scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of theEscherichia colicommon pilus chaperone EcpB

2015 ◽  
Vol 71 (6) ◽  
pp. 676-679 ◽  
Author(s):  
James A. Garnett ◽  
Mamou Diallo ◽  
Steve J. Matthews
Keyword(s):  
Heavy Atom ◽  
Three Dimensional ◽  
Solid Surfaces ◽  
Dimensional Structure ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
The Common ◽  
First Time

Pili are key cell-surface components that allow the attachment of bacteria to both biological and abiotic solid surfaces, whilst also mediating interactions between themselves. InEscherichia coli, the common pilus (Ecp) belongs to an alternative chaperone–usher (CU) pathway that plays a major role in both early biofilm formation and host-cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.4 Å resolution. These crystals belonged to the trigonal space groupP3121 orP3221, with unit-cell parametersa=b= 62.65,c= 121.14 Å and one monomer in the asymmetric unit. Molecular replacement was unsuccessful, but selenomethionine-substituted protein and heavy-atom derivatives are being prepared for phasing. The three-dimensional structure of EcpB will provide invaluable information on the subtle mechanistic differences in biogenesis between the alternative and classical CU pathways. Furthermore, this is the first time that this refolding strategy has been used to purify CU chaperones, and it could be implemented in similar systems where it has not been possible to obtain highly ordered crystals.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of a single variable domain of the immunoglobulin superfamily in amphioxus,Amphi-IgSF-V

2014 ◽  
Vol 70 (8) ◽  
pp. 1072-1075 ◽  
Author(s):  
Bo Jiang ◽  
Yanjie Liu ◽  
Rong Chen ◽  
Zhenbao Wang ◽  
Mansoor Tariq ◽  
...  
Keyword(s):  
Immune System ◽  
Structural Information ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Immunoglobulin Superfamily ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
System A ◽  
Human Immunoglobulins

Amphioxus is regarded as an essential animal model for the study of immune evolution. Discovery of new molecules with the immunoglobulin superfamily (IgSF) variable (V) domain in amphioxus would help in studying the evolution of IgSF V molecules in the immune system. A protein was found which just contains only one IgSF V domain in amphioxus, termedAmphi-IgSF-V; it has over 30% sequence identity to the V domains of human immunoglobulins and mammalian T-cell receptors. In order to clarify the three-dimensional structure of this new molecule in amphioxus,Amphi-IgSF-V was expressed, purified and crystallized, and diffraction data were collected to a resolution of 1.95 Å. The crystal belonged to space groupP3221, with unit-cell parametersa=b= 53.9,c= 135.5 Å. The Matthews coefficient and solvent content were calculated to be 2.58 Å3 Da−1and 52.38%, respectively. The results will provide structural information to study the evolution of IgSF V molecules in the immune system.

scholarly journals Crystallization and preliminary X-ray characterization of MutT2, MSMEG_5148 fromMycobacterium smegmatis

2014 ◽  
Vol 70 (2) ◽  
pp. 190-192 ◽  
Author(s):  
S. M. Arif ◽  
P. B. Sang ◽  
U. Varshney ◽  
M. Vijayan
Keyword(s):  
Mycobacterium Smegmatis ◽  
Three Dimensional ◽  
Protein Molecule ◽  
Dimensional Structure ◽  
Molecular Replacement ◽  
Nudix Hydrolase ◽  
X Ray Diffraction ◽  
Orthorhombic Unit Cell ◽  
X Ray

Crystallization of MutT2, MSMEG_5148 fromMycobacterium smegmatis, has been carried out and the crystals have been characterized using X-ray diffraction. Matthews coefficient calculation suggests the possibility of one protein molecule in the asymmetric unit of the orthorhombic unit cell, space groupP21212 orP2122. Solution of the structure of the protein by molecular replacement using the known three-dimensional structure of a bacterial Nudix hydrolase is envisaged.

scholarly journals Crystallization and preliminary X-ray diffraction studies of the family 54 carbohydrate-binding module from laminarinase (β-1,3-glucanase) Lic16A ofClostridium thermocellum

2015 ◽  
Vol 71 (2) ◽  
pp. 217-220 ◽  
Author(s):  
Yury A. Kislitsyn ◽  
Valeriya R. Samygina ◽  
Igor A. Dvortsov ◽  
Nataliya A. Lunina ◽  
Inna P. Kuranova ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Diffusion Method ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
The Family ◽  
Overexpression System

The crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding module (CBM) from laminarinase Lic16A of the hyperthermophilic anaerobic bacteriumClostridium thermocellum(ctCBM54) are reported. Recombinant ctCBM54 was prepared using anEscherichia coli/pQE30 overexpression system and was crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The crystals belonged to space groupP6322, with unit-cell parametersa=b= 130.15,c= 131.05 Å. The three-dimensional structure of ctCBM54 will provide valuable information about the structure–function relation of the laminarinase Lic16A and will allow the exploitation of this binding module in biotechnological applications.

