scholarly journals Preliminary crystallographic analysis of Xyn52B2, a GH52 β-D-xylosidase fromGeobacillus stearothermophilusT6

2014 ◽  
Vol 70 (12) ◽  
pp. 1675-1682 ◽  
Author(s):  
Roie Dann ◽  
Shifra Lansky ◽  
Noa Lavid ◽  
Arie Zehavi ◽  
Valery Belakhov ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Crystal Form ◽  
Thermophilic Bacterium ◽  
Crystallographic Analysis ◽  
Dimensional Structure ◽  
Glycoside Hydrolase Family ◽  
Data Sets ◽  
X Ray Diffraction ◽  
Cell Parameters ◽  
Average Unit

Geobacillus stearothermophilusT6 is a thermophilic bacterium that possesses an extensive hemicellulolytic system, including over 40 specific genes that are dedicated to this purpose. For the utilization of xylan, the bacterium uses an extracellular xylanase which degrades xylan to decorated xylo-oligomers that are imported into the cell. These oligomers are hydrolyzed by side-chain-cleaving enzymes such as arabinofuranosidases, acetylesterases and a glucuronidase, and finally by an intracellular xylanase and a number of β-xylosidases. One of these β-xylosidases is Xyn52B2, a GH52 enzyme that has already proved to be useful for various glycosynthesis applications. In addition to its demonstrated glycosynthase properties, interest in the structural aspects of Xyn52B2 stems from its special glycoside hydrolase family, GH52, the structures and mechanisms of which are only starting to be resolved. Here, the cloning, overexpression, purification and crystallization of Xyn52B2 are reported. The most suitable crystal form that has been obtained belonged to the orthorhombicP212121space group, with average unit-cell parametersa = 97.7,b= 119.1,c = 242.3 Å. Several X-ray diffraction data sets have been collected from flash-cooled crystals of this form, including the wild-type enzyme (3.70 Å resolution), the E335G catalytic mutant (2.95 Å resolution), a potential mercury derivative (2.15 Å resolution) and a selenomethionine derivative (3.90 Å resolution). These data are currently being used for detailed three-dimensional structure determination of the Xyn52B2 protein.

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 Ribokinase fromLeishmania donovani: purification, characterization and X-ray crystallographic analysis

2018 ◽  
Vol 74 (2) ◽  
pp. 99-104 ◽  
Author(s):  
Santhosh Gatreddi ◽  
Sayanna Are ◽  
Insaf Ahmed Qureshi
Keyword(s):  
Functional Characterization ◽  
Three Dimensional ◽  
Protozoan Parasite ◽  
Crystallographic Analysis ◽  
Size Exclusion ◽  
Dimensional Structure ◽  
Diffusion Method ◽  
Gene Encoding ◽  
Cell Parameters ◽  
Α Helix

Leishmaniais an auxotrophic protozoan parasite which acquires D-ribose by transporting it from the host cell and also by the hydrolysis of nucleosides. The enzyme ribokinase (RK) catalyzes the first step of ribose metabolism by phosphorylating D-ribose using ATP to produce D-ribose-5-phosphate. To understand its structure and function, the gene encoding RK fromL. donovaniwas cloned, expressed and purified using affinity and size-exclusion chromatography. Circular-dichroism spectroscopy of the purified protein showed comparatively more α-helix in the secondary-structure content, and thermal unfolding revealed theTmto be 317.2 K. Kinetic parameters were obtained by functional characterization ofL. donovaniRK, and theKmvalues for ribose and ATP were found to be 296 ± 36 and 116 ± 9.0 µM, respectively. Crystals obtained by the hanging-drop vapour-diffusion method diffracted to 1.95 Å resolution and belonged to the hexagonal space groupP61, with unit-cell parametersa=b= 100.25,c= 126.77 Å. Analysis of the crystal content indicated the presence of two protomers in the asymmetric unit, with a Matthews coefficient (VM) of 2.45 Å3 Da−1and 49.8% solvent content. Further study revealed that human counterpart of this protein could be used as a template to determine the first three-dimensional structure of the RK from trypanosomatid parasites.

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 SpaB, an atypically adhesive basal pilin from the lactobacillar SpaCBA pilus: crystallization and X-ray diffraction analysis

2019 ◽  
Vol 75 (12) ◽  
pp. 731-737 ◽  
Author(s):  
Abhin Kumar Megta ◽  
Airi Palva ◽  
Ingemar von Ossowski ◽  
Vengadesan Krishnan
Keyword(s):  
Lactobacillus Rhamnosus ◽  
Crystal Form ◽  
Data Sets ◽  
Structural Factors ◽  
Lysine Methylation ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Dual Functional ◽  
Anomalous Signal

The SpaB pilin is recognized as the basal subunit of the sortase-dependent SpaCBA pilus, which is known to be produced by the Gram-positive Lactobacillus rhamnosus GG, a gut-adapted commensal advocated to have health benefits. Despite seeming to function as an archetypal basal pilin by serving as the terminal subunit in pilus assembly, SpaB also assumes an atypical role as a mucoadhesive protein. To shed light on the structural factors that contribute to this dual functional behaviour, a recombinant form of the L. rhamnosus GG SpaB pilin was produced and purified for crystallization and X-ray diffraction experiments. The crystallization of SpaB remained particularly challenging until the implementation of a three-pronged crystallization approach involving C-terminal tail truncation, surface lysine methylation and magnesium additives. Ultimately, hexagonal crystals of SpaB were produced and were able to diffract to a resolution of 2.4 Å. This crystal form belonged to space group P6522 or P6122, with unit-cell parameters a = b = 51.53, c = 408.22 Å, α = β = 90.0, γ = 120.0°. Obtaining an interpretable electron-density map via single-wavelength anomalous diffraction (SAD) using iodide-derivative data sets did not succeed owing to the weak anomalous signal. As an alternative, attempts to provide phases by molecular replacement using the iodide-SAD data from SpaB and a collection of distant homology models (<28% sequence identity) are in progress.

