PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-23 (23)
 

Clipboard (0)
None
Journals
Year of Publication
Document Types
1.  Expression, purification, crystallization and preliminary X-ray studies of the TAN1 ortholog from Methanothermobacter thermautotrophicus 
MTH909 is the Methanothermobacter thermautotrophicus ortholog of Saccharomyces cerevisiae TAN1, which is required for N4-acetylcytidine formation in tRNA. The protein consists of an N-terminal near ferredoxin-like domain and a C-terminal THUMP domain. Unlike most other proteins containing the THUMP domain, TAN1 lacks any catalytic domains and has been proposed to form a complex with a catalytic protein capable of making base modifications. MTH909 has been cloned, over-expressed and purified. The molecule exists as a monomer in solution. X-ray data from a native crystal, belonging to the space group P6122 (P6522) with the unit cell dimensions of a = 69.9 Å and c = 408.5 Å, have been collected to 2.85 Å resolution.
Synopsis
MTH909, the Methanothermobacter thermautotrophicus ortholog of Saccharomyces cerevisiae TAN1, has been over-expressed, purified and crystallized. X-ray data from a crystal belonging to the space group P6122 (P6522) have been collected to 2.85 Å resolution.
doi:10.1107/S1744309108034039
PMCID: PMC2581707  PMID: 18997348
THUMP domain; TAN1; RNA-binding
2.  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
3.  Expression, purification and preliminary X-ray analysis of the Neisseria meningitidis outer membrane protein PorB 
The Neisseria meningitidis outer membrane protein PorB was expressed in Escherichia coli and purified from inclusion bodies by denaturation in urea followed by refolding in buffered LDAO on a size exclusion column. PorB has been crystallized in three different crystal forms: C222, R32 and P63. The C222 crystal form may contain either one or two PorB monomers in the asymmetric unit while both the R32 and P63 crystal forms contain one PorB monomer in the asymmetric unit. Of the three, the P63 crystal form had the best diffraction quality, yielding data extending to 2.3 Å resolution.
doi:10.1107/S1744309109032333
PMCID: PMC2765884  PMID: 19851005
outer membrane protein; Neisseria meningitides; denaturation; refolding; detergent; β-barrel membrane protein; porin
4.  Preliminary crystallographic analysis of GpgS, a key glucosyltransferase involved in methylglucose lipopolysaccharides biosynthesis in Mycobacterium tuberculosis 
Synopsis Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharides (MGLP) biosynthesis in Mycobacterium spp. Here we report the crystallization and preliminary crystallographic analysis of GpgS from Mycobacterium tuberculosis and its complex with UDP at 2.6 Å and 3.0 Å resolution, respectively.
Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharides (MGLP) biosynthesis in mycobacteria. These important molecules are believed to be involved in the regulation of fatty acid and mycolic acid synthesis. The enzyme belongs to the recently defined GT81 family of retaining glycosyltransferases (CAZy, Carbohydrate-Active enZymes data base; see www.cazy.org). Here we report the purification, crystallization and preliminary crystallographic analysis of GpgS from Mycobacterium tuberculosis and its complex with UDP. GpgS crystals belong to space group I4, with unit-cell parameters a = 98.85, b = 98.85, c= 127.64 Å, and diffract to 2.6 Å resolution. GpgS-UDP complex crystals belong to space group I4 with unit-cell parameters a= 98.32, b= 98.32, c= 127.96 Å, and diffract to 3.0 Å resolution.
doi:10.1107/S1744309108032892
PMCID: PMC2593697  PMID: 19052364
glycosyltransferase; methylglucose lipopolysaccharides; Mycobacterium; X ray structure
5.  Structure of a 6-pyruvoyltetrahydropterin synthase homolog from Streptomyces coelicolor 
The X-ray crystal structure of the 6-pyruvoyltetrahydropterin synthase (PTPS) homolog from Streptomyces coelicolor, SCO 6650, was solved at 1.5 Å resolution. SCO 6650 forms a hexameric T-fold that closely resembles other PTPS proteins. The biological activity of SCO 6650 is unknown, but it lacks both a required active-site zinc metal ion and the essential catalytic triad and does not catalyze the PTPS reaction. However, SCO 6650 maintains active-site residues consistent with binding a pterin-like substrate.
