Crystallization, X-ray diffraction and preliminary structure analysis of Mycobacterium tuberculosis chaperonin 10

1999 ◽  
Vol 55 (4) ◽  
pp. 910-914 ◽  
Author(s):  
Michael M. Roberts ◽  
Alun R. Coker ◽  
Gianluca Fossati ◽  
Paolo Mascagni ◽  
Anthony R. M. Coates ◽  
...  
Keyword(s):  
Protein Folding ◽  
Mycobacterium Tuberculosis ◽  
Cell Signalling ◽  
Host Cells ◽  
Diffusion Method ◽  
Monoclinic Space Group ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Chaperonin 10

The Mycobacterium tuberculosis chaperonin 10 (Mtcpn10) has been crystallized by the sitting-drop vapour-diffusion method. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 76.5, b = 87.9, c = 124.4 Å, β = 106.8°. X-ray diffraction data were collected to 2.8 Å. The self-rotation function and the molecular-replacement solution show that the asymmetric unit contains a dimer of heptamers related by twofold non-crystallographic symmetry. The two heptamers interact through interleaving flexible loops in a similar fashion to M. leprae and Gp31 cpn10. In addition to its role in protein folding, Mtcpn10 has unique effects on the growth of host cells and is a major immunogen in tuberculosis infections. The structure determination will permit the analysis of the amino acids identified as important for the protein-folding and cell-signalling activity of Mtcpn10.

Crystallization and preliminary X-ray diffraction studies on the N-utilizing substance-B (NusB) from Mycobacterium tuberculosis

2000 ◽  
Vol 56 (1) ◽  
pp. 64-66 ◽  
Author(s):  
B. Gopal ◽  
R. A. Cox ◽  
M. J. Colston ◽  
G. G. Dodson ◽  
S. J. Smerdon ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ribosomal Rna ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Core Complex ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters

N-utilizing substance B (NusB) is a protein which forms part of a complex assembly in transcriptional antitermination in Mycobacterium tuberculosis. It forms a heterodimer with the product of the NusE gene (identical to the ribosomal protein S10) and mediates the process of transcriptional antitermination by forming the core complex with the nut site of the ribosomal RNA along with other protein factors. NusB has been cloned and overexpressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. The space group is P212121, with unit-cell parameters a = 46.6, b = 64.2, c = 90.1 Å. A native data set complete to 1.6 Å resolution has been collected from a single crystal.

scholarly journals Purification, crystallization and preliminary X-ray diffraction studies of UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) fromSynechococcussp. PCC 7942

2014 ◽  
Vol 70 (2) ◽  
pp. 203-205
Author(s):  
Asaithambi Killivalavan ◽  
Ningning Zhuang ◽  
Young Shik Park ◽  
Kon Ho Lee
Keyword(s):  
Size Exclusion ◽  
Diffusion Method ◽  
Monoclinic Space Group ◽  
X Ray Diffraction ◽  
Dispersion Method ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography ◽  
Multi Wavelength ◽  
Pcc 7942

A UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) enzyme was discovered in the cyanobacteriumSynechococcussp. PCC 7942 which transfers a glucose moiety from UDP-glucose to tetrahydrobiopterin (BH4). BGluT protein was overexpressed with selenomethionine labelling for structure determination by the multi-wavelength anomalous dispersion method. The BGluT protein was purified by nickel-affinity and size-exclusion chromatography. It was then crystallized by the hanging-drop vapour-diffusion method using a well solution consisting of 0.1 Mbis-tris pH 5.5, 19%(w/v) polyethylene glycol 3350 with 4%(w/v) D(+)-galactose as an additive. X-ray diffraction data were collected to 1.99 Å resolution using a synchrotron-radiation source. The crystals belonged to the monoclinic space groupC2, with unit-cell parametersa= 171.35,b= 77.99,c = 53.77 Å, β = 90.27°.

