scholarly journals Crystallization and preliminary X-ray diffraction analysis of UspE fromEscherichia coli

2014 ◽  
Vol 70 (12) ◽  
pp. 1640-1642 ◽  
Author(s):  
Yongbin Xu ◽  
Chun-Shan Quan ◽  
Xuanzhen Jin ◽  
Xiaoling Jin ◽  
Jing Zhao ◽  
...  
Keyword(s):  
Stress Proteins ◽  
Gel Filtration ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
E Coli ◽  
Vapour Diffusion ◽  
Induced Genes

Universal stress proteins (Usps) are among the most highly induced genes when bacteria are subjected to several stress conditions such as heat shock, nutrient starvation or the presence of oxidants or other stress agents.Escherichia colihas five small Usps and one tandem-type Usp. UspE (or YdaA) is the tandem-type Usp and consists of two Usp domains arranged in tandem. To date, the structure of UspE remains to be elucidated. To contribute to the molecular understanding of the function of the tandem-type UspE, UspE fromE. coliwas overexpressed and the recombinant protein was purified using Ni–NTA affinity, Q anion-exchange and gel-filtration chromatography. Crystals of UspE were obtained by sitting-drop vapour diffusion. A diffraction data set was collected to a resolution of 3.2 Å from flash-cooled crystals. The crystals belonged to the tetragonal space groupI4122 orI4322, with unit-cell parametersa=b= 121.1,c = 241.7 Å.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of AntE, a crotonyl-CoA carboxylase/reductase fromStreptomycessp. NRRL 2288

2014 ◽  
Vol 70 (6) ◽  
pp. 734-737 ◽  
Author(s):  
Lihan Zhang ◽  
Jing Chen ◽  
Takahiro Mori ◽  
Yan Yan ◽  
Wen Liu ◽  
...  
Keyword(s):  
Diffusion Method ◽  
Antimycin A ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion ◽  
Unsaturated Acyl ◽  
Reductive Carboxylation

AntE fromStreptomycessp. 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 inEscherichia coliwas crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space groupI222 orI212121, with unit-cell parametersa= 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.

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 Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp6

2014 ◽  
Vol 70 (6) ◽  
pp. 794-799 ◽  
Author(s):  
Junko Morita ◽  
Kazuki Kato ◽  
Emiko Mihara ◽  
Ryuichiro Ishitani ◽  
Junichi Takagi ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Choline Metabolism ◽  
Protein Molecules ◽  
Membrane Bound

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 groupP1, with unit-cell parametersa= 63.7,b= 68.8,c= 69.7 Å, α = 60.6, β = 87.0, γ = 68.1°. Assuming the presence of two protein molecules per asymmetric unit, the solvent content was estimated to be 49.5%.

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 Cloning, expression, purification, crystallization and preliminary crystallographic analysis of the C-terminal domain of Par-4 (PAWR)

2014 ◽  
Vol 70 (9) ◽  
pp. 1224-1227 ◽  
Author(s):  
Udaya Kumar Tiruttani Subhramanyam ◽  
Jan Kubicek ◽  
Ulf B. Eidhoff ◽  
Joerg Labahn
Keyword(s):  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Intrinsically Disordered ◽  
Tumour Suppressor Function ◽  
C Terminus ◽  
Apoptotic Protein

Prostate apoptosis response-4 protein is an intrinsically disordered pro-apoptotic protein with tumour suppressor function. Par-4 is known for its selective induction of apoptosis in cancer cells only and its ability to interact with various apoptotic proteinsviaits C-terminus. Par-4, with its unique function and various interacting partners, has gained importance as a potential target for cancer therapy. The C-terminus of the rat homologue of Par-4 was crystallized and a 3.7 Å resolution X-ray diffraction data set was collected. Preliminary data analysis shows the space group to beP41212. The unit-cell parameters area=b= 115.351,c= 123.663 Å, α = β = γ = 90°.

scholarly journals Overproduction, crystallization and preliminary X-ray crystallographic analysis ofEscherichia colitRNAN6-threonylcarbamoyladenosine dehydratase

2014 ◽  
Vol 70 (11) ◽  
pp. 1517-1520 ◽  
Author(s):  
Sunmin Kim ◽  
Keon Young Kim ◽  
Jeong Kuk Park ◽  
Byung Il Lee ◽  
Yun-Gon Kim ◽  
...  
Keyword(s):  
Crystallographic Analysis ◽  
X Rays ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
E Coli ◽  
Protein Mass ◽  
Crystal Volume

