scholarly journals Crystallization and preliminary X-ray analysis of the NAD+-reducing [NiFe] hydrogenase fromHydrogenophilus thermoluteolusTH-1

2015 ◽  
Vol 71 (1) ◽  
pp. 96-99 ◽  
Author(s):  
Midori Taketa ◽  
Hanae Nakagawa ◽  
Mao Habukawa ◽  
Hisao Osuka ◽  
Kiyohito Kihira ◽  
...  
Keyword(s):  
Diffusion Method ◽  
Asymmetric Unit ◽  
Dispersion Method ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion ◽  
Anomalous Signal ◽  
Th 1

NAD+-reducing [NiFe] hydrogenases catalyze the oxidoreduction of dihydrogen concomitant with the interconversion of NAD+and NADH. Here, the isolation, purification and crystallization of the NAD+-reducing [NiFe] hydrogenase fromHydrogenophilus thermoluteolusTH-1 are reported. Crystals of the NAD+-reducing [NiFe] hydrogenase were obtained within one week from a solution containing polyethylene glycol using the sitting-drop vapour-diffusion method and micro-seeding. The crystal diffracted to 2.58 Å resolution and belonged to space groupC2, with unit-cell parametersa= 131.43,b= 189.71,c= 124.59 Å, β = 109.42°. Assuming the presence of two NAD+-reducing [NiFe] hydrogenase molecules in the asymmetric unit,VMwas calculated to be 2.2 Å3 Da−1, which corresponds to a solvent content of 43%. Initial phases were determined by the single-wavelength anomalous dispersion method using the anomalous signal from the Fe atoms.

scholarly journals Crystallization and X-ray analysis ofD-threonine aldolase fromChlamydomonas reinhardtii

2017 ◽  
Vol 73 (2) ◽  
pp. 86-89
Author(s):  
Yuki Hirato ◽  
Masaru Goto ◽  
Mayumi Tokuhisa ◽  
Minoru Tanigawa ◽  
Katsushi Nishimura
Keyword(s):  
Diffusion Method ◽  
Asymmetric Unit ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters ◽  
Threonine Aldolase ◽  
Vapour Diffusion ◽  
Resolution Analysis

D-Threonine aldolase from the green algaChlamydomonas reinhardtii(CrDTA) catalyzes the interconversion of several β-hydroxy-D-amino acids (e.g.D-threonine) and glycine plus the corresponding aldehydes. Recombinant CrDTA was overexpressed inEscherichia coliand purified to homogeneity; it was subsequently crystallized using the hanging-drop vapour-diffusion method at 295 K. Data were collected and processed at 1.85 Å resolution. Analysis of the diffraction pattern showed that the crystal belonged to space groupP1, with unit-cell parametersa= 64.79,b= 74.10,c= 89.94 Å, α = 77.07, β = 69.34, γ = 71.93°. The asymmetric unit contained four molecules of CrDTA. The Matthews coefficient was calculated to be 2.12 Å3 Da−1and the solvent content was 41.9%.

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 Cloning, purification and preliminary X-ray crystallographic analysis of the OmpA-like domain of peptidoglycan-associated lipoprotein fromAcinetobacter baumannii

2012 ◽  
Vol 68 (11) ◽  
pp. 1351-1353 ◽  
Author(s):  
Jung Hyun Song ◽  
Woo Cheol Lee ◽  
Jeong Soon Park ◽  
Seung Il Kim ◽  
Je Chul Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acinetobacter Baumannii ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion ◽  
Strain Bl21

Peptidoglycan-associated lipoprotein (Pal) is one component of the Tol–Pal system that is involved in maintaining the integrity and stability of the outer membrane. The C-terminal OmpA-like domain of Pal interacts noncovalently with peptidoglycan. In this study, the OmpA-like domain of Pal fromAcinetobacter baumanniiwas overexpressed inEscherichia colistrain BL21 (DE3), purified and crystallized using the vapour-diffusion method. A native crystal diffracted to 1.4 Å resolution and belonged to space groupP61orP65, with unit-cell parametersa=b= 72.58,c= 44.65 Å, a calculated Matthews coefficient of 2.64 Å3 Da−1and one molecule per asymmetric unit.

scholarly journals Crystallization and preliminary X-ray analysis of Rv1674c fromMycobacterium tuberculosis

2015 ◽  
Vol 71 (3) ◽  
pp. 354-357 ◽  
Author(s):  
Jincheng Li ◽  
Xudong Wang ◽  
Weimin Gong ◽  
Chunyan Niu ◽  
Min Zhang
Keyword(s):  
Diffusion Method ◽  
Dna Binding Domain ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Protein Molecules ◽  
Vapour Diffusion ◽  
Transcription Regulators ◽  
Interdomain Interaction

Adaptations to hypoxia play an important role inMycobacterium tuberculosispathogenesis. Rv0324, which contains an HTH DNA-binding domain and a rhodanese domain, is one of the key transcription regulators in response to hypoxia.M. tuberculosisRv1674c is a homologue of Rv0324. To understand the interdomain interaction and regulation of the HTH domain and the rhodanese domain, recombinant Rv1674c protein was purified and crystallized by the vapour-diffusion method. The crystals diffracted to 2.25 Å resolution. Preliminary diffraction analysis suggests that the crystals belonged to space groupP3121 orP3221, with unit-cell parametersa=b= 67.8,c= 174.5 Å, α = β = 90, γ = 120°. The Matthews coefficient was calculated to be 2.44 Å3 Da−1, assuming that the crystallographic asymmetric unit contains two protein molecules.

scholarly journals Plant-specific DUF1110 protein fromOryza sativa: expression, purification and crystallization