doi:10.1107/S1744309108027048
PMCID: PMC2564891  PMID: 18931427
6.  Expression, purification, crystallization and preliminary X-ray studies of a proly-4-hydroxylase protein from Bacillus anthracis 
Collagen prolyl-4-hydroxylases (C-P4H) catalyze the hydroxylation of specific proline residues in procollagen, an essential step in collagen biosynthesis. We have recently characterized a new form of P4H from Bacillus anthracis (anthrax-P4H) that shares many characteristics with the type I C-P4H from human. The structure of anthrax-P4H could provide important insights into the chemistry of C-P4Hs and into the function of this unique homodimeric P4H. X-ray diffraction data of selenomethionine-labeled anthrax-P4H recombinantly expressed in E. coli has been collected to 1.4 Å resolution.
doi:10.1107/S1744309108023439
PMCID: PMC2531266  PMID: 18765905
7.  Expression, purification and crystallization and preliminary X-ray studies of histamine dehydrogenase from Nocardioides simplex 
Histamine dehydrogenase (HADH) from Nocardioides simplex catalyzes the oxidative deamination of histamine to produce imidazole acetaldehyde and ammonium. HADH is functionally related to trimethylamine dehydrogenase (TMADH) but HADH has strict substrate specificity towards histamine. HADH is a homodimer with each 76 kDa subunit containing two redox cofactors, a [4Fe-4S] cluster and an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-FMN. In order to understand the substrate specificity of HADH, we seek to determine its structure by X-ray crystallography. This enzyme has been expressed recombinantly in Escherichia coli and successfully crystallized in two forms. Diffraction data were collected to 2.7 Å resolution at the SSRL synchrotron with 99.7% completeness. The crystals belong to the orthorhombic space group P212121 with unit-cell parameters a = 101.14 Å, b = 107.03 Å, and c = 153.35 Å.
doi:10.1107/S1744309108023336
PMCID: PMC2531280  PMID: 18765904
8.  Preliminary time-of-flight neutron diffraction study of human deoxyhemoglobin 
Human hemoglobin (HbA) is an intricate system that has evolved to efficiently transport oxygen molecules (O2) from lung to tissue. Its quaternary structure can fluctuate between two conformations, T (tense or deoxy) and R (relaxed or oxy), which have low and high affinity for O2, respectively. The binding of O2 to the heme sites of HbA is regulated by protons and by inorganic anions. In order to investigate the role of the protonation states of protein residues in O2 binding, large crystals of deoxy HbA (∼20 mm3) were grown in D2O under anaerobic conditions for neutron diffraction studies. A time-of-flight neutron data set was collected to 1.8Å resolution on the Protein Crystallography Station (PCS) at the spallation source run by Los Alamos Neutron Science Center (LANSCE). The HbA tetramer (64.6 kDa; 574 residues excluding the four heme groups) occupies the largest asymmetric unit (space group P21) from which a high-resolution neutron data set has been collected to date.
doi:10.1107/S1744309108005137
PMCID: PMC2374244  PMID: 18391424
9.  Crystallization and preliminary X-ray analysis of the NKX2.5 homeodomain in complex with DNA 
As part of an effort to elucidate the molecular basis for the pathogenesis of NKX2.5 mutations in congenital heart disease using X-ray crystallography, the NKX2.5 homeodomain has been crystallized in complex with a specific DNA element, the −242 promoter region of atrial natriuretic factor. Crystals of the homeodomain–DNA complex diffracted X-rays to 1.7 Å resolution and belonged to space group P65, with unit-cell parameters a = b = 71.5, c = 94.3 Å. The asymmetric unit contained two molecules of the NKX2.5 homeodomain and one double-stranded oligonucleotide.
