scholarly journals Crystallization and preliminary X-ray analysis ofS-ribosylhomocysteinase fromStreptococcus mutans

2012 ◽  
Vol 68 (2) ◽  
pp. 199-202 ◽  
Author(s):  
Hui Li ◽  
Hongyan Zhao ◽  
Laikuan Zhu ◽  
Lihua Hong ◽  
Hong Zhang ◽  
...  
Keyword(s):  
High Yield ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Sensing System ◽  
Sds Page ◽  
Quorum Sensing System

S-Ribosylhomocysteinase (LuxS) encoded by theluxSgene fromStreptococcus mutansplays a crucial role in the quorum-sensing system. LuxS was solubly expressed inEscherichia coliwith high yield. The purity of the purified target protein, which was identified by SDS–PAGE and MALDI–TOF MS analysis, was >95%. The protein was crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as the primary precipitant. X-ray diffraction data were collected at Beijing Synchrotron Radiation Facility (BSRF). Diffraction by the crystal extended to 2.4 Å resolution and the crystal belonged to space groupC2221, with unit-cell parametersa= 55.3,b= 148.7,c= 82.8 Å.

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.

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 Crystallization and preliminary X-ray diffraction analysis of Trap1 complexed with Hsp90 inhibitors

2014 ◽  
Vol 70 (12) ◽  
pp. 1683-1687 ◽  
Author(s):  
Hanbin Jeong ◽  
Byoung Heon Kang ◽  
Changwook Lee
Keyword(s):  
Diffraction Analysis ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Hsp90 Inhibitors ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Middle Domain ◽  
Client Proteins

Hsp90 is a molecular chaperone responsible for the assembly and regulation of many cellular client proteins. In particular, Trap1, a mitochondrial Hsp90 homologue, plays a pivotal role in maintaining mitochondrial integrity, protecting against apoptosis in cancer cells. The N (N-terminal)-M (middle) domain of human Trap1 was crystallized in complex with Hsp90 inhibitors (PU-H71 and BIIB-021) by the hanging-drop vapour-diffusion method at pH 6.5 and 293 K using 15% PEG 8K as a precipitant. Diffraction data were collected from crystals of the Trap1–PU-H71 (2.7 Å) and Trap1–BIIB-021 (3.1 Å) complexes to high resolution at a synchrotron-radiation source. Preliminary X-ray diffraction analysis revealed that both crystals belonged to space groupP41212 orP43212, with unit-cell parametersa=b= 69.2,c= 252.5 Å, and contained one molecule per asymmetric unit according to Matthews coefficient calculations.

scholarly journals Crystallization and preliminary X-ray diffraction analysis ofBacillus subtilisYwfE, anL-amino-acid ligase

2012 ◽  
Vol 68 (2) ◽  
pp. 203-206 ◽  
Author(s):  
Takeo Tsuda ◽  
Tomomi Suzuki ◽  
Shuichi Kojima
Keyword(s):  
Amino Acid ◽  
Diffusion Method ◽  
Dependent Manner ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters

Bacillus subtilisYwfE, an L-amino-acid ligase, catalyzes the formation of an α-dipeptide from L-amino acids in an ATP-dependent manner. In order to elucidate the substrate-recognition mode and the reaction mechanism of this ligase, native and selenomethionine-derivatized (SeMet) crystals of YwfE in the presence of ADP, MgCl2and the dipeptide L-Ala-L-Gln were obtained using the hanging-drop vapour-diffusion method. These crystals diffracted to 1.9 and 2.8 Å resolution, respectively. Preliminary SAD phase calculations using the data set from the SeMet crystal suggested that the crystal belonged to the hexagonal space groupP6522, with unit-cell parametersa=b= 90.85,c = 250.31 Å, and contained one molecule in the asymmetric unit with a solvent content of 57.3%.

scholarly journals Purification, crystallization and preliminary X-ray crystallographic studies of Drep2 CIDE domain

2014 ◽  
Vol 70 (10) ◽  
pp. 1414-1417 ◽  
Author(s):  
Seung Mi Lee ◽  
Hyun Ho Park
Keyword(s):  
Fruit Fly ◽  
Diffusion Method ◽  
Amino Acid Residues ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Protein Protein Interaction

Drep2 is a novel nuclease from the fruit fly that might have a similar function in apoptosis to DFF40 and DFF45, which are primary players in apoptotic DNA fragmentation. Drep2 contains a conserved CIDE domain of ∼90 amino-acid residues that is involved in protein–protein interaction. In this study, the Drep2 CIDE domain was purified and crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were then collected to a resolution of 2.3 Å. The crystals were found to belong to the orthorhombic space groupP212121, with unit-cell parametersa= 50.28,b= 88.70,c= 113.37 Å.

