecule per asymmetric unit, with an estimated solvent content of 56%. Initial phases were obtained by molecular replacement.
doi:10.1107/S1744309110042156
PMCID: PMC3079970  PMID: 21206022
G3BP1; Ras signalling; RasGAP
5.  Crystal structures, dynamics and functional implications of molybdenum-cofactor biosynthesis protein MogA from two thermophilic organisms 
Three crystal structures of the molybdenum-cofactor biosynthesis protein MogA from two highly thermophilic organisms have been determined at high resolution. Comparative analyses revealed the residues involved in oligomerization. In addition, molecular-dynamics and docking studies suggested the binding affinities of several small molecules towards MogA and homologous proteins.
Molybdenum-cofactor (Moco) biosynthesis is an evolutionarily conserved pathway in almost all kingdoms of life, including humans. Two proteins, MogA and MoeA, catalyze the last step of this pathway in bacteria, whereas a single two-domain protein carries out catalysis in eukaryotes. Here, three crystal structures of the Moco-biosynthesis protein MogA from the two thermophilic organisms Thermus thermophilus (TtMogA; 1.64 Å resolution, space group P21) and Aquifex aeolicus (AaMogA; 1.70 Å resolution, space group P21 and 1.90 Å resolution, space group P1) have been determined. The functional roles and the residues involved in oligomerization of the protein molecules have been identified based on a comparative analysis of these structures with those of homologous proteins. Furthermore, functional roles have been proposed for the N- and C-terminal residues. In addition, a possible protein–protein complex of MogA and MoeA has been proposed and the residues involved in protein–protein interactions are discussed. Several invariant water molecules and those present at the subunit interfaces have been identified and their possible structural and/or functional roles are described in brief. In addition, molecular-dynamics and docking studies with several small molecules (including the substrate and the product) have been carried out in order to estimate their binding affinities towards AaMogA and TtMogA. The results obtained are further compared with those obtained for homologous eukaryotic proteins.
doi:10.1107/S1744309110035037
PMCID: PMC3079962  PMID: 21206014
MogA; molybdenum-cofactor biosynthesis proteins
6.  Structures of the SEp22 dodecamer, a Dps-like protein from Salmonella enterica subsp. enterica serovar Enteritidis 
The crystal structure of SEp22, a DNA-binding protein from starved cells from S. enterica subsp. enterica serovar Enteritidis, was determined in two forms: the native state at 1.25 Å resolution and an iron-soaked form at 1.30 Å resolution.
The crystal structure of SEp22, a DNA-binding protein from starved cells from Salmonella enterica subsp. enterica serovar Enteritidis, has been determined in two forms: the native state at 1.25 Å resolution and an iron-soaked form at 1.30 Å resolution. The SEp22 protomers form a dodecameric shell with 23 symmetry and a single iron ion per protomer was found at the ferroxidase centre in the iron-soaked form. Along the threefold axes of the 23 symmetry, hydrophilic Asp channels that consist of Asp146 were found. Iron ions may flow into the cavity of the dodecameric shell through the Asp channels.
doi:10.1107/S1744309110043113
PMCID: PMC3079963  PMID: 21206015
DNA-binding protein from starved cells; ferroxidase centre; iron ions; Salmonella enterica subsp. enterica serovar Enteritidis
7.  Structure of laccase from Streptomyces coelicolor after soaking with potassium hexacyanoferrate and at an improved resolution of 2.3 Å 
The structure of the small laccase from S. coelicolor is reported at improved resolution and in a different space group. The soaked ligand is bound between laccase molecules.
The paper reports the structure of the small laccase from Streptomyces coelicolor determined from a crystal soaked with potassium hexacyanoferrate [K4Fe(CN)6]. The decolorization of the natively blue crystal observed upon soaking indicates the reduction of the enzyme in the crystal. The ligand binds between laccase molecules and stabilizes the crystal. The increased diffraction limit of the diffraction data collected from this crystal enabled the refinement of the small laccase structure at 2.3 Å resolution, which is the highest resolution obtained to date.
doi:10.1107/S1744309110046099
PMCID: PMC3079965  PMID: 21206017
laccases; ligand soaking; oxidoreductases; multicopper blue proteins
8.  Crystallization and preliminary X-ray crystallographic analysis of human quinolinate phosphoribosyltransferase 
H. sapiens quinolinate phosphoribosyltransferase has been expressed, purified and crystallized. A diffraction data set has been collected and processed at 2.8 Å resolution.