doi:10.1107/S1744309108033447
PMCID: PMC2581709  PMID: 18997347
10.  Preliminary neutron and ultrahigh-resolution X-ray diffraction studies of the aspartic proteinase endothiapepsin cocrystallized with a gem-diol inhibitor 
Endothiapepsin has been cocrystallized with the gem-diol inhibitor PD-135,040 in a low solvent-content (39%) unit cell, which is unprecedented for this enzyme—inhibitor complex and enables ultrahigh-resolution (1.0 Å) X-ray diffraction data to be collected. This atomic resolution X-ray data set will be used to deduce the protonation states of the catalytic aspartate residues. A room-temperature neutron data set has also been collected for joint refinement with a room-temperature X-ray data set in order to locate the H/D atoms at the active site.
doi:10.1107/S1744309107061283
PMCID: PMC2344097  PMID: 18084100
11.  Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris 
The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety of toxic organophosphorus nerve agents. DFPase is structurally related to a number of enzymes, such as the medically important paraoxonase (PON). In order to investigate the reaction mechanism of this phosphotriesterase and to elucidate the protonation state of the active-site residues, large-sized crystals of DFPase have been prepared for neutron diffraction studies. Available H atoms have been exchanged through vapour diffusion against D2O-containing mother liquor in the capillary. A neutron data set has been collected to 2.2Å resolution on a relatively small (0.43 mm3) crystal at the spallation source in Los Alamos. The sample size and asymmetric unit requirements for the feasibility of neutron diffraction studies are summarized.
doi:10.1107/S1744309106052924
PMCID: PMC2330113  PMID: 17183172
12.  Purification, crystallization and structure determination of native GroEL from Escherichia coli lacking bound potassium ions 
GroEL is a member of the ATP-dependent chaperonin family that promotes the proper folding of many cytosolic bacterial proteins. The structures of GroEL in a variety of different states have been determined using X-ray crystallography and cryo-electron microscopy. In this study, a 3.02 Å crystal structure of the native GroEL complex from Escherichia coli is presented. The complex was purified and crystallized in the absence of potassium ions, which allowed evaluation of the structural changes that may occur in response to cognate potassium-ion binding by comparison to the previously determined wild-type GroEL structure (PDB code 1xck), in which potassium ions were observed in all 14 subunits. In general, the structure is similar to the previously determined wild-type GroEL crystal structure with some differences in regard to temperature-factor distribution.
doi:10.1107/S1744309107020295
PMCID: PMC2335072  PMID: 17554162
GroEL; 2nwc; r2nwcsf
13.  Cloning, expression, purification, crystallization and initial crystallographic analysis of transcription elongation factors GreB from Escherichia coli and Gfh1 from Thermus thermophilus 
The Escherichia coli gene encoding the transcription cleavage factor GreB and the Thermus thermophilus gene encoding the anti-GreA transcription factor Gfh1 were cloned and expressed and the purified proteins were crystallized by the sitting-drop vapor-diffusion technique. The GreB and Gfh1 crystals, which were improved by macroseeding, belong to space group P41212 (or P43212), with unit-cell parameters a = b = 148, c = 115.2 Å and a = b = 59.3, c = 218.9 Ǻ, respectively. Complete diffraction data sets were collected for the GreB and Gfh1 crystals to 2.6 and 2.8 Å resolution, respectively. Crystals of the selenomethionine proteins were obtained by microseeding using the native protein crystals and diffract as well as the native ones. The structure determination of these proteins is now in progress.
