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 Purification, crystallization and preliminary crystallographic analysis of a 4-thiouridine synthetase–RNA complex

2013 ◽  
Vol 69 (4) ◽  
pp. 421-424 ◽  
Author(s):  
Peter-Thomas Naumann ◽  
Charles T. Lauhon ◽  
Ralf Ficner
Keyword(s):  
Diffraction Data ◽  
Thermotoga Maritima ◽  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Dependent Modification ◽  
Rna Complex

The sulfurtransferase 4-thiouridine synthetase (ThiI) is involved in the ATP-dependent modification of U8 in tRNA. ThiI fromThermotoga maritimawas cloned, overexpressed and purified. A complex comprising ThiI and a truncated tRNA was prepared and crystallized, and X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 102.9,b= 112.8,c= 132.8 Å.

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 Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp6

2014 ◽  
Vol 70 (6) ◽  
pp. 794-799 ◽  
Author(s):  
Junko Morita ◽  
Kazuki Kato ◽  
Emiko Mihara ◽  
Ryuichiro Ishitani ◽  
Junichi Takagi ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Choline Metabolism ◽  
Protein Molecules ◽  
Membrane Bound

Enpp (ectonucleotide phosphodiesterase/pyrophosphatase) 6 is a membrane-bound glycoprotein that hydrolyzes choline-containing compounds such as lysophosphatidylcholine and glycerophosphorylcholine, and presumably participates in choline metabolism. The catalytic domain of mouse Enpp6 was expressed in HEK293T cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 1.8 Å resolution. The crystal belonged to space groupP1, with unit-cell parametersa= 63.7,b= 68.8,c= 69.7 Å, α = 60.6, β = 87.0, γ = 68.1°. Assuming the presence of two protein molecules per asymmetric unit, the solvent content was estimated to be 49.5%.

scholarly journals Purification, crystallization and X-ray crystallographic analysis of human RAB11(S20V), a constitutively active GTP-binding form

2015 ◽  
Vol 71 (10) ◽  
pp. 1247-1250 ◽  
Author(s):  
Chang Min Kim ◽  
Jae Young Choi ◽  
Jong Hwan Yoon ◽  
Hyun Ho Park
Keyword(s):  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Gtp Hydrolysis ◽  
X Ray ◽  
Cell Parameters ◽  
Gtp Binding ◽  
His Tag ◽  
Hydrolysis Activity ◽  
Constitutively Active

RAB11, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking during endosome recycling. Substitution of Ser20 by Val20 in Rab11 [RAB11(S20V)] inhibits its GTP hydrolysis activity and produces a constitutively active GTP-binding form. In this study, the RAB11(S20V) mutant was overexpressed inEscherichia coliwith an engineered C-terminal His tag. RAB11(S20V) was then purified to homogeneity and was crystallized at 293 K. X-ray diffraction data were collected to a resolution of 2.4 Å from a crystal belonging to space groupI4, with unit-cell parametersa = 74.11,b= 74.11,c= 149.44 Å. The asymmetric unit was estimated to contain two molecules of RAB11(S20V).

scholarly journals Cloning, expression, purification, crystallization and preliminary crystallographic analysis of the C-terminal domain of Par-4 (PAWR)

2014 ◽  
Vol 70 (9) ◽  
pp. 1224-1227 ◽  
Author(s):  
Udaya Kumar Tiruttani Subhramanyam ◽  
Jan Kubicek ◽  
Ulf B. Eidhoff ◽  
Joerg Labahn
Keyword(s):  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Intrinsically Disordered ◽  
Tumour Suppressor Function ◽  
C Terminus ◽  
Apoptotic Protein

Prostate apoptosis response-4 protein is an intrinsically disordered pro-apoptotic protein with tumour suppressor function. Par-4 is known for its selective induction of apoptosis in cancer cells only and its ability to interact with various apoptotic proteinsviaits C-terminus. Par-4, with its unique function and various interacting partners, has gained importance as a potential target for cancer therapy. The C-terminus of the rat homologue of Par-4 was crystallized and a 3.7 Å resolution X-ray diffraction data set was collected. Preliminary data analysis shows the space group to beP41212. The unit-cell parameters area=b= 115.351,c= 123.663 Å, α = β = γ = 90°.

scholarly journals Cloning, purification, crystallization and preliminary X-ray studies of the putative type VI secretion immunity protein Tli5 (PA5088) fromPseudomonas aeruginosa

2014 ◽  
Vol 70 (7) ◽  
pp. 903-905 ◽  
Author(s):  
Zhen Chen ◽  
Zengqiang Gao ◽  
Haidai Hu ◽  
Jianhua Xu ◽  
Heng Zhang ◽  
...  
Keyword(s):  
Crystallographic Analysis ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Immunity Protein ◽  
X Ray ◽  
Cell Parameters ◽  
Putative Protein ◽  
Type Vi Secretion

The putative protein PA5089 fromPseudomonas aeruginosahas recently been identified as a Tle5 phospholipase effector from a type VI secretion system (T6SS), and its toxicity can be neutralized by the cognate immunity protein Tli5 (PA5088). Here, the expression, purification, crystallization and preliminary crystallographic analysis of PA5088 are reported. X-ray diffraction data were collected from selenomethionine-derivatized PA5088 crystals to a resolution of 2.55 Å. The crystals belonged to space groupP21, with unit-cell parametersa= 64.002,b= 104.744,c= 90.168 Å.