Quinolinate phosphoribosyltransferase (QPRTase) is a key NAD-biosynthetic enzyme which catalyzes the transfer of quinolinic acid to 5-phosphoribosyl-1-pyrophosphate, yielding nicotinic acid mononucleotide. Homo sapiens QPRTase (Hs-QPRTase) appeared as a hexamer during purification and the protein was crystallized. Diffraction data were collected and processed at 2.8 Å resolution. Native Hs-QPRTase crystals belonged to space group P21, with unit-cell parameters a = 76.2, b = 137.1, c = 92.7 Å, β = 103.8°. Assuming the presence of six molecules in the asymmetric unit, the calculated Matthews coefficient is 2.46 Å3 Da−1, which corresponds to a solvent content of 49.9%.
doi:10.1107/S1744309110041011
PMCID: PMC3079967  PMID: 21206019
quinolinate phosphoribosyltransferase; NAD biosynthesis; NadC
9.  Crystallization and preliminary X-ray studies of native and mutant intimin from enterohaemorrhagic Escherichia coli  
Crystals of native intimin and its N916Y mutant from enterohaemorrhagic E. coli O157:H7 diffracted to 2.8 and 2.6 Å resolution, respectively.
Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a primarily food-borne bacterial pathogen that is capable of causing life-threatening human infections and poses a serious challenge to public health worldwide. The bacterial outer-membrane protein intimin plays a key role in the initiation process of EHEC infection. In this study, intimin from EHEC O157:H7 (Int188) and its N916Y mutant (IntN916Y) were purified and crystals of both were obtained using the hanging-drop vapour-diffusion method at 291 K. Data were collected from Int188 and IntN916Y crystals to 2.8 and 2.6 Å resolution, respectively. The crystal of Int188 belonged to the orthorhombic space group C2, with unit-cell parameters a = 235.16, b = 44.81, c = 129.12 Å, α = γ = 90, β = 97.53°. The crystal of IntN916Y belonged to space group P212121, with unit-cell parameters a = 43.78, b = 92.49, c = 100.05 Å, α = β = γ = 90°.
doi:10.1107/S1744309110036432
PMCID: PMC3079968  PMID: 21206020
EHEC O157:H7; intimin; Tir proteins
10.  Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of the co-chaperonin XoGroES from Xanthomonas oryzae pv. oryzae  
Bacterial blight, an infectious disease caused by X. oryzae pv. oryzae (Xoo), causes huge rice-production losses in most rice-cultivating countries. The co-chaperonin of Xoo, XoGroES, an important protein for protein folding, was cloned from Xoo, purified and crystallized for atomic resolution structure determination.
Bacterial blight (BB), a devastating disease caused by Xanthomonas oryzae pv. oryzae (Xoo), causes serious production losses of rice in Asian countries. Protein misfolding may interfere with the function of proteins in all living cells and must be prevented to avoid cellular disaster. All cells naturally contain molecular chaperones that assist the unfolded proteins in folding into the native structure. One of the well characterized chaperone complexes is GroEL–GroES. GroEL, which consists of two chambers, captures misfolded proteins and refolds them. GroES is a co-chaperonin protein that assists the GroEL protein as a lid that temporarily closes the chamber during the folding process. Xoo4289, the GroES gene from Xoo, was cloned and expressed for X-ray crystallographic study. The purified protein (XoGroES) was crystallized using the hanging-drop vapour-diffusion method and a crystal diffracted to 2.0 Å resolution. The crystal belonged to the hexagonal space group P61, with unit-cell parameters a = 64.4, c = 36.5 Å. The crystal contains a single molecule in the asymmetric unit, with a corresponding V M of 2.05 Å3 Da−1 and a solvent content of 39.9%.
doi:10.1107/S1744309110038820
PMCID: PMC3079969  PMID: 21206021
Xanthomonas oryzae pv. oryzae; bacterial blight; co-chaperonins; Xoo4289
11.  Purification, crystallization and preliminary X-ray diffraction analysis of the thiaminase type II from Staphylococcus aureus  
Crystals of the thiaminase type II from S. aureus are orthorhombic, belonging to space group P212121 with unit-cell parameters a = 103.5, b = 104.1, c = 109.6 Å, and diffracted to 2.6 Å resolution.