Crystallization and preliminary X-ray diffraction studies of a Bowman–Birk inhibitor from Vigna unguiculata seeds

1999 ◽  
Vol 55 (11) ◽  
pp. 1920-1922 ◽  
Author(s):  
K. N. Rao ◽  
S. S. Hegde ◽  
R. J. Lewis ◽  
C. G. Suresh
Keyword(s):  
Vigna Unguiculata ◽  
Diffusion Method ◽  
Monoclinic Space Group ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Method ◽  
Molecular Replacement Method ◽  
Citrate Phosphate

A Bowman–Birk type trypsin/chymotrypsin inhibitor isolated from Vigna unguiculata seeds has been crystallized. Crystals were grown using the vapour-diffusion method at pH 4.0 using citrate/phosphate as a buffer and 30% saturated ammonium sulfate as precipitant. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 32.4, b = 61.8, c = 32.9 Å, β = 114.5°. The Matthews coefficient calculated assuming two molecules in the asymmetric unit was 1.95 Å3 Da−1, which corresponds to a 37% solvent content. X-ray data were collected to 2.5 Å resolution from a flash-frozen crystal. The structure was solved using the molecular-replacement method using tracy soybean inhibitor structure (PDB entry 1pi2) as a model.

Crystallization and preliminary X-ray diffraction studies of undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26

1999 ◽  
Vol 55 (9) ◽  
pp. 1606-1607 ◽  
Author(s):  
Masahiro Fujihashi ◽  
Naoto Shimizu ◽  
Yuan-Wei Zhang ◽  
Tanetoshi Koyama ◽  
Kunio Miki
Keyword(s):  
Micrococcus Luteus ◽  
Crystal Structure Analysis ◽  
Diffusion Method ◽  
Monoclinic Space Group ◽  
Lithium Sulfate ◽  
X Rays ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters

Undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26, one of the Z-prenyl chain-elongating enzymes, was crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate and lithium sulfate as precipitants. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 127.2, b = 60.2, c = 75.7 Å, β = 105.6°. The crystals diffract X-rays to at least 2.2 Å resolution using synchrotron radiation and are suitable for high-resolution crystal structure analysis.

scholarly journals In situproteolysis, crystallization and preliminary X-ray diffraction analysis of a VHH that binds listeria internalin B

2014 ◽  
Vol 70 (11) ◽  
pp. 1532-1535 ◽  
Author(s):  
Ian Huh ◽  
Robert Gene ◽  
Jyothi Kumaran ◽  
C. Roger MacKenzie ◽  
Cory L. Brooks
Keyword(s):  
Gel Filtration ◽  
Antibody Fragment ◽  
Variable Region ◽  
Host Cells ◽  
Monoclinic Space Group ◽  
Crystal Formation ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Internalin B

The variable region of camelid heavy-chain antibodies produces the smallest known antibody fragment with antigen-binding capability (a VHH). The VHH R303 binds internalin B (InlB), a virulence factor expressed by the pathogenListeria monocytogenes. InlB is critical for initiation ofListeriainfection, as it binds a receptor (c-Met) on epithelial cells, triggering the entry of bacteria into host cells. InlB is surface-exposed and is required for virulence, hence a VHH targeting InlB has potential applications for pathogen detection or therapeutic intervention. Here, the expression, purification, crystallization and X-ray diffraction of R303 are reported. Crystals of R303 were obtained followingin situproteolysis with trypsin. Gel filtration and SDS–PAGE revealed that trypsin removed the C-terminal tag region of R303, facilitating crystal formation. Crystals of R303 diffracted to 1.3 Å resolution and belonged to the monoclinic space groupP21, with unit-cell parametersa= 46.4,b= 31.2,c= 74.8 Å, β = 93.8°. The crystals exhibited a Matthews coefficient of 1.95 Å3 Da−1with two molecules in the asymmetric unit.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of the Csu pili CsuC–CsuA/B chaperone–major subunit pre-assembly complex fromAcinetobacter baumannii

