scholarly journals Expression, purification, crystallization and preliminary X-ray analysis of full-length human RIG-I

2014 ◽  
Vol 70 (2) ◽  
pp. 248-251
Author(s):  
Jane Kwok ◽  
Kenrie P. Y. Hui ◽  
Julien Lescar ◽  
Masayo Kotaka
Keyword(s):  
Pattern Recognition ◽  
Ebola Virus ◽  
Pattern Recognition Receptors ◽  
Full Length ◽  
Viral Rna ◽  
Microbial Pathogens ◽  
Diffusion Method ◽  
X Ray ◽  
Cell Parameters ◽  
Molecular Patterns

The human innate immune system can detect invasion by microbial pathogens through pattern-recognition receptors that recognize structurally conserved pathogen-associated molecular patterns. Retinoic acid-inducible gene I (RIG-I)-like helicases (RLHs) are one of the two major families of pattern-recognition receptors that can detect viral RNA. RIG-I, belonging to the RLH family, is capable of recognizing intracellular viral RNA from RNA viruses, including influenza virus and Ebola virus. Here, full-length human RIG-I (hRIG-I) was cloned inEscherichia coliand expressed in a recombinant form with a His-SUMO tag. The protein was purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.85 Å resolution; the crystal belonged to space groupF23, with unit-cell parametersa = b=c= 216.43 Å, α = β = γ = 90°.

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 Crystallization and preliminary X-ray crystallographic analysis of carboxyl-terminal region 4 of SigR fromStreptomyces coelicolorA3(2)

2014 ◽  
Vol 70 (6) ◽  
pp. 747-749
Author(s):  
Keon Young Kim ◽  
Sunmin Kim ◽  
Jeong Kuk Park ◽  
HyoJin Song ◽  
SangYoun Park
Keyword(s):  
Streptomyces Coelicolor ◽  
Crystallographic Analysis ◽  
Terminal Region ◽  
Full Length ◽  
Spectrometric Analysis ◽  
X Rays ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Carboxyl Terminal

Full-length SigR fromStreptomyces coelicolorA3(2) was overexpressed inEscherichia coli, purified and submitted to crystallization trials using either polyethylene glycol 3350 or 4000 as a precipitant. X-ray diffraction data were collected to 2.60 Å resolution under cryoconditions using synchrotron X-rays. The crystal packs in space groupP43212, with unit-cell parametersa=b= 42.14,c= 102.02 Å. According to the Matthews coefficient, the crystal asymmetric unit cannot contain the full-length protein. Molecular replacement with the known structures of region 2 and region 4 as independent search models indicates that the crystal contains only the −35 element-binding carboxyl-terminal region 4 of full-length SigR. Mass-spectrometric analysis of the harvested crystal confirms this, suggesting a crystal volume per protein weight (VM) of 2.24 Å3 Da−1and 45.1% solvent content.

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.

scholarly journals Preliminary X-ray diffraction analysis of a thermophilic β-1,3–1,4-glucanase fromClostridium thermocellum

2014 ◽  
Vol 70 (7) ◽  
pp. 946-948 ◽  
Author(s):  
Lilan Zhang ◽  
Puya Zhao ◽  
Chun-Chi Chen ◽  
Chun-Hsiang Huang ◽  
Tzu-Ping Ko ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
High Specific Activity ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Glycosidic Bonds ◽  
Hydrolysis Of

β-1,3–1,4-Glucanases catalyze the specific hydrolysis of internal β-1,4-glycosidic bonds adjacent to the 3-O-substituted glucose residues in mixed-linked β-glucans. The thermophilic glycoside hydrolase CtGlu16A fromClostridium thermocellumexhibits superior thermal profiles, high specific activity and broad pH adaptability. Here, the catalytic domain of CtGlu16A was expressed inEscherichia coli, purified and crystallized in the trigonal space groupP3121, with unit-cell parametersa=b= 74.5,c= 182.9 Å, by the sitting-drop vapour-diffusion method and diffracted to 1.95 Å resolution. The crystal contains two protein molecules in an asymmetric unit. Further structural determination and refinement are in progress.

scholarly journals Broad and direct interaction between TLR and Siglec families of pattern recognition receptors and its regulation by Neu1