2016 ◽  
Vol 72 (6) ◽  
pp. 480-484 ◽  
Author(s):  
Kenichi Harada ◽  
Eiki Yamashita ◽  
Kento Inoue ◽  
Koji Yamaguchi ◽  
Toshimichi Fujiwara ◽  
...  
Keyword(s):  
Diffusion Method ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Sequence Identity ◽  
Vapour Diffusion ◽  
Domain Of Unknown Function

The Os01T0156300 protein fromOryza sativahas been classified into the domain of unknown function (DUF) family DUF1110. DUF1110 family members exist in monocotyledons but not in dicotyledons, and share no sequence identity with proteins for which structures have been reported. In this study, the Os01T0156300 protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.84 Å resolution. The crystal belonged to space groupP21, with unit-cell parametersa= 89.9,b= 89.8,c= 107.1 Å, β = 106.6°. The asymmetric unit was estimated to contain 6–11 molecules.

scholarly journals Cloning, expression, crystallization and preliminary X-ray diffraction studies of staphylococcal superantigen-like protein 1 (SSL1)

2014 ◽  
Vol 70 (5) ◽  
pp. 600-603 ◽  
Author(s):  
Debabrata Dutta ◽  
Anirudha Dutta ◽  
Atanu Bhattacharjee ◽  
Amit Basak ◽  
Amit Kumar Das
Keyword(s):  
Expression Vector ◽  
Diffusion Method ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Vapour Diffusion ◽  
Structural Homologues

Staphylococcus aureusproduces a family of exotoxins which are structural homologues of superantigens and thus are called staphylococcal superantigen-like proteins (SSLs). Amongst the 14 SSL genes,ssl1(SAOUHSC_00383) has been cloned in the pQE30 expression vector, overexpressed inEscherichia coliM15 (pREP4) cells and the protein purified to homogeneity. The protein was crystallized using 6% Tacsimate pH 6.0, 0.1 MMES pH 6.0, 25%(w/v) polyethylene glycol 3350, 100 mMNDSB 256 at 298 K by the sitting-drop vapour-diffusion method. The crystals belonged to space groupP21, with unit-cell parametersa= 77.9,b= 70.5,c= 126.5 Å, β = 106.2°. X-ray diffraction data were collected and processed to a maximum resolution of 2.5 Å. The crystal contains six molecules in the asymmetric unit.

scholarly journals Crystallographic analysis of FAD-dependent glucose dehydrogenase

2015 ◽  
Vol 71 (8) ◽  
pp. 1017-1019 ◽  
Author(s):  
Hirofumi Komori ◽  
Koji Inaka ◽  
Naoki Furubayashi ◽  
Michinari Honda ◽  
Yoshiki Higuchi
Keyword(s):  
Aspergillus Terreus ◽  
Glucose Dehydrogenase ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Data Set ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters

An FAD-dependent glucose dehydrogenase (GDH) fromAspergillus terreuswas purified and crystallized at 293 K using the sitting-drop vapour-diffusion method. A data set was collected to a resolution of 1.6 Å from a single crystal at 100 K using a rotating-anode X-ray source. The crystal belonged to space groupP21, with unit-cell parametersa= 56.56,b= 135.74,c= 74.13 Å, β = 90.37°. The asymmetric unit contained two molecules of GDH. The Matthews coefficient was calculated to be 2.2 Å3 Da−1and the solvent content was estimated to be 44%.

scholarly journals Crystallographic analysis of NosA, which catalyzes terminal amide formation in the biosynthesis of nosiheptide

2015 ◽  
Vol 71 (8) ◽  
pp. 1033-1037 ◽  
Author(s):  
Yanting Wang ◽  
Shanshan Liu ◽  
Pengfei Yao ◽  
Yi Yu ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Diffraction Data ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
Cell Parameters ◽  
Vapour Diffusion ◽  
Streptomyces Actuosus

Nosiheptide is a member of the thiopeptide family of antibiotics which demonstrates potent activities against various bacterial pathogens. The formation of its C-terminal amide is catalysed by NosA in an unusual strategy for maturating certain thiopeptides by processing precursor peptides featuring a serine extension. Here, a recombinant C-terminally truncated selenomethionine-derivatized NosA1–111variant fromStreptomyces actuosusconsisting of residues 1–111, named SeMet NosA1–111, was crystallized using the sitting-drop vapour-diffusion method. Diffraction data were collected to 2.40 Å resolution using synchrotron radiation. The crystals belonged to the primitive cubic space groupP4132, with unit-cell parametersa=b=c= 143.3 Å. Assuming the presence of three molecules in the asymmetric unit, the calculated Matthews coefficient was 3.94 Å3 Da−1and the corresponding solvent content was 40.3%.

Crystallization and preliminary X-ray crystallographic analysis of a vancomycin–N-acetyl-D-Ala-D-Ala complex

1999 ◽  
Vol 55 (2) ◽  
pp. 534-535 ◽  
Author(s):  
Deborah McPhail ◽  
Alan Cooper ◽  
Andy Freer
Keyword(s):  
Room Temperature ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
The Novel ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters ◽  
Crystal Density

A vancomycin–N-acetyl-D-Ala-D-Ala complex has been crystallized by the sitting-drop vapour-diffusion method using imidazole maleic buffer at pH 7.6. The novel crystals obtained belong to the space group P6322 with unit-cell parameters a = b = 73.43 (1), c = 277.17 (4) Å, γ = 120°. The crystal density was determined as 1.106 g cm−3 which gives a supercell of 24 molecules (12 dimers) per asymmetric unit for an acceptable Matthews number and an estimated solvent content of 42%. Data were collected at room temperature to 2.8 Å.

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.

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