Crystallization and preliminary X-ray characterization of the full-length bacteriophytochrome from the plant pathogenXanthomonas campestrispv.campestris

Phytochromes give rise to the largest photosensor family known to date. However, they are underrepresented in the Protein Data Bank. Plant, cyanobacterial, fungal and bacterial phytochromes share a canonical architecture consisting of an N-terminal photosensory module (PAS2–GAF–PHY domains) and a C-terminal variable output module. The bacteriumXanthomonas campestrispv.campestris, a worldwide agricultural pathogen, codes for a single bacteriophytochrome (XccBphP) that has this canonical architecture, bearing a C-terminal PAS9 domain as the output module. Full-length XccBphP was cloned, expressed and purified to homogeneity by nickel–NTA affinity and size-exclusion chromatography and was then crystallized at room temperature bound to its cofactor biliverdin. A complete native X-ray diffraction data set was collected to a maximum resolution of 3.25 Å. The crystals belonged to space groupP43212, with unit-cell parametersa=b= 103.94,c= 344.57 Å and a dimer in the asymmetric unit. Refinement is underway after solving the structure by molecular replacement.

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Purification, crystallization and preliminary X-ray diffraction analysis of a soluble variant of the monoglyceride lipase Yju3p from the yeastSaccharomyces cerevisiae

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15001557 ◽

2015 ◽

Vol 71 (2) ◽

pp. 243-246 ◽

Cited By ~ 2

Author(s):

Srinivasan Rengachari ◽

Philipp Aschauer ◽

Christian Sturm ◽

Monika Oberer

Keyword(s):

Crystal Form ◽

Size Exclusion ◽

X Ray Diffraction ◽

Unit Cell Parameters ◽

Orthorhombic Space Group ◽

Data Set ◽

X Ray ◽

Cell Parameters ◽

Monoglyceride Lipase ◽

Exclusion Chromatography

The protein Yju3p is the orthologue of monoglyceride lipases in the yeastSaccharomyces cerevisiae. A soluble variant of this lipase termed s-Yju3p (38.3 kDa) was generated and purified to homogeneity by affinity and size-exclusion chromatography. s-Yju3p was crystallized in a vapour-diffusion setup at 293 K and a complete data set was collected to 2.4 Å resolution. The crystal form was orthorhombic (space groupP212121), with unit-cell parametersa= 77.2,b= 108.6,c= 167.7 Å. The asymmetric unit contained four molecules with a solvent content of 46.4%.

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Purification, crystallization and preliminary X-ray diffraction analysis of 3-ketoacyl-CoA thiolase A1887 fromRalstonia eutrophaH16

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15007888 ◽

2015 ◽

Vol 71 (6) ◽

pp. 758-762

Author(s):

Jieun Kim ◽

Kyung-Jin Kim

Keyword(s):

Ralstonia Eutropha ◽

Size Exclusion ◽

Asymmetric Unit ◽

X Ray Diffraction ◽

Unit Cell Parameters ◽

Data Set ◽

X Ray ◽

Cell Parameters ◽

Exclusion Chromatography ◽

Fourth Step

The gene product of A1887 fromRalstonia eutropha(ReH16_A1887) has been annotated as a 3-ketoacyl-CoA thiolase, an enzyme that catalyzes the fourth step of β-oxidation degradative pathways by converting 3-ketoacyl-CoA to acyl-CoA.ReH16_A1887 was overexpressed and purified to homogeneity by affinity and size-exclusion chromatography. The degradative thiolase activity of the purifiedReH16_A1887 was measured and enzyme-kinetic parameters for the protein were obtained, withKm,Vmaxandkcatvalues of 158 µM, 32 mM min−1and 5 × 106 s−1, respectively. TheReH16_A1887 protein was crystallized in 17% PEG 8K, 0.1 MHEPES pH 7.0 at 293 K and a complete data set was collected to 1.4 Å resolution. The crystal belonged to space groupP43212, with unit-cell parametersa=b= 129.52,c= 114.13 Å, α = β = γ = 90°. The asymmetric unit contained two molecules, with a solvent content of 58.9%.