Thiaminase type II (TenA) catalyzes the deamination of aminopyrimidines, including the cleavage of thiamine to 4-amino-5-hydroxymethyl-2-methyl­pyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole in the metabolism of thiamine (vitamin B1), in Staphylococcus aureus (Sa). SaTenA was crystallized by the vapour-diffusion method and the resulting crystal diffracted to 2.6 Å resolution usng synchrotron radiation. The crystal is orthorhombic, belonging to space group P212121 with unit-cell parameters a = 103.5, b = 104.1, c = 109.6 Å. With four molecules in the asymmetric unit, the Matthews coefficient is 2.85 Å3 Da−1. Initial attempts to solve the structure by molecular-replacement techniques were successful.
doi:10.1107/S1744309110043174
PMCID: PMC3079971  PMID: 21206023
thiaminase type II; TenA; Staphylococcus aureus
12.  Crystallization and preliminary crystallographic characterization of three peptidic inhibitors in complex with α-thrombin 
Human α-thrombin was crystallized in complex with specific peptide inhibitors of general sequence d-Phe-Pro-d-Arg-P1′-CONH2. The crystals belonged to the orthorhombic space group P212121 and diffracted to beyond 1.3 Å resolution.
The serine protease thrombin plays a major role in thrombosis and haemostasis. This has driven interest in thrombin inhibitors as potential antithrombotic drugs. Here, the crystallization and preliminary crystallographic analysis of human α-­thrombin in complex with three noncovalent peptide inhibitors of the general sequence d-Phe-Pro-d-Arg-P1′-CONH2 are reported. The crystals belonged to the orthorhombic space group P212121 and diffracted to beyond 1.3 Å resolution.
doi:10.1107/S1744309110043472
PMCID: PMC3079972  PMID: 21206024
thrombin; blood coagulation; peptide inhibitors; noncovalent
13.  Crystallization and preliminary X-ray analysis of the vWA domain of human anthrax toxin receptor 1 
The vWA domain of human anthrax toxin receptor 1 was overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 1.8 Å resolution.
The Gram-positive spore-forming bacterium Bacillus anthracis causes anthrax by secreting anthrax toxin, which consists of protective antigen (PA), lethal factor and oedema factor. Binding of PA to receptors triggers the multi-step process of anthrax toxin entry into target cells. Two distinct cellular receptors, ANTXR1 (also known as tumour endothelial marker 8; TEM8) and ANTXR2 (also known as capillary morphogenesis protein 2; CMG2), for anthrax toxin have been identified. Although the crystal structure of the extracellular von Willebrand factor A (vWA) domain of CMG2 has been reported, the difference between the vWA domains of TEM8 and CMG2 remains unclear because there are no structural data for the TEM8 vWA domain. In this report, the TEM8 vWA domain was expressed, purified and crystallized. X-ray diffraction data were collected to 1.8 Å resolution from a single crystal, which belonged to space group P1 with unit-cell parameters a = 65.9, b = 66.1, c = 74.4 Å, α = 63.7, β = 88.2, γ = 59.9°.
doi:10.1107/S1744309110043770
PMCID: PMC3079974  PMID: 21206026
human anthrax toxin receptor 1; von Willibrand factor A domain; tumour endothelial marker 8
14.  Crystallization and preliminary crystallographic analysis of the glycoside hydrolase family 115 α-­glucuronidase from Streptomyces pristinaespiralis  
α-Glucuronidase from S. pristinaespiralis was crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group R3 and diffracted to a resolution of 1.9 Å.
α-Glucuronidase from Streptomyces pristinaespiralis (SpGlcA115A) is com­posed of a single-chain peptide containing a catalytic domain belonging to glycosyl hydrolase family 115, a novel family of hemicellulolytic α-glucuronidases. The enzyme catalyzes the hydrolysis of α-linked 4-O-methylglucuronosyl and glucuronosyl residues from both polymeric xylans and oligosaccharides. SpGlcA115A was crystallized at 293 K using the sitting-drop vapour-diffusion method. The crystals belonged to space group R3 and diffracted to a resolution of 1.9 Å.
doi:10.1107/S1744309110043721
PMCID: PMC3079975  PMID: 21206027
α-glucuronidase; glycosyl hydrolase family 115; Streptomyces pristinaespiralis
15.  Expression, purification, crystallization and preliminary X-ray analysis of wild-type and of an active-site mutant of glyceraldehyde-3-phosphate dehydrogenase from Campylobacter jejuni  
The cloning, expression, purification, crystallization and preliminary X-ray analysis of wild-type and of an active-site mutant of C. jejuni glyceraldehyde-3-phosphate dehydrogenase is reported.
