scholarly journals Expression, purification and preliminary crystallographic analysis of the cryptic polo-box domain ofCaenorhabditis elegansZYG-1

2014 ◽  
Vol 70 (10) ◽  
pp. 1346-1350
Author(s):  
Ekaterina Shimanovskaya ◽  
Gang Dong
Keyword(s):  
Crystallographic Analysis ◽  
Structural Knowledge ◽  
Diffusion Method ◽  
Lysine Methylation ◽  
Wild Type ◽  
Data Set ◽  
Cell Parameters ◽  
Anomalous Data ◽  
Strain Bl21 ◽  
Shed Light

ZYG-1 is a polo-like kinase essential for centriole assembly inCaenorhabditis elegans. The targeting of ZYG-1 to nascent centrioles isviaits central cryptic polo-box (CPB) domain. To shed light on the molecular basis of ZYG-1 recruitment, it is necessary to obtain structural knowledge of the ZYG-1 CPB. Here, the expression, purification and preliminary crystallographic analysis of the ZYG-1 CPB are reported. The protein was overexpressed inEscherichia colistrain BL21 (DE3), purified by multi-step chromatography and crystallized using the vapour-diffusion method. Crystals of the wild-type protein exhibited an order–disorder pathology, which was solved by reductive lysine methylation. A complete anomalous data set was collected to 2.54 Å resolution at the Se Kedge (λ = 0.9792 Å). The crystal belonged to space groupP2, with unit-cell parametersa= 53.3,b= 60.09,c= 87.51 Å, β = 93.31°. There were two molecules in the asymmetric unit.

scholarly journals Cloning, expression, purification, crystallization and preliminary crystallographic analysis of 5-aminolaevulinic acid dehydratase fromBacillus subtilis

2010 ◽  
Vol 66 (9) ◽  
pp. 1053-1055 ◽  
Author(s):  
Qianda Lu ◽  
Jinming Ma ◽  
Hui Rong ◽  
Jun Fan ◽  
Ye Yuan ◽  
...  
Keyword(s):  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Gene Encoding ◽  
Data Set ◽  
X Ray ◽  
Aminolaevulinic Acid ◽  
Acid Dehydratase ◽  
Strain Bl21 ◽  
Aminolaevulinic Acid Dehydratase

5-Aminolaevulinic acid dehydratase (ALAD), a crucial enzyme in the biosynthesis of tetrapyrrole, catalyses the condensation of two 5-aminolaevulinic acid (ALA) molecules to form porphobilinogen (PBG). The gene encoding ALAD was amplified from genomic DNA ofBacillus subtilisand the protein was overexpressed inEscherichia colistrain BL21 (DE3). The protein was purified and crystallized with an additional MGSSHHHHHHSSGLVPRGSH– tag at the N-terminus of the target protein. Diffraction-quality single crystals were obtained by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected at a resolution of 2.7 Å.

scholarly journals Cloning, purification and preliminary crystallographic analysis of theHelicobacter pyloripseudaminic acid biosynthesisN-acetyltransferase PseH

2014 ◽  
Vol 70 (9) ◽  
pp. 1276-1279 ◽  
Author(s):  
Yu C. Liu ◽  
Abu I. Ud-Din ◽  
Anna Roujeinikova
Keyword(s):  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Data Set ◽  
Cell Parameters ◽  
Nonulosonic Acid ◽  
Pseudaminic Acid ◽  
The Common ◽  
H Pylori ◽  
Development Of Gastric Cancer

Helicobacter pyloriinfection is the common cause of gastritis and duodenal and stomach ulcers, which have been linked to a higher risk of the development of gastric cancer. The motility that facilitates persistent infection requires functional flagella that are heavily glycosylated with 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid). Pseudaminic acid biosynthesis protein H (PseH) catalyzes the third step in its biosynthetic pathway, producing UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. Crystals ofH. pyloriPseH have been grown by the hanging-drop vapour-diffusion method using diammonium tartrate as a precipitating agent. The crystals belonged to space groupI222 orI212121, with unit-cell parametersa= 107.8,b= 145.4,c= 166.3 Å. A complete X-ray diffraction data set has been collected to 2.5 Å resolution using cryocooling conditions and synchrotron radiation.