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Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease fromPseudomonas aeruginosa

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14017865 ◽

2014 ◽

Vol 70 (10) ◽

pp. 1362-1367 ◽

Cited By ~ 3

Author(s):

Emmanuel Nji ◽

Dianfan Li ◽

Declan A. Doyle ◽

Martin Caffrey

Keyword(s):

Metal Ion ◽

Cell Volume Regulation ◽

Ion Concentration ◽

Size Exclusion ◽

Substrate Selectivity ◽

X Ray Diffraction ◽

Unit Cell Parameters ◽

X Ray ◽

Cell Parameters ◽

Exclusion Chromatography

The prokaryotic lysine-specific permease (LysP) belongs to the amino acid–polyamine–organocation (APC) transporter superfamily. In the cell, members of this family are responsible for the uptake and recycling of nutrients, for the maintenance of a constant internal ion concentration and for cell volume regulation. The detailed mechanism of substrate selectivity and transport of L-lysine by LysP is not understood. A high-resolution crystal structure would enormously facilitate such an understanding. To this end, LysP fromPseudomonas aeruginosawas recombinantly expressed inEscherichia coliand purified to near homogeneity by immobilized metal ion-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Hexagonal- and rod-shaped crystals were obtained in the presence of L-lysine and the L-lysine analogue L-4-thialysine by vapour diffusion and diffracted to 7.5 Å resolution. The diffraction data were indexed in space groupP21, with unit-cell parametersa= 169.53,b= 169.53,c= 290.13 Å, γ = 120°.

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VirB8 homolog TraE from plasmid pKM101 forms a hexameric ring structure and interacts with the VirB6 homolog TraD

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1802501115 ◽

2018 ◽

Vol 115 (23) ◽

pp. 5950-5955 ◽

Cited By ~ 6

Author(s):

Bastien Casu ◽

Charline Mary ◽

Aleksandr Sverzhinsky ◽

Aurélien Fouillen ◽

Antonio Nanci ◽

...

Keyword(s):

Molecular Mass ◽

Secretion System ◽

High Molecular Mass ◽

Full Length ◽

Size Exclusion ◽

Cross Linking ◽

X Ray ◽

Exclusion Chromatography ◽

Plasmid Pkm101 ◽

Membrane Bound

Type IV secretion systems (T4SSs) are multiprotein assemblies that translocate macromolecules across the cell envelope of bacteria. X-ray crystallographic and electron microscopy (EM) analyses have increasingly provided structural information on individual T4SS components and on the entire complex. As of now, relatively little information has been available on the exact localization of the inner membrane-bound T4SS components, notably the mostly periplasmic VirB8 protein and the very hydrophobic VirB6 protein. We show here that the membrane-bound, full-length version of the VirB8 homolog TraE from the plasmid pKM101 secretion system forms a high-molecular-mass complex that is distinct from the previously characterized periplasmic portion of the protein that forms dimers. Full-length TraE was extracted from the membranes with detergents, and analysis by size-exclusion chromatography, cross-linking, and size exclusion chromatography (SEC) multiangle light scattering (MALS) shows that it forms a high-molecular-mass complex. EM and small-angle X-ray scattering (SAXS) analysis demonstrate that full-length TraE forms a hexameric complex with a central pore. We also overproduced and purified the VirB6 homolog TraD and show by cross-linking, SEC, and EM that it binds to TraE. Our results suggest that TraE and TraD interact at the substrate translocation pore of the secretion system.

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PsEst3, a new psychrophilic esterase from the Arctic bacterium Paenibacillus sp. R4: crystallization and X-ray crystallographic analysis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18007525 ◽

2018 ◽

Vol 74 (6) ◽

pp. 367-372

Author(s):

Hyun Kim ◽

Ae Kyung Park ◽

Jun Hyuck Lee ◽

Seung Chul Shin ◽

Hyun Park ◽

...