The genome of the enteric pathogen Campylobacter jejuni encodes a single glyceraldehyde-3-phosphate dehydrogenase that can utilize either NADP+ or NAD+ as coenzymes for the oxidative phosphorylation of glyceraldehyde-3-­phosphate to 1,3-diphosphoglycerate. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of both the wild type and an active-site mutant of the enzyme are presented. Preliminary X-ray analysis revealed that in both cases the crystals diffracted to beyond 1.9 Å resolution. The space group is shown to be I4122, with unit-cell parameters a = 90.75, b = 90.75, c = 225.48 Å, α = 90.46, β = 90.46, γ = 222.79°; each asymmetric unit contains only one subunit of the tetrameric enzyme.
doi:10.1107/S1744309110044465
PMCID: PMC3079976  PMID: 21206028
glyceraldehyde-3-phosphate dehydrogenase; Campylobacter jejuni
16.  Purification, crystallization and preliminary crystallographic analysis of SMU.1108c protein from Streptococcus mutans  
SMU.1108c, a putative uncharacterized protein from S. mutans, was crystallized and X-ray diffraction data were collected to a resolution of 2.2 Å.
Streptococcus mutans SMU.1108c (KEGG database) encodes a functionally uncharacterized protein consisting of 270 amino-acid residues. This protein is predicted to have a haloacid dehalogenase hydrolase-like domain and is a homologue of haloacid dehalogenase phosphatases that catalyze phosphoryl-transfer reactions. In this work, SMU.1108c was cloned into the pET28a vector and overexpressed in Escherichia coli strain BL21 (DE3). The protein was purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The best crystal diffracted to 2.0 Å resolution and belonged to space group C2, with unit-cell parameters a = 77.1, b = 80.2, c = 47.9 Å, β = 99.5°.
doi:10.1107/S174430911004457X
PMCID: PMC3079977  PMID: 21206029
SMU.1108c; Streptococcus mutans; haloacid dehalogenase superfamily
17.  Purification and crystallization of RNase HIII from Staphylococcus aureus  
The purification, crystallization and preliminary X-ray diffraction analysis of RNase HIII from S. aureus is presented. Crystals that diffracted to 2.6 Å resolution in space group P212121 were only obtained after removal of the hexahistidine tag.
As part of collaborative efforts to characterize virulence factors from Staphylococcus aureus, methods for the large-scale recombinant production of RNase HIII from S. aureus subspecies MRSA252 (Sa-RNase HIII) have been developed. RNase HIII-type ribonucleases are poorly characterized members of the RNase H group of endonucleases which hydrolyze RNA from RNA/DNA hybrids and are thought to be involved in DNA replication and repair. They are characterized by N-terminal extensions of unknown function that do not share sequence homology with the N-terminal extensions of bacterial RNases HI and RNases HII. Sa-RNase HIII was crystallized in the orthorhombic space group P212121, with unit-cell parameters a = 48.9, b = 74.2, c = 127.5 Å, and diffracted to 2.6 Å resolution.
doi:10.1107/S1744309110045616
PMCID: PMC3079978  PMID: 21206030
RNase HIII; Staphylococcus aureus; RNases ; MRSA
18.  Cloning, purification, crystallization and preliminary X-ray analysis of ESX-1-secreted protein regulator (EspR) from Mycobacterium tuberculosis  
ESX-1 secreted protein regulator (EspR, Rv3849) from M. tuberculosis has been purified and crystallized, and diffracted to 3.2 Å resolution at wavelength 0.97625 Å.