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Guo-Yun Chen ◽  
Nicholas K Brown ◽  
Wei Wu ◽  
Zahra Khedri ◽  
Hai Yu ◽  
...  
Keyword(s):  
Pattern Recognition ◽  
Direct Interaction ◽  
Pattern Recognition Receptors ◽  
Immunoglobulin Superfamily ◽  
Lectin Receptors ◽  
Sialidase Inhibitor ◽  
Tlr4 Ligand ◽  
Tlr4 Activation ◽  
Molecular Patterns ◽  
Damage Associated Molecular Patterns

Both pathogen- and tissue damage-associated molecular patterns induce inflammation through toll-like receptors (TLRs), while sialic acid-binding immunoglobulin superfamily lectin receptors (Siglecs) provide negative regulation. Here we report extensive and direct interactions between these pattern recognition receptors. The promiscuous TLR binders were human SIGLEC-5/9 and mouse Siglec-3/E/F. Mouse Siglec-G did not show appreciable binding to any TLRs tested. Correspondingly, Siglece deletion enhanced dendritic cell responses to all microbial TLR ligands tested, while Siglecg deletion did not affect the responses to these ligands. TLR4 activation triggers Neu1 translocation to cell surface to disrupt TLR4:Siglec-E interaction. Conversely, sialidase inhibitor Neu5Gc2en prevented TLR4 ligand-induced disruption of TLR4:Siglec E/F interactions. Absence of Neu1 in hematopoietic cells or systematic treatment with sialidase inhibitor Neu5Gc2en protected mice against endotoxemia. Our data raised an intriguing possibility of a broad repression of TLR function by Siglecs and a sialidase-mediated de-repression that allows positive feedback of TLR activation during infection.

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.

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, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module (CBM_E1) derived from sugarcane soil metagenome

2014 ◽  
Vol 70 (9) ◽  
pp. 1232-1235 ◽  
Author(s):  
Bruna Medeia Campos ◽  
Thabata Maria Alvarez ◽  
Marcelo Vizona Liberato ◽  
Igor Polikarpov ◽  
Harry J. Gilbert ◽  
...  
Keyword(s):  
Fossil Fuels ◽  
Plant Biomass ◽  
Metagenomic Library ◽  
Glycoside Hydrolases ◽  
Diffusion Method ◽  
Carbohydrate Binding ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Carbohydrate Binding Modules

In recent years, owing to the growing global demand for energy, dependence on fossil fuels, limited natural resources and environmental pollution, biofuels have attracted great interest as a source of renewable energy. However, the production of biofuels from plant biomass is still considered to be an expensive technology. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases for polysaccharide degradation, is attracting growing attention. Aiming at the identification of new CBMs, a sugarcane soil metagenomic library was analyzed and an uncharacterized CBM (CBM_E1) was identified. In this study, CBM_E1 was expressed, purified and crystallized. X-ray diffraction data were collected to 1.95 Å resolution. The crystals, which were obtained by the sitting-drop vapour-diffusion method, belonged to space groupI23, with unit-cell parametersa=b=c= 88.07 Å.

scholarly journals Crystallization and preliminary X-ray crystallographic analysis of a nonstructural protein 15 mutant fromHuman coronavirus 229E

2015 ◽  
Vol 71 (9) ◽  
pp. 1156-1160 ◽  
Author(s):  
Tong Huo ◽  
Xiang Liu
Keyword(s):  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Replication Process ◽  
Reading Frame ◽  
X Ray ◽  
Cell Parameters ◽  
Human Coronavirus 229E ◽  
Important Enzyme

Nonstructural protein 15 (nsp15), also called endoribonuclease, is a gene product of open reading frame 1b (ORF 1b) in coronaviruses. It is an important enzyme in the transcription/replication process involved in discontinuous negative-strand RNA synthesis. In this work, mutants of nsp15 fromHuman coronavirus 229E(HCoV-229E) were made based on structural analysis of the homologous nsp15s inSevere acute respiratory syndrome coronavirus(SARS-CoV) andMouse hepatitis virus(MHV). The I26A/N52A mutant of nsp15 was overexpressed, purified and crystallized, and this mutant led to a trimeric form rather than hexamers or monomers. Crystals of trimeric nsp15 were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant and diffracted to 2.5 Å resolution. The crystals belonged to space groupC2221, with unit-cell parametersa= 85.9,b= 137.5,c= 423.1 Å, α = β = γ = 90°.

Sign in / Sign up

Export Citation Format

Share Document