Keyword(s):

Expression System ◽

Crystallographic Analysis ◽

Size Exclusion ◽

The Arctic ◽

Diffusion Method ◽

X Ray ◽

Cell Parameters ◽

Paenibacillus Sp ◽

Exclusion Chromatography ◽

And Function

Esterases are very useful biocatalysts in industry: they hydrolyze esters and split them into a carboxylic acid and an alcohol. The psychrophilic esterase PsEst3 was obtained from Paenibacillus sp. R4, which was isolated from the active layer of the permafrost in Council, Alaska. PsEst3 was successfully overexpressed using a psychrophilic chaperonin co-expression system and was purified by nickel-affinity and size-exclusion chromatography. Recombinant PsEst3 was crystallized at 290 K using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.1 Å resolution. The crystal was determined to belong to space group P4132 or P4332, with unit-cell parameters a = b = c = 145.33 Å. Further crystallographic analysis needs to be conducted to investigate the structure and function of this esterase.

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Purification, crystallization and preliminary X-ray diffraction studies of UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) fromSynechococcussp. PCC 7942

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x13034298 ◽

2014 ◽

Vol 70 (2) ◽

pp. 203-205

Author(s):

Asaithambi Killivalavan ◽

Ningning Zhuang ◽

Young Shik Park ◽

Kon Ho Lee

Keyword(s):

Size Exclusion ◽

Diffusion Method ◽

Monoclinic Space Group ◽

X Ray Diffraction ◽

Dispersion Method ◽

X Ray ◽

Cell Parameters ◽

Exclusion Chromatography ◽

Multi Wavelength ◽

Pcc 7942

A UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) enzyme was discovered in the cyanobacteriumSynechococcussp. PCC 7942 which transfers a glucose moiety from UDP-glucose to tetrahydrobiopterin (BH4). BGluT protein was overexpressed with selenomethionine labelling for structure determination by the multi-wavelength anomalous dispersion method. The BGluT protein was purified by nickel-affinity and size-exclusion chromatography. It was then crystallized by the hanging-drop vapour-diffusion method using a well solution consisting of 0.1 Mbis-tris pH 5.5, 19%(w/v) polyethylene glycol 3350 with 4%(w/v) D(+)-galactose as an additive. X-ray diffraction data were collected to 1.99 Å resolution using a synchrotron-radiation source. The crystals belonged to the monoclinic space groupC2, with unit-cell parametersa= 171.35,b= 77.99,c = 53.77 Å, β = 90.27°.

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The Pseudomonas syringae pv. actinidiae chemoreceptor protein F (PscF) periplasmic sensor domain: cloning, purification and X-ray crystallographic analysis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17016831 ◽

2017 ◽

Vol 73 (12) ◽

pp. 701-705 ◽

Cited By ~ 2

Author(s):

Tifany Oulavallickal ◽

Jodi L. Brewster ◽

James L. O. McKellar ◽

Michael J. Fairhurst ◽

Nicholas A. Tenci ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Klebsiella Oxytoca ◽

Crystallographic Analysis ◽

Size Exclusion ◽

Protein Crystal ◽

Data Set ◽

Space Group ◽

Maximum Resolution ◽

Exclusion Chromatography ◽

Nitrate And Nitrite

Nitrate- and nitrite-sensing (NIT) domains are found associated with a wide variety of bacterial receptors, including chemoreceptors. However, the structure of a chemoreceptor-associated NIT domain has not yet been characterized. Recently, a chemoreceptor named PscF was identified from the plant pathogen Pseudomonas syringae pv. actinidiae that is predicted to contain a periplasmic NIT domain. The PscF sensor domain (PscF-SD; residues 42–332) was cloned into an appropriate expression vector, recombinantly produced in Escherichia coli BL21-Gold(DE3) cells and purified via immobilized metal-affinity and size-exclusion chromatography. Purified PscF-SD was screened for crystallization; the best crystal diffracted to a maximum resolution of 1.46 Å in space group P212121. However, the data could not be phased using the only available NIT-domain structure (Klebsiella oxytoca NasR; PDB entry 4akk) as the search model. Therefore, a data set from a selenomethionine-labelled protein crystal was also collected. The selenomethionine-labelled protein crystal diffracted to a resolution of 2.46 Å in space group P212121. These data will be used to attempt to solve the structure using the single-wavelength anomalous diffraction technique. The structure is expected to provide insights into the ligand specificity of NIT domains and the role of NIT domains in chemotaxis.