2015 ◽  
Vol 71 (6) ◽  
pp. 770-774 ◽  
Author(s):  
Natalia Pakharukova ◽  
Minna Tuittila ◽  
Sari Paavilainen ◽  
Anton Zavialov
Keyword(s):  
Diffusion Method ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Gram Negative ◽  
X Ray ◽  
Cell Parameters ◽  
Abiotic Surfaces ◽  
Vapour Diffusion ◽  
Fimbrial Adhesins

The attachment of many Gram-negative pathogens to biotic and abiotic surfaces is mediated by fimbrial adhesins, which are assembledviathe classical, alternative and archaic chaperone–usher (CU) pathways. The archaic CU fimbrial adhesins have the widest phylogenetic distribution, yet very little is known about their structure and mechanism of assembly. To elucidate the biogenesis of archaic CU systems, structural analysis of the Csu fimbriae, which are used byAcinetobacter baumanniito form stable biofilms and cause nosocomial infection, was focused on. The major fimbriae subunit CsuA/B complexed with the CsuC chaperone was purified from the periplasm ofEscherichia colicells co-expressing CsuA/B and CsuC, and the complex was crystallized in PEG 3350 solution using the hanging-drop vapour-diffusion method. Selenomethionine-labelled CsuC–CsuA/B complex was purified and crystallized under the same conditions. The crystals diffracted to 2.40 Å resolution and belonged to the hexagonal space groupP6422, with unit-cell parametersa=b= 94.71,c = 187.05 Å, α = β = 90, γ = 120°. Initial phases were derived from a single anomalous diffraction (SAD) experiment using the selenomethionine derivative.

scholarly journals Crystallization and X-ray diffraction analysis of native and selenomethionine-substituted PhyH-DI fromBacillussp. HJB17

2017 ◽  
Vol 73 (11) ◽  
pp. 607-611
Author(s):  
Fang Lu ◽  
Bei Zhang ◽  
Yong Liu ◽  
Ying Song ◽  
Gangxing Guo ◽  
...  
Keyword(s):  
Unit Cell ◽  
Diffusion Method ◽  
Data Sets ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
Space Group ◽  
X Ray ◽  
Cell Parameters

Phytases are phosphatases that hydrolyze phytates to less phosphorylatedmyo-inositol derivatives and inorganic phosphate. β-Propeller phytases, which are very diverse phytases with improved thermostability that are active at neutral and alkaline pH and have absolute substrate specificity, are ideal substitutes for other commercial phytases. PhyH-DI, a β-propeller phytase fromBacillussp. HJB17, was found to act synergistically with other single-domain phytases and can increase their efficiency in the hydrolysis of phytate. Crystals of native and selenomethionine-substituted PhyH-DI were obtained using the vapour-diffusion method in a condition consisting of 0.2 Msodium chloride, 0.1 MTris pH 8.5, 25%(w/v) PEG 3350 at 289 K. X-ray diffraction data were collected to 3.00 and 2.70 Å resolution, respectively, at 100 K. Native PhyH-DI crystals belonged to space groupC121, with unit-cell parametersa = 156.84,b = 45.54,c = 97.64 Å, α = 90.00, β = 125.86, γ = 90.00°. The asymmetric unit contained two molecules of PhyH-DI, with a corresponding Matthews coefficient of 2.17 Å3 Da−1and a solvent content of 43.26%. Crystals of selenomethionine-substituted PhyH-DI belonged to space groupC2221, with unit-cell parametersa = 94.71,b= 97.03,c= 69.16 Å, α = β = γ = 90.00°. The asymmetric unit contained one molecule of the protein, with a corresponding Matthews coefficient of 2.44 Å3 Da−1and a solvent content of 49.64%. Initial phases for PhyH-DI were obtained from SeMet SAD data sets. These data will be useful for further studies of the structure–function relationship of PhyH-DI.

