Purification, crystallization and preliminary X-ray diffraction analysis of theN-acetyltransferase SAV0826 fromStaphylococcus aureus

Staphylococcus aureusis a prevalent microorganism that is capable of causing a wide range of infections and diseases. Several strains of this bacterial species have developed antibiotic resistance to methicillin and vancomycin, and higher death rates are still being reported each year owing to multidrug-resistant strains. Certain GCN5-relatedN-acetyltransferases (GNATs) exhibit a broad substrate range, including aminoglycosides, histones, other proteins and serotonin, and have been implicated in antibiotic drug resistance. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis of a GNAT fromS. aureus(SaNAT) are reported. SaNAT was recombinantly expressed and crystallized by the hanging-drop vapour-diffusion method at 296 K, and the crystals diffracted to 1.7 Å resolution on the MX2 beamline at the Australian Synchrotron. The crystals belonged to space groupP43212, with unit-cell parametersa=b= 84.86,c= 49.06 Å, α = β = γ = 90°. A single molecule is likely to be present in the asymmetric unit. A full structural and functional analysis is currently being undertaken to provide novel insights into the protein function, which in turn may provide a basis for drug design.

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Crystallization and preliminary X-ray diffraction analysis of Trap1 complexed with Hsp90 inhibitors

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14024959 ◽

2014 ◽

Vol 70 (12) ◽

pp. 1683-1687 ◽

Cited By ~ 3

Author(s):

Hanbin Jeong ◽

Byoung Heon Kang ◽

Changwook Lee

Keyword(s):

Diffraction Analysis ◽

Diffusion Method ◽

X Ray Diffraction ◽

Hanging Drop ◽

Hsp90 Inhibitors ◽

Unit Cell Parameters ◽

X Ray ◽

Cell Parameters ◽

Middle Domain ◽

Client Proteins

Hsp90 is a molecular chaperone responsible for the assembly and regulation of many cellular client proteins. In particular, Trap1, a mitochondrial Hsp90 homologue, plays a pivotal role in maintaining mitochondrial integrity, protecting against apoptosis in cancer cells. The N (N-terminal)-M (middle) domain of human Trap1 was crystallized in complex with Hsp90 inhibitors (PU-H71 and BIIB-021) by the hanging-drop vapour-diffusion method at pH 6.5 and 293 K using 15% PEG 8K as a precipitant. Diffraction data were collected from crystals of the Trap1–PU-H71 (2.7 Å) and Trap1–BIIB-021 (3.1 Å) complexes to high resolution at a synchrotron-radiation source. Preliminary X-ray diffraction analysis revealed that both crystals belonged to space groupP41212 orP43212, with unit-cell parametersa=b= 69.2,c= 252.5 Å, and contained one molecule per asymmetric unit according to Matthews coefficient calculations.

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Crystallization and preliminary X-ray diffraction analysis of the homing endonuclease I-CvuI fromChlorella vulgarisin complex with its target DNA

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x1400065x ◽

2014 ◽

Vol 70 (2) ◽

pp. 256-259 ◽

Cited By ~ 2

Author(s):

Pilar Redondo ◽

Nekane Merino ◽

Maider Villate ◽

Francisco J. Blanco ◽

Guillermo Montoya ◽

...

Keyword(s):

Diffraction Analysis ◽

Homing Endonuclease ◽

Homing Endonucleases ◽

Resolution Limit ◽

X Ray Diffraction ◽

Orthorhombic Space Group ◽

Strand Breaks ◽

X Ray ◽

Cell Parameters ◽

Wide Range

Homing endonucleases are highly specific DNA-cleaving enzymes that recognize long stretches of DNA. The engineering of these enzymes provides novel instruments for genome modification in a wide range of fields, including gene targeting, by inducing specific double-strand breaks. I-CvuI is a homing endonuclease from the green algaChlorella vulgaris. This enzyme was purified after overexpression inEscherichia coli. Crystallization experiments of I-CvuI in complex with its DNA target in the presence of Mg2+yielded crystals suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 62.83,b= 83.56,c= 94.40 Å. The self-rotation function and the Matthews coefficient suggested the presence of one protein–DNA complex per asymmetric unit. The crystals diffracted to a resolution limit of 1.9 Å using synchrotron radiation.