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Structure of the Prx6-subfamily 1-Cys peroxiredoxin from Sulfolobus islandicus

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x19006472 ◽

2019 ◽

Vol 75 (6) ◽

pp. 428-434 ◽

Cited By ~ 2

Author(s):

Sander Stroobants ◽

Inge Van Molle ◽

Queen Saidi ◽

Karl Jonckheere ◽

Dominique Maes ◽

...

Keyword(s):

Crystal Structure ◽

Quaternary Structure ◽

Size Exclusion ◽

Molecular Replacement ◽

Oligomeric State ◽

Data Set ◽

Cell Parameters ◽

Aeropyrum Pernix ◽

Sulfolobus Islandicus ◽

Exclusion Chromatography

Aerobic thermoacidophilic archaea belonging to the genus Sulfolobus harbor peroxiredoxins, thiol-dependent peroxidases that assist in protecting the cells from oxidative damage. Here, the crystal structure of the 1-Cys peroxiredoxin from Sulfolobus islandicus, named 1-Cys SiPrx, is presented. A 2.75 Å resolution data set was collected from a crystal belonging to space group P212121, with unit-cell parameters a = 86.8, b = 159.1, c = 189.3 Å, α = β = γ = 90°. The structure was solved by molecular replacement using the homologous Aeropyrum pernix peroxiredoxin (ApPrx) structure as a search model. In the crystal structure, 1-Cys SiPrx assembles into a ring-shaped decamer composed of five homodimers. This quaternary structure corresponds to the oligomeric state of the protein in solution, as observed by size-exclusion chromatography. 1-Cys SiPrx harbors only a single cysteine, which is the peroxidatic cysteine, and lacks both of the cysteines that are highly conserved in the C-terminal arm domain in other archaeal Prx6-subfamily proteins such as ApPrx and that are involved in the association of dimers into higher-molecular-weight decamers and dodecamers. It is thus concluded that the Sulfolobus Prx6-subfamily protein undergoes decamerization independently of arm-domain cysteines.

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Purification, crystallization and preliminary X-ray diffraction analysis of ArsH fromSynechocystissp. strain PCC 6803

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14004865 ◽

2014 ◽

Vol 70 (4) ◽

pp. 497-500 ◽

Cited By ~ 2

Author(s):

Xiao Zhang ◽

Xi-Mei Xue ◽

Yu Yan ◽

Jun Ye

Keyword(s):

Sinorhizobium Meliloti ◽

Size Exclusion ◽

Diffusion Method ◽

Flavin Mononucleotide ◽

Molecular Replacement ◽

X Ray Diffraction ◽

X Ray ◽

Cell Parameters ◽

Exclusion Chromatography ◽

Pcc 6803

ArsH is an NADPH-dependent flavin mononucleotide reductase and is frequently encoded as part of anarsoperon. The function of thearsHgene remains to be characterized. Crystallization and structural studies may contribute to elucidating the specific biological function of ArsH associated with arsenic resistance. ArsH fromSynechocystissp. strain PCC 6803 was overproduced, purified and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 1.6 Å. The crystals belonged to the tetragonal space groupI4122, with unit-cell parametersa=b= 127.94,c= 65.86 Å and one molecule in the asymmetric unit. Size-exclusion chromatography and molecular-replacement results showed that the ArsH formed a tetramer. Further structural analysis and comparison with ArsH fromSinorhizobium melilotiwill provide information about the oligomerization of ArsH.

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Crystallographic studies of SarV, a global regulator fromStaphylococcus aureus

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15011097 ◽

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.

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