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 crystallographic analysis of Pz peptidase B fromGeobacillus collagenovoransMO-1

2012 ◽  
Vol 68 (7) ◽  
pp. 757-759 ◽  
Author(s):  
Hiroaki Nakano ◽  
Allin Hosokawa ◽  
Ryuji Tagawa ◽  
Koji Inaka ◽  
Kazunori Ohta ◽  
...  
Keyword(s):  
Space Station ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Unit Cell Parameters ◽  
Data Set ◽  
Peptide Substrates ◽  
X Ray ◽  
Cell Parameters ◽  
Experiment Module ◽  
Counter Diffusion

Pz peptidase B is an intracellular M3 metallopeptidase that is found together with Pz peptidase A in the thermophileGeobacillus collagenovoransMO-1 and recognizes collagen-specific tripeptide units (-Gly-Pro-X-). These peptidases have low homology in their primary structures; however, their cleavage patterns towards peptide substrates are similar. In this work, Pz peptidase B was crystallized using the counter-diffusion method. Data were collected to a resolution of 1.6 Å at 100 K from a crystal obtained in the Japanese Experiment Module (JEM; also known as `Kibo') at the International Space Station (ISS). The crystal belonged to the trigonal space groupP3121, with unit-cell parametersa=b= 87.64,c= 210.5 Å. A complete data set was also obtained from crystals of selenomethionine-substituted protein.

scholarly journals Cloning, expression, refolding, purification and preliminary crystallographic analysis of the sensory domain of theCampylobacterchemoreceptor for aspartate A (CcaA)

2015 ◽  
Vol 71 (1) ◽  
pp. 110-113 ◽  
Author(s):  
Mayra A. Machuca ◽  
Yu C. Liu ◽  
Anna Roujeinikova
Keyword(s):  
Campylobacter Jejuni ◽  
Inclusion Bodies ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Data Set ◽  
Cell Parameters ◽  
Extracellular Signals ◽  
Denatured Protein

InCampylobacter jejuni, chemotaxis and motility have been identified as important virulence factors that are required for host colonization and invasion. Chemotactic recognition of extracellular signals is mediated by the periplasmic sensory domains of its transducer-like proteins (Tlps). In this study, the sensory domain of theC. jejunichemoreceptor for aspartate A (CcaA) has been expressed inEscherichia coliand purified from inclusion bodies. The urea-denatured protein was refolded and then crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as a precipitating agent. A complete data set has been collected to 1.4 Å resolution using cryocooling conditions and synchrotron radiation. The crystals belonged to space groupP1, with unit-cell parametersa= 39.3,b= 43.3,c= 50.9 Å, α = 92.5, β = 111.4, γ = 114.7°.

scholarly journals Crystallization and preliminary crystallographic analysis of Axe2, an acetylxylan esterase fromGeobacillus stearothermophilus

2013 ◽  
Vol 69 (4) ◽  
pp. 430-434 ◽  
Author(s):  
Shifra Lansky ◽  
Onit Alalouf ◽  
Vered Solomon ◽  
Anat Alhassid ◽  
Lata Govada ◽  
...  
Keyword(s):  
Diffraction Data ◽  
Biochemical Characterization ◽  
Plant Biomass ◽  
Crystallographic Analysis ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Data Set ◽  
Cell Parameters ◽  
Acetylxylan Esterase

Acetylxylan esterases are part of the hemi-cellulolytic system of many microorganisms which utilize plant biomass for growth. Xylans, which are polymeric sugars that constitute a significant part of the plant biomass, are usually substituted with acetyl side groups attached at position 2 or 3 of the xylose backbone units. Acetylxylan esterases hydrolyse the ester linkages of the xylan acetyl groups and thus improve the ability of main-chain hydrolysing enzymes to break down the sugar backbone units. As such, these enzymes play an important part in the hemi-cellulolytic utilization system of many microorganisms that use plant biomass for growth. Interest in the biochemical characterization and structural analysis of these enzymes stems from their numerous potential biotechnological applications. An acetylxylan esterase (Axe2) of this type fromGeobacillus stearothermophilusT-6 has recently been cloned, overexpressed, purified, biochemically characterized and crystallized. One of the crystal forms obtained (RB1) belonged to the tetragonal space groupI422, with unit-cell parametersa=b= 110.2,c= 213.1 Å. A full diffraction data set was collected to 1.85 Å resolution from flash-cooled crystals of the wild-type enzyme at 100 K using synchrotron radiation. A selenomethionine derivative of Axe2 has also been prepared and crystallized for single-wavelength anomalous diffraction experiments. The crystals of the selenomethionine-derivatized Axe2 appeared to be isomorphous to those of the wild-type enzyme and enabled the measurement of a full 1.85 Å resolution diffraction data set at the selenium absorption edge and a full 1.70 Å resolution data set at a remote wavelength. These data are currently being used for three-dimensional structure determination of the Axe2 protein.