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Xylanase B fromClostridium cellulovorans743B: overexpression, purification, crystallization and X-ray diffraction analysis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18000341 ◽

2018 ◽

Vol 74 (2) ◽

pp. 113-116 ◽

Cited By ~ 1

Author(s):

Daichi Nakajima ◽

Akihiko Nagano ◽

Toshiyuki Shibata ◽

Reiji Tanaka ◽

Kouichi Kuroda ◽

...

Keyword(s):

Diffraction Analysis ◽

Catalytic Domain ◽

Cellulosic Biomass ◽

Diffusion Method ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

X Ray Diffraction ◽

X Ray ◽

Cell Parameters ◽

Peg 4000

Clostridium cellulovoransproduces multi-enzyme complexes called cellulosomes capable of efficiently degrading cellulosic biomass. There are three xylanase genes containing a sequence corresponding to a dockerin domain that are necessary for constructing cellulosomes in the genome. Among the xylanases encoded by these genes, xylanase B (XynB) contains a catalytic domain belonging to glycoside hydrolase family 10 and a carbohydrate-binding module (CBM) at the N-terminus, making it a member of CBM family 22. In this study, XynB was cloned, overexpressed, purified and crystallized. XynB was crystallized using the hanging-drop vapour-diffusion method in the presence of 0.2 Msodium acetate trihydrate, 0.1 MTris–HCl pH 8.5, 32%(w/v) PEG 4000 at 293 K. X-ray diffraction analysis revealed that the crystal diffracted to 1.95 Å resolution and belonged to space groupP212121, with unit-cell parametersa = 74.28,b= 77.55,c= 88.20 Å, α = β = γ = 90°. The data-evaluation statistics revealed high quality of the collected data, thereby establishing a solid basis for determination of the structure of cellulosomal xylanase fromC. cellulovorans.

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Crystallization and X-ray structure determination of a thermoalkalophilic lipase fromGeobacillusSBS-4S

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309113004910 ◽

2013 ◽

Vol 69 (4) ◽

pp. 355-357

Author(s):

Muhammad Tayyab ◽

Naeem Rashid ◽

Clement Angkawidjaja ◽

Shigenori Kanaya ◽

Muhammad Akhtar

Keyword(s):

Diffusion Method ◽

Molecular Replacement ◽

X Ray Diffraction ◽

Orthorhombic Space Group ◽

X Ray ◽

Cell Parameters ◽

Replacement Method ◽

Wide Range ◽

Molecular Replacement Method

A thermoalkalophilic lipase (LIPSBS) from the newly isolatedGeobacillusstrain SBS-4S which hydrolyzes a wide range of fatty acids has been characterized. In the present study, the crystallization of purified LIPSBSusing the sitting-drop vapour-diffusion method and its X-ray diffraction studies are described. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 55.13,b= 71.75,c= 126.26 Å. The structure was determined at 1.6 Å resolution by the molecular-replacement method using the lipase fromG. stearothermophilusL1 as a model.

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Crystallization and preliminary X-ray diffraction analysis of the Csu pili CsuC–CsuA/B chaperone–major subunit pre-assembly complex fromAcinetobacter baumannii

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15007955 ◽

2015 ◽

Vol 71 (6) ◽

pp. 770-774 ◽

Cited By ~ 6

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.

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Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF fromMicrocystis aeruginosa, a putative reductase participating in aeruginosin biosynthesis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15005063 ◽

2015 ◽

Vol 71 (4) ◽

pp. 466-470 ◽

Cited By ~ 1

Author(s):

Ruyi Ding ◽

Cui Xu ◽

Xu Chen ◽

Mengyun Bao ◽

Xiaoting Qiu

Keyword(s):

Diffraction Analysis ◽

Biosynthetic Pathway ◽

Molecular Replacement ◽

X Ray Diffraction ◽

Antithrombotic Activity ◽

X Ray ◽

Cell Parameters ◽

Maximum Resolution ◽

Replacement Method ◽

Molecular Replacement Method

The 2-carboxy-6-hydroxyoctahydroindole moiety is an essential residue for the antithrombotic activity of aeruginosins, which are a class of cyanobacteria-derived bioactive linear tetrapeptides. The biosynthetic pathway of the 2-carboxy-6-hydroxyoctahydroindole moiety has not yet been resolved. AerF was indicated to be involved in the biosynthesis of the 2-carboxy-6-hydroxyoctahydroindole moiety. This study reports the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF fromMicrocystis aeruginosawith a C-terminal His6tag. The crystal diffracted to a maximum resolution of 1.38 Å and belonged to the tetragonal space groupP4322, with unit-cell parametersa=b= 101.581,c= 116.094 Å. The calculated Matthews coefficient and solvent content of the crystal were 2.47 Å3 Da−1and 50.32%, respectively. The initial model of the structure was obtained by the molecular-replacement method and refinement of the structure is in progress.