scholarly journals Expression, purification, crystallization and X-ray diffraction studies of the molecular chaperone prefoldin fromHomo sapiens

2015 ◽  
Vol 71 (9) ◽  
pp. 1189-1193 ◽  
Author(s):  
Yoshiki Aikawa ◽  
Hiroshi Kida ◽  
Yuichi Nishitani ◽  
Kunio Miki
Keyword(s):  
Crystal Structure ◽  
Protein Folding ◽  
Diffraction Data ◽  
Molecular Chaperone ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Native Proteins ◽  
Group Ii Chaperonin

Proper protein folding is an essential process for all organisms. Prefoldin (PFD) is a molecular chaperone that assists protein folding by delivering non-native proteins to group II chaperonin. A heterohexamer of eukaryotic PFD has been shown to specifically recognize and deliver non-native actin and tubulin to chaperonin-containing TCP-1 (CCT), but the mechanism of specific recognition is still unclear. To determine its crystal structure, recombinant human PFD was reconstituted, purified and crystallized. X-ray diffraction data were collected to 4.7 Å resolution. The crystals belonged to space groupP21212, with unit-cell parametersa= 123.2,b= 152.4,c= 105.9 Å.

scholarly journals A putative siderophore-interacting protein from the marine bacteriumShewanella frigidimarinaNCIMB 400: cloning, expression, purification, crystallization and X-ray diffraction analysis

2016 ◽  
Vol 72 (9) ◽  
pp. 667-671 ◽  
Author(s):  
Inês B. Trindade ◽  
Bruno M. Fonseca ◽  
Pedro M. Matias ◽  
Ricardo O. Louro ◽  
Elin Moe
Keyword(s):  
Iron Acquisition ◽  
Crystallographic Analysis ◽  
Monoclinic Space Group ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Interacting Protein ◽  
X Ray ◽  
Cell Parameters ◽  
Unit Structure ◽  
Shewanella Frigidimarina

Siderophore-binding proteins (SIPs) perform a key role in iron acquisition in multiple organisms. In the genome of the marine bacteriumShewanella frigidimarinaNCIMB 400, the gene tagged as SFRI_RS12295 encodes a protein from this family. Here, the cloning, expression, purification and crystallization of this protein are reported, together with its preliminary X-ray crystallographic analysis to 1.35 Å resolution. The SIP crystals belonged to the monoclinic space groupP21, with unit-cell parametersa= 48.04,b= 78.31,c= 67.71 Å, α = 90, β = 99.94, γ = 90°, and are predicted to contain two molecules per asymmetric unit. Structure determination by molecular replacement and the use of previously determined ∼2 Å resolution SIP structures with ∼30% sequence identity as templates are ongoing.

scholarly journals Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal DUF490963–1138domain of TamB fromEscherichia coli

2014 ◽  
Vol 70 (9) ◽  
pp. 1272-1275 ◽  
Author(s):  
Inokentijs Josts ◽  
Rhys Grinter ◽  
Sharon M. Kelly ◽  
Khedidja Mosbahi ◽  
Aleksander Roszak ◽  
...  
Keyword(s):  
Recombinant Expression ◽  
Diffusion Method ◽  
Gram Negative Bacteria ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Partner Protein ◽  
Experimental Phasing ◽  
Inner Membrane Protein ◽  
K 12

TamB is a recently described inner membrane protein that, together with its partner protein TamA, is required for the efficient secretion of a subset of autotransporter proteins in Gram-negative bacteria. In this study, the C-terminal DUF490963–1138domain of TamB was overexpressed inEscherichia coliK-12, purified and crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to the primitive trigonal space groupP3121, with unit-cell parametersa=b= 57.34,c= 220.74 Å, and diffracted to 2.1 Å resolution. Preliminary secondary-structure and X-ray diffraction analyses are reported. Two molecules are predicted to be present in the asymmetric unit. Experimental phasing using selenomethionine-labelled protein will be undertaken in the future.

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