scholarly journals Recombinant ACHT1 fromArabidopsis thaliana: crystallization and X-ray crystallographic analysis

2017 ◽  
Vol 73 (7) ◽  
pp. 382-385 ◽  
Author(s):  
Weimin Pan ◽  
Junchao Wang ◽  
Ye Yang ◽  
Lin Liu ◽  
Min Zhang
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Disulfide Bonds ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion

Thioredoxins (Trxs) play important roles in chloroplasts by linking photosynthetic light reactions to a series of plastid functions. They execute their function by regulating the oxidation and reduction of disulfide bonds. ACHT1 (atypical cysteine/histidine-rich Trx1) is a thylakoid-associated thioredoxin-type protein found in theArabidopsis thalianachloroplast. Recombinant ACHT1 protein was overexpressed inEscherichia coli, purified and crystallized by the vapour-diffusion method. The crystal diffracted to 1.7 Å resolution and a complete X-ray data set was collected. Preliminary crystallographic analysis suggested that the crystals belonged to space groupC2221, with unit-cell parametersa = 102.7,b= 100.6,c= 92.8 Å.

scholarly journals Cloning, refolding, purification and preliminary crystallographic analysis of the sensory domain of theCampylobacterchemoreceptor for multiple ligands (CcmL)

2015 ◽  
Vol 71 (2) ◽  
pp. 211-216 ◽  
Author(s):  
Mayra A. Machuca ◽  
Yu C. Liu ◽  
Simone A. Beckham ◽  
Anna Roujeinikova
Keyword(s):  
Polyethylene Glycol ◽  
Campylobacter Jejuni ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Data Set ◽  
Cell Parameters ◽  
Vapour Diffusion ◽  
Multiple Ligands

A periplasmic sensory domain of theCampylobacter jejunichemoreceptor for multiple ligands (CcmL) has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 as a precipitating agent. A complete data set was collected to 1.3 Å resolution using cryocooling conditions and synchrotron radiation. The crystals belonged to space groupP21, with unit-cell parametersa= 42.6,b= 138.0,c= 49.0 Å, β = 94.3°.

scholarly journals Preparation, purification, crystallization and preliminary crystallographic analysis of dual-domain β-propeller phytase fromBacillussp. HJB17

2014 ◽  
Vol 70 (12) ◽  
pp. 1671-1674 ◽  
Author(s):  
Fang Lu ◽  
Gangxin Guo ◽  
Qianqian Li ◽  
Duo Feng ◽  
Yong Liu ◽  
...  
Keyword(s):  
Fold Increase ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Sodium Acetate Trihydrate ◽  
Data Set ◽  
Cell Parameters ◽  
Acetate Trihydrate ◽  
C Terminus ◽  
Phytate Hydrolysis ◽  
Dual Domain

β-Propeller phytases (BPPs) are abundant in nature. Recently, dual-domain BPPs have been found in which the typical BPP domain is responsible for phytate hydrolysis. The dual-domain BPP (PhyH) fromBacillussp. HJB17 was obtained with an incomplete N-terminal BPP domain (PhyH-DI; residues 41–318) and a typical BPP domain (PhyH-DII; residues 319–644) at the C-terminus. PhyH-DI was found to act synergistically (with a 1.2–2.5-fold increase in phosphate release) with PhyH-DII, other BPPs (PhyP and 168PhyA) and a histidine acid phosphatase. The structure of PhyH was therefore studied with the aim of explaining these functions. PhyH with the secreted signal peptide of the first 40 amino acids deleted (PhyHT) was cloned and expressed inEscherichia coli. Purified and active PhyHT protein was obtained by refolding from the precipitant. PhyHT was crystallized using the vapour-diffusion method. The crystal grew in a condition consisting of 0.2 Msodium acetate trihydrate, 0.1 MTris–HCl pH 9.5, 25%(w/v) polyethylene glycol 4000 using 1 mg ml−1protein solution at 289 K. A complete data set was collected from a crystal to 2.85 Å resolution using synchrotron radiation at 100 K. The crystal belonged to space groupP1211, with unit-cell parametersa= 46.82,b= 140.19,c= 81.94 Å, α = 90.00, β = 92.00, γ = 90.00°. The asymmetric unit was estimated to contain one molecule of PhyHT.

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%.

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