scholarly journals Crystallization and X-ray analysis of Borrelia burgdorferi β-barrel assembly machinery A

2020 ◽  
Vol 76 (6) ◽  
pp. 235-240
Author(s):  
Shishang Dong ◽  
Hongguan Chu ◽  
Kangning Wen ◽  
Qianqian Yu ◽  
Hui Li ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Outer Membrane Proteins ◽  
Homology Model ◽  
Molecular Replacement ◽  
Transport Function ◽  
Biological Functions ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Bam Complex

Mitochondria, chloroplasts and several species of bacteria have outer membrane proteins (OMPs) that perform many essential biological functions. The β-barrel assembly machinery (BAM) complex is one of the OMPs of Borrelia burgdorferi, the pathogenic spirochete that causes Lyme disease, and its BamA component (BbBamA) includes a C-terminal β-barrel domain and five N-terminal periplasmic polypeptide-transport-associated (POTRA) domains, which together perform a central transport function. In the current work, the production, crystallization and X-ray analysis of the three N-terminal POTRA domains of BbBamA (BbBamA-POTRA P1–P3; residues 30–273) were carried out. The crystals of BbBamA-POTRA P1–P3 belonged to space group P21, with unit-cell parameters a = 45.353, b = 111.538, c = 64.376 Å, β = 99.913°. The Matthews coefficient was calculated to be 2.92 Å3 Da−1, assuming the presence of two molecules per asymmetric unit, and the corresponding solvent content was 57.9%. Owing to the absence of an ideal homology model, numerous attempts to solve the BbBamA-POTRA P1–P3 structure using molecular replacement (MR) failed. In order to solve the structure, further trials using selenomethionine derivatization are currently being carried out.

scholarly journals Crystallization and X-ray diffraction studies of arginine kinase from the white Pacific shrimpLitopenaeus vannamei

2012 ◽  
Vol 68 (7) ◽  
pp. 783-785 ◽  
Author(s):  
Alonso A. Lopez-Zavala ◽  
Rogerio R. Sotelo-Mundo ◽  
Karina D. Garcia-Orozco ◽  
Felipe Isac-Martinez ◽  
Luis G. Brieba ◽  
...  
Keyword(s):  
Arginine Kinase ◽  
Homology Model ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
The Pacific ◽  
Crystallization Conditions ◽  
Reported Data

Crystals of an unligated monomeric arginine kinase from the Pacific whiteleg shrimpLitopenaeus vannamei(LvAK) were successfully obtained using the microbatch method. Crystallization conditions and preliminary X-ray diffraction analysis to 1.25 Å resolution are reported. Data were collected at 100 K on NSLS beamline X6A. The crystals belonged to space groupP212121, with unit-cell parametersa= 56.5,b= 70.2,c= 81.7 Å. One monomer per asymmetric unit was found, with a Matthews coefficient (VM) of 2.05 Å3 Da−1and 40% solvent content. Initial phases were determined by molecular replacement using a homology model ofLvAK as the search model. Refinement was performed withPHENIX, with finalRworkandRfreevalues of 0.15 and 0.19, respectively. Biological analysis of the structure is currently in progress.

Crystallization and preliminary X-ray studies of sialidase L from the leech Macrobdella decora

1998 ◽  
Vol 54 (1) ◽  
pp. 111-113 ◽  
Author(s):  
Yu Luo ◽  
Min-yuan Chou ◽  
Su-chen Li ◽  
Yu-teh Li ◽  
Ming Luo
Keyword(s):  
Escherichia Coli ◽  
Unit Cell ◽  
Data Sets ◽  
Molecular Replacement ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters ◽  
Mercury Derivative ◽  
Macrobdella Decora

Functional monomeric 83 kDa sialidase L, a NeuAcα2→3Gal-specific sialidase from Macrobdella leech, was expressed in Escherichia coli and readily crystallized by a macroseeding technique. The crystal belongs to space group P1 with unit-cell parameters a = 46.4, b = 69.3, c = 72.5 Å, α = 113.5, β = 95.4 and γ = 107.3°. There is one molecule per unit cell, giving a Vm = 2.4 Å3 Da−1 and a solvent content of 40%. Native and mercury-derivative data sets were collected to 2.0 Å resolution. Threading and molecular-replacement calculations confirmed the existence of a bacterial sialidase-like domain.