scholarly journals Overproduction, crystallization and preliminary X-ray crystallographic analysis ofEscherichia colitRNAN6-threonylcarbamoyladenosine dehydratase

2014 ◽  
Vol 70 (11) ◽  
pp. 1517-1520 ◽  
Author(s):  
Sunmin Kim ◽  
Keon Young Kim ◽  
Jeong Kuk Park ◽  
Byung Il Lee ◽  
Yun-Gon Kim ◽  
...  
Keyword(s):  
Crystallographic Analysis ◽  
X Rays ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
E Coli ◽  
Protein Mass ◽  
Crystal Volume

Escherichia colitRNAN6-threonylcarbamoyladenosine dehydratase (TcdA), previously called CsdL or YgdL, was overproduced and purified fromE. coliand crystallized using polyethylene glycol 3350 as a crystallizing agent. X-ray diffraction data were collected to 2.70 Å resolution under cryoconditions using synchrotron X-rays. The crystals belonged to space groupP21, with unit-cell parametersa= 65.4,b= 96.8,c= 83.3 Å, β = 111.7°. According to the Matthews coefficient, the asymmetric unit may contain up to four subunits of the monomeric protein, with a crystal volume per protein mass (VM) of 2.12 Å3 Da−1and 42.1% solvent content.

scholarly journals Purification and X-ray crystallographic analysis of 7-keto-8-aminopelargonic acid (KAPA) synthase fromMycobacterium smegmatis

2014 ◽  
Vol 70 (10) ◽  
pp. 1372-1375 ◽  
Author(s):  
Shanghua Fan ◽  
Defeng Li ◽  
Joy Fleming ◽  
Yuan Hong ◽  
Tao Chen ◽  
...  
Keyword(s):  
Crystallographic Analysis ◽  
Unit Cell ◽  
Monoclinic Space Group ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Essential Enzyme ◽  
Mycobacterial Growth

7-Keto-8-aminopelargonic acid synthase (KAPA synthase; BioF) is an essential enzyme for mycobacterial growth that catalyses the first committed step in the biotin-synthesis pathway. It is a pyridoxal 5′-phosphate (PLP)-dependent enzyme and is a potential drug target. Here, the cloning, expression, purification and crystallization of KAPA synthase fromMycobacterium smegmatis(MsBioF) and the characterization of MsBioF crystals using X-ray diffraction are described. The crystals diffracted to 2.3 Å resolution and belonged to the monoclinic space groupP21, with unit-cell parametersa= 70.88,b= 91.68,c= 109.84 Å, β = 97.8°. According to the molecular weight of MsBioF, the unit-cell parameters and the self-rotation function map, four molecules are present in each asymmetric unit with aVMvalue of 2.06 Å3 Da−1and a solvent content of 40.20%.

scholarly journals Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of SF173 fromShigella flexneri

2015 ◽  
Vol 71 (1) ◽  
pp. 54-56 ◽  
Author(s):  
Ha-Neul Kim ◽  
Jeong-Gi An ◽  
Yoo-Sup Lee ◽  
Seung-Hyeon Seok ◽  
Hee-Seop Yoo ◽  
...  
Keyword(s):  
Anaerobic Bacterium ◽  
Shigella Flexneri ◽  
Hypothetical Protein ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Gram Negative ◽  
X Ray ◽  
Cell Parameters

Shigella flexneriis a Gram-negative, anaerobic bacterium in the genusShigellathat can cause diarrhoea in humans. SF173, a hypothetical protein fromS. flexneri5a strain M90T, has been cloned, overexpressed, purified and crystallized as a part of laboratory-scale structural genomics project. The SF173 protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 0.8 Msuccinic acid pH 7.0 at 293 K. Preliminary X-ray diffraction analysis revealed that the crystal diffracted to 1.47 Å resolution and belonged to space groupI432, with unit-cell parametersa=b=c= 110.245 Å.

scholarly journals Crystallization and preliminary X-ray crystallographic analysis of the CARD domain of apoptosis repressor with CARD (ARC)

2015 ◽  
Vol 71 (1) ◽  
pp. 82-85
Author(s):  
Seong Hyun Kim ◽  
Hyun Ho Park
Keyword(s):  
Diffraction Data ◽  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Extrinsic Pathway ◽  
X Ray ◽  
Cell Parameters ◽  
Rich Domain ◽  
Card Domain ◽  
Extrinsic Apoptosis

Apoptosis repressor with caspase-recruiting domain (ARC) is an apoptosis repressor that inhibits both intrinsic and extrinsic apoptosis signalling. Human ARC contains an N-terminal caspase-recruiting domain (CARD domain) and a C-terminal proline- and glutamic acid-rich (P/E-rich) domain. The CARD domain in ARC is the domain that is directly involved in inhibition of the extrinsic pathway. In this study, the N-terminal CARD domain of ARC was overexpressed, purified and crystallized. X-ray diffraction data were collected to a resolution of 2.1 Å and the crystals were found to belong to space groupP61orP65, with unit-cell parametersa= 98.28,b= 98.28, c = 51.86 Å, α = 90, β = 90, γ = 120°.

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