scholarly journals Cloning, purification, crystallization and preliminary X-ray diffraction crystallographic study of acyl-protein thioesterase 1 fromSaccharomyces cerevisiae

2012 ◽  
Vol 68 (7) ◽  
pp. 775-777
Author(s):  
Ye Yuan ◽  
Xiao Wang ◽  
Xu Li ◽  
Maikun Teng ◽  
Liwen Niu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Modification ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Crystallographic Study ◽  
Preliminary Model

Palmitoylation/depalmitoylation plays an important role in protein modification. yApt1 is the only enzyme inSaccharomyces cerevisiaethat catalyses depalmitoylation. In the present study, recombinant full-length yApt1 was cloned, expressed, purified and crystallized. The crystals diffracted to 2.40 Å resolution and belonged to space groupP42212, with unit-cell parametersa = b = 146.43,c = 93.29 Å. A preliminary model of the three-dimensional structure has been built and further refinement is ongoing.

scholarly journals Crystallization and preliminary X-ray diffraction data for a purple acid phosphatase from sweet potato

1999 ◽  
Vol 55 (12) ◽  
pp. 2051-2052 ◽  
Author(s):  
Gerhard Schenk ◽  
Lyle E. Carrington ◽  
Susan E. Hamilton ◽  
John de Jersey ◽  
Luke W. Guddat
Keyword(s):  
Acid Phosphatase ◽  
Sweet Potato ◽  
Diffraction Data ◽  
Three Dimensional ◽  
Purple Acid Phosphatase ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
The Subject

Purple acid phosphatase from sweet potato is a homodimer of 110 kDa. Two forms of the enzyme have been characterized. One contains an Fe–Zn centre similar to that previously reported for red kidney bean purple acid phosphatase. Another isoform, the subject of this work, is the first confirmed example of an Fe–Mn-containing enzyme. Crystals of this protein have been grown from PEG 6000. They have unit-cell parameters a = b = 118.4, c = 287.4 Å and have the symmetry of space group P6522, with one dimer per asymmetric unit. Diffraction data collected using a conventional X-ray source from a cryocooled crystal extend to 2.90 Å resolution. The three-dimensional structure of the enzyme will provide insight into the coordination of this novel binuclear metal centre.

scholarly journals Crystallization and preliminary X-ray diffraction studies of mammalian purple acid phosphatase

1999 ◽  
Vol 55 (8) ◽  
pp. 1462-1464 ◽  
Author(s):  
Luke W. Guddat ◽  
Alan S. McAlpine ◽  
David Hume ◽  
John de Jersey ◽  
Susan Hamilton ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Rational Design ◽  
Three Dimensional ◽  
Allantoic Fluid ◽  
Bone Diseases ◽  
Purple Acid Phosphatase ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters

The oxidized form of purple acid phosphatase from pig allantoic fluid has been crystallized in the presence of phosphate using the hanging-drop technique. The crystals belong to the space group P212121 and have unit-cell parameters a = 66.8, b = 70.3, c = 78.7 Å. Diffraction data collected from a cryocooled crystal using a conventional X-ray source extend to 1.55 Å resolution. A knowledge of the three-dimensional structure of mammalian purple acid phosphatase will aid in understanding the substrate specificity of the enzyme and will be important in the rational design of inhibitors, with potential in the treatment of bone diseases.

scholarly journals Crystallization and preliminary crystallographic analysis of LipC12, a true lipase isolated through a metagenomics approach

2012 ◽  
Vol 68 (2) ◽  
pp. 175-177 ◽  
Author(s):  
V. P. Martini ◽  
A. Glogauer ◽  
J. Iulek ◽  
E. M. Souza ◽  
F. O. Pedrosa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Three Dimensional ◽  
Sodium Formate ◽  
Crystallographic Analysis ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Sequence Identity

LipC12, a true lipase from family I.1 of bacterial lipases which was previously isolated through a metagenomics approach, contains 293 amino acids. Among lipases of known three-dimensional structure, it has a sequence identity of 47% to the lipase fromPseudomonas aeruginosaPAO1. Recombinant N-terminally His6-tagged LipC12 protein was expressed inEscherichia coli, purified in a homogenous form and crystallized in several conditions, with the best crystals being obtained using 2.0 Msodium formate and 0.1 Mbis-tris propane pH 7.0. X-ray diffraction data were collected to 2.70 Å resolution. The crystals belonged to the tetragonal space groupP4122, with unit-cell parametersa=b= 58.62,c = 192.60 Å.