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 Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of a ferredoxin/flavodoxin-NADP(H) oxidoreductase (Bc0385) fromBacillus cereus

2014 ◽  
Vol 70 (6) ◽  
pp. 777-780 ◽  
Author(s):  
Silje Skråmo ◽  
Hans-Petter Hersleth ◽  
Marta Hammerstad ◽  
K. Kristoffer Andersson ◽  
Åsmund K. Røhr
Keyword(s):  
Electron Transfer ◽  
Thioredoxin Reductase ◽  
Free Form ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters

Ferredoxin/flavodoxin-NADP(H) oxidoreductases (FNRs) are key enzymes involved in catalysing electron transfer between ferredoxins/flavodoxins and NAD(P)H/NAD(P)+. InBacillus cereusthere are three genes that may encode FNRs, and the Bc0385 FNR has been cloned, overexpressed, purified and successfully crystallized in its NADPH/NADP+-free form. Diffraction data have been collected to 2.5 Å resolution from crystals belonging to the orthorhombic space groupP21212, with unit-cell parametersa= 57.2,b= 164.3,c= 95.0 Å, containing two FNR molecules in the asymmetric unit. The structure of the Bc0385 FNR has been solved by molecular replacement, and is a member of the homodimeric thioredoxin reductase-like class of FNRs.

scholarly journals Expression, purification, crystallization and preliminary X-ray analysis of glucose-1-phosphate uridylyltransferase (GalU) fromErwinia amylovora

2014 ◽  
Vol 70 (9) ◽  
pp. 1249-1251 ◽  
Author(s):  
Mirco Toccafondi ◽  
Michele Cianci ◽  
Stefano Benini
Keyword(s):  
Erwinia Amylovora ◽  
Search Model ◽  
Data Sets ◽  
Molecular Replacement ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
E Coli ◽  
Maximum Resolution ◽  
His Tag

Glucose-1-phosphate uridylyltransferase fromErwinia amylovoraCFPB1430 was expressed as a His-tag fusion protein inEscherichia coli. After tag removal, the purified protein was crystallized from 100 mMTris pH 8.5, 2 Mammonium sulfate, 5% ethylene glycol. Diffraction data sets were collected to a maximum resolution of 2.46 Å using synchrotron radiation. The crystals belonged to the hexagonal space groupP62, with unit-cell parametersa= 80.67,b= 80.67,c = 169.18. The structure was solved by molecular replacement using the structure of theE. colienzyme as a search model.

Crystallization and Preliminary X-ray Diffraction Analysis of Cold-Active β-Galactosidase from Arthrobacter sp. C2-2

2005 ◽  
Vol 70 (1) ◽  
pp. 124-132 ◽  
Author(s):  
Hana Petroková ◽  
Eva Vondráčková ◽  
Tereza Skálová ◽  
Jan Dohnálek ◽  
Petra Lipovová ◽  
...  
Keyword(s):  
Structure Refinement ◽  
Monoclinic Space Group ◽  
Molecular Replacement ◽  
Phase Problem ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Psychrotrophic Bacteria ◽  
Cold Active

β-Galactosidase from psychrotrophic bacteria strainArthrobactersp. C2-2 catalyzes cleavage of β-D-galactosyl moieties from β-D-galactosides and is interesting for its activity at low temperatures. Various types of crystals with dimensions of up to 0.8 mm were obtained and X-ray diffraction data up to 1.9 Å were collected. The crystals belong to the monoclinic space groupP21with unit-cell parametersa= 140.1 Å,b= 205.7 Å,c= 140.5 Å and β = 102.3°. The enzyme (molecular weight of a monomer is 111 kDa) forms hexamers in the crystal structure (one hexamer per asymmetric unit). The phase problem was solved by molecular replacement. Structure refinement is in progress.

Crystallization and preliminary crystallographic analysis of the snake muscle fructose 1,6-bisphosphatase

1999 ◽  
Vol 55 (7) ◽  
pp. 1342-1344 ◽  
Author(s):  
D.-W. Zhu ◽  
G.-J. Xu ◽  
P. H. Rehse ◽  
A. Azzi ◽  
F.-K. Zhao ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
Magnesium Chloride ◽  
Crystallographic Analysis ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Allosteric Enzyme ◽  
X Ray ◽  
Cell Parameters ◽  
Fructose 1,6 Bisphosphatase