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Crystallization and X-ray diffraction analysis of native and selenomethionine-substituted PhyH-DI fromBacillussp. HJB17

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17015102 ◽

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.

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Purification, crystallization and preliminary X-ray diffraction analysis of Imp3 in complex with an Mpp10 peptide involved in yeast ribosome biogenesis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14010905 ◽

2014 ◽

Vol 70 (7) ◽

pp. 918-921 ◽

Cited By ~ 10

Author(s):

Sanduo Zheng ◽

Keqiong Ye

Keyword(s):

Diffraction Analysis ◽

Associated Factors ◽

Ribosome Biogenesis ◽

X Ray Diffraction ◽

Unit Cell Parameters ◽

Vast Number ◽

X Ray ◽

Cell Parameters ◽

Early Processing ◽

Ribosomal Particle

Eukaryotic ribosome synthesis requires a vast number of transiently associated factors. Mpp10, Imp3 and Imp4 form a protein complex in the 90S pre-ribosomal particle that conducts early processing of 18S rRNA. Here, a short fragment of Mpp10 was identified to associate with and increase the solubility of Imp3. An Imp3–Mpp10 complex was co-expressed, co-purified and co-crystallized. Preliminary X-ray diffraction analysis revealed that the crystal diffracted to 2.1 Å resolution and belonged to space groupP212121, with unit-cell parametersa= 51.6,b= 86.9,c= 88.7 Å.

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Synthesis, growth, structure and characterization of molybdenum zinc thiourea complex crystals

Acta Crystallographica Section B Structural Science Crystal Engineering and Materials ◽

10.1107/s2052520615005922 ◽

2015 ◽

Vol 71 (3) ◽

pp. 285-292 ◽

Cited By ~ 1

Author(s):

M. Rajasekar ◽

K. Muthu ◽

A. Aditya Prasad ◽

R. Agilandeshwari ◽

SP Meenakshisundaram

Keyword(s):

Single Crystal ◽

Diffraction Analysis ◽

Intermolecular Interactions ◽

Crystal Packing ◽

Second Harmonic ◽

Morphological Changes ◽

Strong Interactions ◽

X Ray Diffraction ◽

X Ray ◽

Cell Parameters

Single crystals of molybdenum-incorporated tris(thiourea)zinc(II) sulfate (MoZTS) are grown by the slow evaporation solution growth technique. Crystal composition as determined by single-crystal X-ray diffraction analysis reveals that it belongs to the orthorhombic system with space groupPca21and cell parametersa= 11.153 (2),b= 7.7691 (14),c= 15.408 (3) Å,V= 1335.14 (4) Å3andZ= 4. The surface morphological changes are studied by scanning electron microscopy. The vibrational patterns in FT–IR are used to identify the functional group and TGA/DTA (thermogravimetric analysis/differential thermal analysis) indicates the stability of the material. The structure and the crystallinity of the material were confirmed by powder X-ray diffraction analysis and the simulated X-ray diffraction (XRD) closely matches the experimental one with varied intensity patterns. The band gap energy is estimated using diffuse reflectance data by the application of the Kubelka–Munk algorithm. The relative second harmonic generation (SHG) efficiency measurements reveal that MoZTS has an efficiency comparable to that of tris(thiourea)zinc(II) sulfate (ZTS). Hirshfeld surfaces were derived using single-crystal X-ray diffraction data. Investigation of the intermolecular interactions and crystal packingviaHirshfeld surface analysis reveal that the close contacts are associated with strong interactions. Intermolecular interactions as revealed by the fingerprint plot and close packing could be the possible reasons for facile charge transfer leading to SHG activity.

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Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal DUF490963–1138domain of TamB fromEscherichia coli

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14017403 ◽

2014 ◽

Vol 70 (9) ◽

pp. 1272-1275 ◽

Cited By ~ 4

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.

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