Crystallization

2015 ◽  
Author(s):  
Roger G. Harrison ◽  
Paul W. Todd ◽  
Scott R. Rudge ◽  
Demetri P. Petrides
Keyword(s):  
Large Scale ◽  
Three Dimensional ◽  
Crystal Form ◽  
Solid Particles ◽  
Dimensional Structure ◽  
Internal Quality ◽  
Small Scale ◽  
X Ray Diffraction ◽  
Production Scale ◽  
Homogeneous Phase

Crystallization is the process of producing crystals from a homogeneous phase. For biochemicals, the homogeneous phase from which crystals are obtained is always a solution. Crystallization is similar to precipitation in that solid particles are obtained from a solution. However, precipitates have poorly defined morphology, while in crystals the constituent molecules are arranged in three-dimensional arrays called space lattices. In comparison to crystallization, precipitation occurs at much higher levels of supersaturation and rates of nucleation but lower solubilities. These and other differences between crystallization and precipitation are highlighted in Table 9.1. Because of these differences and because the theory of crystallization that has been developed is different from that for precipitation, crystallization is considered separately from precipitation. Crystallization is capable of producing bioproducts at very high purity (say, 99.9%) and is considered to be both a polishing step and a purification step. Polishing refers to a process needed to put the bioproduct in its final form for use. For some bioproducts, such as antibiotics, this final form must be crystalline, and sometimes it is even necessary that a specific crystal form be obtained. In some instances, the purification that can be achieved by crystallization is so significant that other more expensive purification steps such as chromatography can be avoided. There are actually two very different applications of crystallization in biotechnology and bioproduct engineering: crystallization for polishing and purification, and crystallization for crystallography. In the latter case, the goal is a small number of crystals with good size (0.2–0.9 mm) and internal quality. Although it has become common to crystallize proteins for characterization of their three-dimensional structure by x-ray diffraction, this is performed only at small scale in the laboratory, and the knowledge about how to crystallize proteins at large scale in a production process is less developed. However, many antibiotics and other small biomolecules are routinely crystallized in production scale processes. This chapter is oriented toward the use of crystallization in processes that can be scaled up.

scholarly journals Crystallization and preliminary crystallographic studies of PotA, a membrane-associated ATPase of the spermidine-preferential uptake system inThermotoga maritima

2014 ◽  
Vol 70 (6) ◽  
pp. 738-741 ◽  
Author(s):  
Shigeru Sugiyama ◽  
Keiko Kashiwagi ◽  
Keisuke Kakinouchi ◽  
Hideyuki Tomitori ◽  
Ken Kanai ◽  
...  
Keyword(s):  
Thermotoga Maritima ◽  
Three Dimensional ◽  
Crystallographic Analysis ◽  
Uptake System ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Polyamine Uptake ◽  
Preferential Uptake

A membrane-associated ATPase, PotA, is a component of the spermidine-preferential uptake system in prokaryotes that plays an important role in normal cell growth by regulating the cellular polyamine concentration. No three-dimensional structures of membrane-associated ATPases in polyamine-uptake systems have been determined to date. Here, the crystallization and preliminary X-ray diffraction analysis of PotA fromThermotoga maritimaare reported. Diffraction data were collected and processed to 2.7 Å resolution from both native and selenomethionine-labelled crystals. Preliminary crystallographic analysis revealed that the crystals belonged to the hexagonal space groupP3112 (orP3212), with unit-cell parametersa=b= 88.9,c= 221.2 Å, α = 90, β = 90, γ = 120°, indicating that a dimer was present in the asymmetric unit.

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 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 studies of La1 fromLiocheles australasiae

2014 ◽  
Vol 70 (7) ◽  
pp. 915-917 ◽  
Author(s):  
Saori Kamachi ◽  
Junya Nagao ◽  
Masahiro Miyashita ◽  
Yoshiaki Nakagawa ◽  
Hisashi Miyagawa ◽  
...  
Keyword(s):  
Biological Function ◽  
Three Dimensional ◽  
Scorpion Venom ◽  
Dimensional Structure ◽  
Diffusion Method ◽  
Monoclinic Space Group ◽  
X Ray Diffraction ◽  
Cell Parameters ◽  
Factor Type ◽  
Von Willebrand

A novel scorpion venom peptide, La1 fromLiocheles australasiae, with a molecular weight of 7.8 kDa, is presumed to possess a single von Willebrand factor type C (VWC) domain, a common protein module, based on the position of eight Cys residues in its sequence. The biological function of La1 is still unknown. Deciphering its three-dimensional structure will be helpful in understanding its biological function. La1 was crystallized by the sitting-drop vapour-diffusion method using magnesium sulfate as a precipitant. The crystals belonged to the monoclinic space groupC2, with unit-cell parametersa= 63.0,b= 30.2,c= 32.3 Å, β = 108.5°, and diffracted to 1.9 Å resolution. The calculatedVMbased on one molecule per asymmetric unit was 1.87 Å3 Da−1. The solvent content was 34.1%.

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.

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