The snake muscle fructose 1,6-bisphosphatase, a typical allosteric enzyme which plays important roles in gluconeogenesis, was crystallized in the presence of polyethylene glycol 3350 and magnesium chloride at pH 8.5. The crystals diffract to 2.3 Å on a rotating-anode X-ray source. The space group was determined to be either P3121 or its enantiomorph P3221, with unit-cell parameters a = b = 83.7, c = 202.41 Å, α = β = 90 and γ = 120°. There are two subunits in the asymmetric unit. Preliminary molecular-replacement studies indicate that the first enantiomorph is the correct one.

scholarly journals The LRR-Roc-COR module of theChlorobium tepidumRoco protein: crystallization and X-ray crystallographic analysis

2017 ◽  
Vol 73 (9) ◽  
pp. 520-524 ◽  
Author(s):  
Egon Deyaert ◽  
Arjan Kortholt ◽  
Wim Versées
Keyword(s):  
Protein Crystallization ◽  
Crystallographic Analysis ◽  
Chlorobium Tepidum ◽  
Molecular Replacement ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Protein Dimer ◽  
Map Analysis ◽  
Lrr Domain

Roco proteins are characterized by the presence of a Roc-COR supradomain harbouring GTPase activity, which is often preceded by an LRR domain. The most notorious member of the Roco protein family is the Parkinson's disease-associated LRRK2. The Roco protein from the bacteriumChlorobium tepidumhas been used as a model system to investigate the structure and mechanism of this class of enzymes. Here, the crystallization and crystallographic analysis of the LRR-Roc-COR construct of theC. tepidumRoco protein is reported. The LRR-Roc-COR crystals belonged to space groupP212121, with unit-cell parametersa= 95.6,b= 129.8,c= 179.5 Å, α = β = γ = 90°, and diffracted to a resolution of 3.3 Å. Based on the calculated Matthews coefficient, Patterson map analysis and an initial molecular-replacement analysis, one protein dimer is present in the asymmetric unit. The crystal structure of this protein will provide valuable insights into the interaction between the Roc-COR and LRR domains within Roco proteins.

scholarly journals Expression, purification, crystallization and preliminary X-ray diffraction analysis of a ribokinase from the thermohalophileHalothermothrix orenii

2012 ◽  
Vol 68 (2) ◽  
pp. 240-243 ◽  
Author(s):  
Lokesh D. Kori ◽  
Andreas Hofmann ◽  
Bharat K. C. Patel
Keyword(s):  
Metal Ion ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Solution ◽  
Metal Ion Affinity Chromatography

A ribokinase gene (rbk) from the anaerobic halothermophilic bacteriumHalothermothrix oreniiwas cloned and overexpressed inEscherichia coli. The recombinant protein (Ho-Rbk) was purified using immobilized metal-ion affinity chromatography and crystals were obtained using the sitting-drop method. Diffraction data were collected to a resolution of 3.1 Å using synchrotron radiation. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 45.6,b= 61.1,c= 220.2, and contained two molecules per asymmetric unit. A molecular-replacement solution has been found and attempts are currently under way to build a model of the ribokinase. Efforts to improve crystal quality so that higher resolution data can be obtained are also being considered.

scholarly journals Purification, crystallization and X-ray crystallographic studies of aBacillus cereusMepR-like transcription factor, BC0657

2015 ◽  
Vol 71 (6) ◽  
pp. 731-734 ◽  
Author(s):  
Min Uk Cho ◽  
Meong Il Kim ◽  
Minsun Hong
Keyword(s):  
Significant Contribution ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Biological Functions ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Antibiotic Drugs ◽  
Transcriptional Regulatory ◽  
Transcriptional Regulatory Mechanisms

Transcription factors of the MarR family respond to internal and external changes and regulate a variety of biological functions through ligand association with microorganisms. MepR belongs to the MarR family, and its mutations are associated with the development of multidrug resistance inStaphylococcus aureus, which has caused a growing health problem. In this study, aBacillus cereusMepR-like transcription regulator, BC0657, was crystallized. The BC0657 crystals diffracted to 2.05 Å resolution and belonged to either space groupP6222 orP6422, with unit-cell parametersa= 110.57,b= 110.57,c= 67.29 Å. There was one molecule per asymmetric unit. Future comparative structural studies on BC0657 would extend knowledge of ligand-induced transcriptional regulatory mechanisms in the MarR family and would make a significant contribution to the design of antibiotic drugs against multidrug-resistant bacteria.

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