ESX-1-secreted protein regulator (EspR; Rv3849) is a key regulator in Mycobacterium tuberculosis that delivers bacterial proteins into the host cell during infection. EspR binds directly to the Rv3616c-Rv3614c promoter and activates transcription and secretes itself from the bacterial cell by the ESX-1 system. The three-dimensional structure of EspR will aid in understanding the mechanisms by which it binds to the Rv3616c-Rv3614c promoter and is involved in transcriptional activation. This study will significantly aid in the development of EspR-based therapeutics against M. tuberculosis. The full-length EspR gene from M. tuberculosis (H37Rv strain) was cloned and overexpressed as a soluble protein in Escherichia coli. The protein was purified by affinity chromatography using His-tagged protein followed by size-exclusion chromatography. EspR was crystallized using polyethylene glycol 3350 as precipitant. The crystals diffracted to 3.2 Å resolution using synchrotron radiation of wavelength 0.97625 Å. The crystal belonged to space group P3121 and contained three monomers in the asymmetric unit. Native and heavy-atom-derivatized data sets were collected from EspR crystals for use in ab initio structure-solution techniques.
doi:10.1107/S174430911004618X
PMCID: PMC3079979  PMID: 21206031
ESX-1-secreted protein regulator; Rv3849; Mycobacterium tuberculosis
19.  Purification, crystallization and X-ray diffraction study of basic 7S globulin from soybean 
Crystallization of basic 7S globulin from soybean is presented.
Basic 7S globulin (Bg7S) is expressed by soybeans in response to biotic or abiotic stress. Bg7S is capable of binding to a 4 kDa protein which is supposedly involved in cell proliferation. Bg7S is widely found not only in legumes, but also in other plants; however, its function is still unclear. Here, Bg7S was successfully crystallized. Orthorhombic and monoclinic crystals of Bg7S were obtained under different conditions and belonged to space groups P21212, with unit-cell parameters a = 111.9, b = 130.1, c = 287.8 Å, and P21, with unit-cell parameters a = 85.3, b = 137.6, c = 162.1 Å, β = 91.2°, respectively.
doi:10.1107/S1744309110046178
PMCID: PMC3079980  PMID: 21206032
basic 7S globulin; soybean
20.  Crystallization of an apo form of human arginase: using all the tools in the toolbox simultaneously 
An apo form of human arginase I which is suitable for soaking experiments has been crystallized.
Arginase (EC 3.5.3.1) is an aminohydrolase that acts on l-arginine to produce urea and ornithine. Two isotypes of the enzyme are found in humans. Type I is predominantly produced in the liver and is a homotrimer of 35 kDa subunits. Human arginase (hArginase) I is seen to be up-regulated in many diseases and is a potential therapeutic target for many diverse indications. Previous reports of crystallization and structure determination of hArginase have always included inhibitors of the enzyme: here, the first case of a true apo crystal form of the enzyme which is suitable for small-molecule soaking is reported. The crystals belonged to space group P212121 and have approximate unit-cell parameters a = 53, b = 67.5, c = 250 Å. The crystals showed slightly anisotropic diffraction to beyond 2.0 Å resolution.
doi:10.1107/S1744309110046208
PMCID: PMC3079981  PMID: 21206033
arginase; seeding; alternative reservoirs
21.  Crystallization and preliminary X-ray crystallographic analysis of the NmrA-like DDB_G0286605 protein from the social amoeba Dictyostelium discoideum  
In order to investigate its structure and function, the NmrA-like domain-containing DDB_G0286605 protein from D. discoideum was expressed, purified and crystallized. X-ray diffraction analysis is reported to a resolution of 1.64 Å.
The DDB_G0286605 gene product from Dictyostelium discoideum, an NmrA-like protein that belongs to the short-chain dehydrogenase/reductase family, has been crystallized by the hanging-drop vapour-diffusion method at 295 K. A 1.64 Å resolution data set was collected using synchrotron radiation. The DDB_G0286605 protein crystals belonged to space group P21, with unit-cell parameters a = 67.598, b = 54.935, c = 84.219 Å, β = 109.620°. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 43.25% with 99% probability. Molecular-replacement trials were attempted with three NmrA-like proteins, NmrA, HSCARG and QOR2, as search models, but failed. This may be a consequence of the low sequence identity between the DDB_G0286605 protein and the search models (DDB_G0286605 has a primary-sequence identity of 28, 32 and 19% to NmrA, HCARG and QOR2, respectively).
doi:10.1107/S1744309110046580
PMCID: PMC3079982  PMID: 21206034
SDR superfamily; NmrA-like proteins; Dictyostelium discoideum
22.  Crystallization and preliminary X-ray crystallographic analysis of the short-chain dehydrogenase/reductase-type DDB_G0291732 protein from Dictyostelium discoideum  
In order to investigate its structure and function, the NmrA-like short-chain dehydrogenase/reductase-type DDB_G0291732 protein from D. discoideum was expressed, purified and crystallized. X-ray diffraction analysis is reported to a resolution of 1.65 Å.