doi:10.1107/S1744309105040297
PMCID: PMC1401493  PMID: 16511259
14.  Crystallization of Hepatocyte Nuclear Factor 1β in Complex with DNA 
Hepatocyte nuclear factor 1β (HNF1β) is a member of the POU transcriptionfactor family and binds the target DNA as a dimer with nanomolar affinity. The HNF1β-DNA complex has been prepared and crystallized by hanging-drop vapor diffusion in 6%(v/v) PEG 300, 5%(w/v) PEG 8000, 8%(v/v) glycerol and 0.1 M Tris pH 8.0. The crystals diffracted to 3.2Å (93.9% completeness) using a synchrotron-radiation source under cryogenic (100 K) conditions and belong to space group R3, with unit-cell parameters a = b = 172.69, c = 72.43Å. A molecular-replacement solution has been obtained and structure refinement is in progress. This structure will shed light on the molecular mechanism of promoter recognition by HNF1β and the molecular basis of the disease-causing mutations found in it.
doi:10.1107/S1744309106015168
PMCID: PMC1581457  PMID: 16754972
15.  Crystallization and preliminary x-ray analysis of a phosphopentomutase from Bacillus cereus 
Phosphopentomutases (PPMs) interconvert d-ribose-5-phosphate and α-d-ribose-1-phosphate to link glucose and nucleotide metabolism. PPM from Bacillus cereus was overexpressed in Escherichia coli, purified to homogeneity, and crystallized. Bacterial PPMs are predicted to contain a di-metal reaction center, but the catalytically relevant metal had not previously been identified. Sparse matrix crystallization screening was performed in the presence or absence of 50 mM MnCl2. This strategy resulted in the formation of two crystal forms from two chemically distinct conditions. Crystals that formed with 50 mM MnCl2 were more easily manipulated and diffracted to higher resolution. These results suggest that even if the catalytically-relevant metal is not known, crystallization of putative metalloproteins may still benefit from supplementation of the crystallization screens with potential catalytic metals.
doi:10.1107/S1744309110017549
PMCID: PMC2898468  PMID: 20606280
Phosphopentomutase; alkaline phosphatase superfamily; ribose-5-phosphate; ribose-1-phosphate; sparse matrix screening; metalloproteins; metalloenzymes
16.  Crystallization and preliminary X-ray diffraction analysis of the barley yellow dwarf virus cap-independent translation element 
Barley yellow dwarf virus (BYDV) RNA lacks a 5′ m7GTP cap, yet it is translated efficiently because it contains a 105-base BYDV-like cap-independent translation element (BTE) in the 3′ untranslated region (UTR). To understand how the BTE outcompetes the host mRNA for protein-synthesis machinery, its three-dimensional structure is being determined at high resolution. The purification using transcription from DNA containing 2′-O-methyl nucleotides and preliminary crystallographic analyses of the BTE RNA are presented here. After varying the BTE sequence and crystallization-condition optimization, crystals were obtained that diffracted to below 5 Å resolution, with a complete data set being collected to 6.9 Å resolution. This crystal form indexes with an Rmerge of 0.094 in the monoclinic space group C2, with unit-cell parameters a = 316.6, b = 54.2, c = 114.5 Å, α = γ = 90, β = 105.1°.
doi:10.1107/S1744309111007196
PMCID: PMC3087640  PMID: 21543861
17.  An inhibited conformation for the protein kinase domain of the Saccharomyces cerevisiae AMPK homolog Snf1 
AMP-activated protein kinase (AMPK) is a master metabolic regulator for controlling cellular energy homeostasis. Its homolog in yeast, SNF1, is activated in response to glucose depletion and other stresses. The catalytic (α) subunit of AMPK/SNF1, Snf1 in yeast, contains a protein Ser/Thr kinase domain (KD), an auto-inhibitory domain (AID), and a region that mediates interactions with the two regulatory (β and γ) subunits. Here we report the crystal structure at 2.4 Å resolution of residues 41–440 of Snf1, which include the KD and AID. The AID is completely disordered in the crystal. A new, inhibited conformation of the KD is observed, in a DFG-out conformation and with the glycine-rich loop adopting a structure that blocks ATP binding to the active site.