scholarly journals Recombinant production, crystallization and X-ray crystallographic structure determination of peptidyl-tRNA hydrolase fromSalmonella typhimurium

2014 ◽  
Vol 70 (7) ◽  
pp. 872-877 ◽  
Author(s):  
Venugopal Vandavasi ◽  
Kasey Taylor-Creel ◽  
Robert L. McFeeters ◽  
Leighton Coates ◽  
Hana McFeeters
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Crystallographic Structure ◽  
Molecular Replacement ◽  
Hanging Drop ◽  
X Ray ◽  
Cell Parameters ◽  
Recombinant Production ◽  
Reservoir Solution

Peptidyl-tRNA hydrolase (Pth; EC 3.1.1.29) from the pathogenic bacteriumSalmonella typhimuriumhas been cloned, expressed inEscherichia coliand crystallized for X-ray analysis. Crystals were grown using hanging-drop vapor diffusion against a reservoir solution consisting of 0.03 Mcitric acid, 0.05 Mbis-tris propane, 1% glycerol, 3% sucrose, 25% PEG 6000 pH 7.6. Crystals were used to obtain the three-dimensional structure of the native protein at 1.6 Å resolution. The structure was determined by molecular replacement of the crystallographic data processed in space groupP212121with unit-cell parametersa= 62.1,b= 64.9,c= 110.5 Å, α = β = γ = 90°. The asymmetric unit of the crystallographic lattice was composed of two copies of the enzyme molecule with a 51% solvent fraction, corresponding to a Matthews coefficient of 2.02 Å3 Da−1. The structural coordinates reported serve as a foundation for computational and structure-guided efforts towards novel small-molecule Pth1 inhibitors and potential antibacterial development.

scholarly journals Crystallographic studies of SarV, a global regulator fromStaphylococcus aureus

2015 ◽  
Vol 71 (8) ◽  
pp. 1038-1041
Author(s):  
Yang Song ◽  
Fan Zhang ◽  
Xu Li ◽  
Jianye Zang ◽  
Xuan Zhang
Keyword(s):  
Heavy Atom ◽  
Size Exclusion ◽  
Monoclinic Space Group ◽  
Molecular Replacement ◽  
Global Regulator ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography ◽  
Heavy Atom Method

SarV, a member of the SarA protein family, is a global transcriptional regulator which has been reported to be involved in the regulation of autolysis inStaphylococcus aureus. In this study, SarV fromS. aureuswas successfully cloned, expressed, purified and crystallized. X-ray diffraction data were collected to 2.10 Å resolution. The crystals of SarV belonged to the monoclinic space groupP21, with unit-cell parametersa= 36.40,b= 119.64,c= 66.80 Å, α = γ = 90, β = 98.75°. The Matthews coefficient and the solvent content were estimated to be 2.57 Å3 Da−1and 52%, respectively, suggesting the presence of four molecules in the asymmetric unit. The results of size-exclusion chromatography (SEC) indicated thatS. aureusSarV exists as a homodimer in solution. Unfortunately, the structure cannot be solved by molecular replacement because of the low sequence identity ofS. aureusSarV to known structures. Further phase determination by selenomethionine single-wavelength anomalous dispersion (SAD) and the heavy-atom method is in progress.

scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of a hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) fromCoffea canephorainvolved in chlorogenic acid biosynthesis

2012 ◽  
Vol 68 (7) ◽  
pp. 824-828 ◽  
Author(s):  
Laura A. Lallemand ◽  
James G. McCarthy ◽  
Sean McSweeney ◽  
Andrew A. McCarthy
Keyword(s):  
Structural Data ◽  
Coffea Canephora ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Key Enzyme ◽  
Substrate Specifities ◽  
Drop Technique ◽  
Better Than

Chlorogenic acids (CGAs) are a group of soluble phenolic compounds that are produced by a variety of plants, includingCoffea canephora(robusta coffee). The last step in CGA biosynthesis is generally catalysed by a specific hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferase (HQT), but it can also be catalysed by the more widely distributed hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT). Here, the cloning and overexpression of HCT fromC. canephorainEscherichia colias well as its purification and crystallization are presented. Crystals were obtained by the sitting-drop technique at 293 K and X-ray diffraction data were collected on the microfocus beamline ID23-2 at the ESRF. The HCT crystals diffracted to better than 3.0 Å resolution, belonged to space groupP42212 with unit-cell parametersa=b= 116.1,c= 158.9 Å and contained two molecules in the asymmetric unit. The structure was solved by molecular replacement and is currently under refinement. Such structural data are needed to decipher the molecular basis of the substrate specifities of this key enzyme, which belongs to the large plant acyl-CoA-dependent BAHD acyltransferase superfamily.

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