Crystallization and preliminary X-ray diffraction analysis of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26

1999 ◽  
Vol 55 (6) ◽  
pp. 1231-1233 ◽  
Author(s):  
Ivana Smatanová ◽  
Yuji Nagata ◽  
L. Anders Svensson ◽  
Masamichi Takagi ◽  
Jaromír Marek
Keyword(s):  
Diffusion Method ◽  
Sphingomonas Paucimobilis ◽  
Asymmetric Unit ◽  
Haloalkane Dehalogenase ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters

Haloalkane hydrolytic dehalogenase LinB from Sphingomonas paucimobilis UT26, an enzyme which releases chloride or bromide anion from n-halogenated alkanes and has a broad range of substrate specificity, was crystallized using the hanging-drop vapour-diffusion method at 278 K. The best crystals were obtained by microseeding with a precipitant containing 18–20%(w/v) PEG 6000, 0.2 M calcium acetate and 0.1 M Tris–HCl pH 8.9. The crystals diffract to at least 1.60 Å using synchrotron X-ray under cryogenic (100 K) conditions. They belong to the orthorhombic space group P21212 with unit-cell parameters a = 50.29, b = 71.70, c = 72.73 Å. The asymmetric unit contains one molecule of the enzyme.

scholarly journals Plant-specific DUF1110 protein fromOryza sativa: expression, purification and crystallization

2016 ◽  
Vol 72 (6) ◽  
pp. 480-484 ◽  
Author(s):  
Kenichi Harada ◽  
Eiki Yamashita ◽  
Kento Inoue ◽  
Koji Yamaguchi ◽  
Toshimichi Fujiwara ◽  
...  
Keyword(s):  
Diffusion Method ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Sequence Identity ◽  
Vapour Diffusion ◽  
Domain Of Unknown Function

The Os01T0156300 protein fromOryza sativahas been classified into the domain of unknown function (DUF) family DUF1110. DUF1110 family members exist in monocotyledons but not in dicotyledons, and share no sequence identity with proteins for which structures have been reported. In this study, the Os01T0156300 protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.84 Å resolution. The crystal belonged to space groupP21, with unit-cell parametersa= 89.9,b= 89.8,c= 107.1 Å, β = 106.6°. The asymmetric unit was estimated to contain 6–11 molecules.

scholarly journals Crystallization and preliminary X-ray diffraction analyses of the redox-controlled complex of terminal oxygenase and ferredoxin components in the Rieske nonhaem iron oxygenase carbazole 1,9a-dioxygenase

2014 ◽  
Vol 70 (10) ◽  
pp. 1406-1409
Author(s):  
Jun Matsuzawa ◽  
Hiroki Aikawa ◽  
Takashi Umeda ◽  
Yuji Ashikawa ◽  
Chiho Suzuki-Minakuchi ◽  
...  
Keyword(s):  
Diffusion Method ◽  
Initial Reaction ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Anaerobic Conditions ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Electron Transfer Complex

The initial reaction in bacterial carbazole degradation is catalyzed by carbazole 1,9a-dioxygenase, which consists of terminal oxygenase (Oxy), ferredoxin (Fd) and ferredoxin reductase components. The electron-transfer complex between reduced Oxy and oxidized Fd was crystallized at 293 K using the hanging-drop vapour-diffusion method with PEG 3350 as the precipitant under anaerobic conditions. The crystal diffracted to a maximum resolution of 2.25 Å and belonged to space groupP21, with unit-cell parametersa= 97.3,b= 81.6,c= 116.2 Å, α = γ = 90, β = 100.1°. TheVMvalue is 2.85 Å3 Da−1, indicating a solvent content of 56.8%.

scholarly journals A thermostable and alkaline GDSL-motif esterase from Bacillus sp. K91: crystallization and X-ray crystallographic analysis

2018 ◽  
Vol 74 (2) ◽  
pp. 117-121 ◽  
Author(s):  
Junmei Ding ◽  
Hujie Zhu ◽  
Yujia Ye ◽  
Jie Li ◽  
Nanyu Han ◽  
...  
Keyword(s):  
Thermophilic Bacterium ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Crystallization Solution ◽  
Gdsl Family

The esterase Est8 from the thermophilic bacterium Bacillus sp. K91 belongs to the GDSL family and is active on a variety of acetylated compounds, including 7-aminocephalosporanic acid. In contrast to other esterases of the GDSL family, the catalytic residues Asp182 and His185 were more pivotal for the catalytic activity of Est8 than the Ser11 residue. To better understand the biochemical and enzymatic properties of Est8, recombinant Est8 protein was purified and crystallized. Crystals of Est8 were obtained by the hanging-drop vapour-diffusion method using 2.0 M ammonium sulfate, 5%(v/v) 2-propanol as the crystallization solution. X-ray diffraction data were collected to a resolution of 2.30 Å with an R merge of 16.4% from a crystal belonging to space group P41212 or P43212, with unit-cell parameters a = b = 68.50, c = 79.57 Å.

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.

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