doi:10.1107/S1744309110028265
PMCID: PMC2935214  PMID: 20823513
fatty acid metabolism; metabolic syndrome; protein kinase; DFG-out conformation; reductive methylation
18.  Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgX 
AlgX is a periplasmic protein required for the production of the exopolysaccharide alginate in Pseudomonas sp. and Azotobacter vinelandii. AlgX has been overexpressed and purified and diffraction-quality crystals have been grown using iterative seeding and the hanging-drop vapor-diffusion method. The crystals grew as flat plates with unit-cell parameters a = 46.4, b = 120.6, c = 86.9 Å, β = 95.7°. The crystals exhibited the symmetry of space group P21 and diffracted to a minimum d-spacing of 2.1 Å. On the basis of the Matthews coefficient (VM = 2.25 Å3 Da−1), two molecules were estimated to be present in the asymmetric unit.
doi:10.1107/S1744309110011851
PMCID: PMC2864699  PMID: 20445266 CAMSID: cams1418
19.  Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of a family 101 glycoside hydrolase from Streptococcus pneumoniae 
Streptococcus pneumoniae is a serious human pathogen that is responsible for a wide range of diseases including pneumonia, meningitis, septicemia, and otitis media. Full virulence of this bacterium is reliant on carbohydrate processing and metabolism as revealed by biochemical and genetic studies. One carbohydrate processing enzyme is a family 101 glycoside hydrolase (SpGH101) responsible for catalyzing the liberation of galactosyl β1,3-N-acetyl-D-galactosamine (Galβ1,3GalNAc) α-linked to serine or threonine residues from mucin-type glycoproteins. The 124 kDa catalytic module of this enzyme (SpGH101CM) was cloned and overproduced in Escherichia coli and purified. Crystals were obtained in spacegroup P21 and diffracted to a resolution of 2.0Å with unit cell dimensions of a = 81.86 b = 88.91 c = 88.77′, and β = 112.46°. The SpGH101CM also qualitatively displayed good activity on the synthetic substrate p-nitrophenyl-2-acetamido-2-deoxy-3-O-(β-D-galactopyranosyl)-α-D-galactopyranoside which is consistent with the classification of this enzyme as an endo-α-N-acetylgalactosaminidase.
doi:10.1107/S1744309108042474
PMCID: PMC2635869  PMID: 19194003 CAMSID: cams1343
Streptococcus pneumoniae; glycoside hydrolase; carbohydrate; endo-α-N-acetylgalactosaminidase; family 101; structure
20.  Z-isomer of 2,4-Diaminofuro[2,3-d]pyrimidine Antifolate Promotes Unusual Crystal Packing in Human Dihydrofolate Reductase Ternary Complex 
The crystal structure of human dihydrofolate reductase (hDHFR) ternary complex with NADPH and the Z-isomer of 2,4-diamino-5-[2-(2’-methoxyphenyl)-propenyl]-furo[2,3-d]pyrimidine (Z1) shows that the Z-isomer binds in the normal antifolate orientation in which the furo oxygen occupies the 8-amino position observed in the binding of 2,4-diaminopteridine antifolates such as methotrexate and with the methoxyphenyl moiety cis and coplanar with the furo[2,3-d]pyrimidine ring. The hDHFR ternary complex was refined to 1.7Ǻ resolution and crystallized in the orthorhombic space group, P212121. Although other hDHFR complexes crystallize in this space group, these data provide only the second example of an unusual packing arrangement in which the conserved active site Arg70 forms a salt bridge to the side chain of Glu44 from a symmetry-related molecule. As a result, the conformations of Phe31 and Gln35 shift with respect to those observed in the structure of mouse DHFR bound to Z1 that crystallizes in the monoclinic space group P21 and shows that Gln35 interacts with Arg70.