Escherichia colitRNAN6-threonylcarbamoyladenosine dehydratase (TcdA), previously called CsdL or YgdL, was overproduced and purified fromE. coliand crystallized using polyethylene glycol 3350 as a crystallizing agent. X-ray diffraction data were collected to 2.70 Å resolution under cryoconditions using synchrotron X-rays. The crystals belonged to space groupP21, with unit-cell parametersa= 65.4,b= 96.8,c= 83.3 Å, β = 111.7°. According to the Matthews coefficient, the asymmetric unit may contain up to four subunits of the monomeric protein, with a crystal volume per protein mass (VM) of 2.12 Å3 Da−1and 42.1% solvent content.

scholarly journals Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis ofHaemophilus influenzaeBamD and BamCD complex

2015 ◽  
Vol 71 (2) ◽  
pp. 234-238
Author(s):  
Jintang Lei ◽  
Xun Cai ◽  
Xiaodan Ma ◽  
Li Zhang ◽  
Yuwen Li ◽  
...  
Keyword(s):  
Diffraction Analysis ◽  
Recombinant Expression ◽  
Outer Membrane Proteins ◽  
Gel Filtration ◽  
Unit Cell ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Space Group ◽  
X Ray ◽  
Cell Parameters

The Bam machinery, which is highly conserved from bacteria to humans, is well recognized as the apparatus responsible for the insertion and folding of most outer membrane proteins in Gram-negative bacteria. InEscherichia coli, the Bam machinery consists of five components (i.e.BamA, BamB, BamC, BamD and BamE). In comparison, there are only four partners inHaemophilus influenzae: a BamB homologue is not found in its genome. In this study, the recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis ofH. influenzaeBamD and BamCD complex are reported. The genes encoding BamC and BamD were cloned into a pET vector and expressed inE. coli. Affinity, ion-exchange and gel-filtration chromatography were used to obtain high-purity protein for further crystallographic characterization. Using the hanging-drop vapour-diffusion technique, BamD and BamCD protein crystals of suitable size were obtained using protein concentrations of 70 and 50 mg ml−1, respectively. Preliminary X-ray diffraction analysis showed that the BamD crystals diffracted to 4.0 Å resolution and belonged to space groupP212121, with unit-cell parametersa= 54.5,b= 130.5,c= 154.7 Å. The BamCD crystals diffracted to 3.8 Å resolution and belonged to space groupI212121, with unit-cell parametersa= 101.6,b= 114.1,c= 234.9 Å.

scholarly journals Expression, purification, crystallization and preliminary X-ray diffraction analysis of the effector-interaction domain of the resistance protein RGA5-A fromOryza sativaL.japonica

2015 ◽  
Vol 71 (2) ◽  
pp. 171-174 ◽  
Author(s):  
Dan Huang ◽  
Yanan Zhang ◽  
Yanxiang Zhao ◽  
Junfeng Liu ◽  
You-Liang Peng
Keyword(s):  
Ammonium Citrate ◽  
Diffusion Method ◽  
Effector Protein ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Interaction Domain ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion ◽  
Resistance Protein

RGA5-A, a component of the Pia resistance-protein complex (RGA4/RGA5-A) fromOryza sativaL.japonica, has the ability to interact physically with the effector protein AVR-Pia fromMagnaporthe oryzaeviaits effector-interaction domain RGA5-A_S. The interaction between RGA5-A and AVR-Pia relieves the repression of RGA4, leading to AVR-independent cell death by the freed RGA4. To further understand the details of this interaction, the effector-interaction domain RGA5-A_S was expressed inEscherichia coliand purified to homogeneity. The purified recombinant protein His-RGA5-A_S was successfully crystallized using the sitting-drop vapour-diffusion method. A single crystal obtained using 0.2Mammonium citrate, 25%(w/v) PEG 3350 diffracted to 2.43 Å resolution. It belonged to space groupP4122 orP4322, with unit-cell parametersa=b= 55.2,c= 78.2 Å, α = β = γ = 90°.

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 Recombinant ACHT1 fromArabidopsis thaliana: crystallization and X-ray crystallographic analysis

2017 ◽  
Vol 73 (7) ◽  
pp. 382-385 ◽  
Author(s):  
Weimin Pan ◽  
Junchao Wang ◽  
Ye Yang ◽  
Lin Liu ◽  
Min Zhang
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Disulfide Bonds ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion

Thioredoxins (Trxs) play important roles in chloroplasts by linking photosynthetic light reactions to a series of plastid functions. They execute their function by regulating the oxidation and reduction of disulfide bonds. ACHT1 (atypical cysteine/histidine-rich Trx1) is a thylakoid-associated thioredoxin-type protein found in theArabidopsis thalianachloroplast. Recombinant ACHT1 protein was overexpressed inEscherichia coli, purified and crystallized by the vapour-diffusion method. The crystal diffracted to 1.7 Å resolution and a complete X-ray data set was collected. Preliminary crystallographic analysis suggested that the crystals belonged to space groupC2221, with unit-cell parametersa = 102.7,b= 100.6,c= 92.8 Å.

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