The DDB_G0291732 gene product from Dictyostelium discoideum, which is a NmrA-like protein that belongs to the short-chain dehydrogenase/reductase superfamily but shows deviations in conserved sequence regions, has been crystallized by the hanging-drop vapour-diffusion method at 295 K. A 1.65 Å resolution data set was collected using synchrotron radiation. The crystals of DDB_G0291732 protein belonged to space group P21, with unit-cell parameters a = 38.5, b = 63.7, c = 56.0 Å, β = 91.7°. Assuming the presence of one molecule in the asymmetric unit, the solvent content was estimated to be about 38.1%.
doi:10.1107/S1744309110046932
PMCID: PMC3079983  PMID: 21206035
SDR superfamily; Rossmann fold; Dictyostelium discoideum
23.  Crystallization and preliminary X-ray analysis of isopentenyl diphosphate isomerase from Methanocaldococcus jannaschii  
Isopentenyl diphosphate isomerase from M. jannaschii has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.08 Å resolution.
Type 2 isopentenyl diphosphate isomerase (IDI-2) is a flavoprotein. Recently, flavin has been proposed to play a role as a general acid–base catalyst with no redox role during the enzyme reaction. To clarify the detailed enzyme reaction mechanism of IDI-2 and the unusual role of flavin, structural analysis of IDI-2 from Methanocaldococcus jannaschii (MjIDI) was performed. Recombinant MjIDI was crystallized at 293 K using calcium acetate as a precipitant. The diffraction of the crystal extended to 2.08 Å resolution at 100 K. The crystal belonged to the tetragonal space group I422, with unit-cell parameters a = 126.46, c = 120.03 Å. The presence of one monomer per asymmetric unit gives a crystal volume per protein weight (V M) of 3.0 Å3 Da−1 and a solvent constant of 59.0% by volume.
doi:10.1107/S1744309110046944
PMCID: PMC3079984  PMID: 21206036
isopentenyl diphosphate isomerase; flavoproteins; no net redox reaction
24.  Expression, purification and preliminary crystallographic analysis of the recombinant β-­glucosidase (BglA) from the halothermophile Halothermothrix orenii  
A β-glucosidase A (BglA) from the thermophile Halothermothrix orenii has been cloned, purified and crystallized in an orthorhombic space group. X-ray diffraction data have been collected to 3.5 Å resolution, and the structure was solved by molecular replacement, revealing the presence of two molecules in the asymmetric unit.
The β-glucosidase A gene (bglA) has been cloned from the halothermophilic bacterium Halothermothrix orenii and the recombinant enzyme (BglA; EC 3.2.1.21) was bacterially expressed, purified using metal ion-affinity chromatography and subsequently crystallized. Orthorhombic crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystal structure with two molecules in the asymmetric unit was solved by molecular replacement using a library of known glucosidase structures. Attempts to collect higher resolution diffraction data from crystals grown under different conditions and structure refinement are currently in progress.
doi:10.1107/S1744309110046981
PMCID: PMC3079986  PMID: 21206038
β-glucosidases; halothermophiles; Halothermothrix orenii; BglA
25.  Crystallization of Escherichia coli maltoporin in the trigonal space group R3 
The crystallization of E. coli maltoporin in a new crystal form that diffracts to high resolution is reported.
Maltoporin is an outer-membrane protein that forms a β-barrel composed of three monomers and ensures the transport of maltose and maltodextrin in Gram-negative bacteria. Previously, the crystallization of Escherichia coli or Salmonella typhimurium maltoporin has been achieved in the presence of a mixture of the detergents β-decylmaltoside and dodecyl nonaoxyethylene. These crystals all belonged to the orthorhombic space group C2221 and gave rise to several structures of maltoporin in complex with different carbohydrates determined at resolutions between 3.2 and 2.4 Å. Here, the crystallization of E. coli maltoporin in a new crystal form is reported; the crystals belonged to the trigonal R3 space group and diffracted to 1.9 Å resolution. These crystals were obtained using n-dodecyl-β-d-maltoside as a detergent. Crystals with a lens or pyramidal morphology could be obtained using sitting or hanging drops, respectively, and despite their very different shapes they presented the same space group and very similar unit-cell parameters.
doi:10.1107/S1744309110047305
PMCID: PMC3079987  PMID: 21206039
outer-membrane proteins; maltoporin; carbohydrates

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