doi:10.1107/S1744309109025548
PMCID: PMC2720327  PMID: 19652333
21.  Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle 
A monodisperse truncation mutant of MxiH, the subunit of the needle from the Shigella flexneri type III secretion system (TTSS), has been overexpressed and purified. Crystals were grown of native and selenomethionine-labelled MxiHCΔ5 and diffraction data were collected to 1.9 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 183.4, b = 28.1, c = 27.8 Å, β= 96.5°. An anomalous difference Patterson map calculated with the data from the SeMet-labelled crystals revealed a single peak on the Harker section v = 0. Inspection of a uranyl derivative also revealed one peak in the isomorphous difference Patterson map on the Harker section v = 0. Analysis of the self-rotation function indicates the presence of a twofold non-crystallographic symmetry axis approximately along a. The calculated Matthews coefficient is 1.9 Å3 Da−1 for two molecules per asymmetric unit, corresponding to a solvent content of 33%.
doi:10.1107/S1744309106006555
PMCID: PMC1894743  PMID: 16511329
22.  Expression, Limited Proteolysis and Preliminary Crystallographic Analysis of IpaD, a Component of the Shigella Flexneri Type III Secretion System 
IpaD, the putative needle-tip protein of the Shigella flexneri type III secretion system, has been overexpressed and purified. Crystals were grown of the native protein in space group P212121, with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 Å, and data were collected to 2.9 Å resolution. Analysis of the native Patterson map revealed a peak at 50% of the origin on the Harker section v = 0.5, suggesting twofold non-crystallographic symmetry parallel to the b crystallographic axis. As attempts to derivatize or grow selenomethionine-labelled protein crystals failed, in-drop proteolysis was used to produce new crystal forms. A trace amount of subtilisin Carlsberg was added to IpaD before sparse-matrix screening, resulting in the production of several new crystal forms. This approach produced SeMet-labelled crystals and diffraction data were collected to 3.2 Å resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 Å, β = 107.9°. An anomalous difference Patterson map revealed peaks on the Harker section v = 0, while the self-rotation function indicates the presence of a twofold noncrystallographic symmetry axis, which is consistent with two molecules per asymmetric unit.
doi:10.1107/S1744309106027047
PMCID: PMC1894744  PMID: 16946465
23.  Expression, Purification, Crystallization and Preliminary Crystallographic Analysis of BipD, a Component of the Burkholderia Pseudomallei Type III Secretion System 
A construct consisting of residues 10–310 of BipD, a component of the Burkholderia pseudomallei type III secretion system (T3SS), has been overexpressed as a GST fusion, cleaved from the GST tag and purified. Crystals were grown of native and selenomethionine-labelled BipD. The crystals grow in two different polymorphs from the same condition. The first polymorph belongs to space group C222, with unit-cell parameters a = 103.98, b = 122.79, c = 49.17 Å, a calculated Matthews coefficient of 2.4 Å3 Da−1 (47% solvent content) and one molecule per asymmetric unit. The second polymorph belongs to space group P21212, with unit-cell parameters a = 136.47, b = 89.84, c = 50.15 Å, and a calculated Matthews coefficient of 2.3 Å3 Da−1 (45% solvent content) for two molecules per asymmetric unit (analysis of the self-rotation function indicates the presence of a weak twofold non-crystallographic symmetry axis in this P21212 form). The native crystals of both forms give diffraction data to 2.7 Å resolution, while the SeMet-labelled P21212 crystals diffract to 3.3 Å resolution. A K2PtCl4 derivative of the P21212 form was also obtained and data were collected to 2.7 Å with radiation of wavelengt λ = 0.933 Å. The Pt-derivative anomalous difference Patterson map revealed two self-peaks on the Harker sections.
doi:10.1107/S1744309106027035
PMCID: PMC1894745  PMID: 16946464

Results 1-23 (23)