scholarly journals Crystallization of uridine phosphorylase fromShewanella oneidensisMR-1 in the laboratory and under microgravity and preliminary X-ray diffraction analysis

2012 ◽  
Vol 68 (11) ◽  
pp. 1387-1389 ◽  
Author(s):  
Tatyana N. Safonova ◽  
Nadezhda N. Mordkovich ◽  
Konstantin M. Polyakov ◽  
Valentin A. Manuvera ◽  
Vladimir P. Veiko ◽  
...  
Keyword(s):  
Space Station ◽  
Shewanella Oneidensis ◽  
Free Form ◽  
Diffusion Method ◽  
Data Sets ◽  
Uridine Phosphorylase ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Key Enzyme

Uridine phosphorylase (UDP, EC 2.4.2.3), a key enzyme in the pyrimidine salvage pathway, catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The gene expression of UDP fromShewanella oneidensisMR-1 was performed in the recipient strainEscherichia coli. The UDP protein was crystallized on earth (in the free form and in complex with uridine as the substrate) by the hanging-drop vapour-diffusion method at 296 K and under microgravity conditions (in the free form) aboard the Russian Segment of the International Space Station by the capillary counter-diffusion method. The data sets were collected to a resolution of 1.9 Å from crystals of the free form grown on earth, 1.6 Å from crystals of the complex with uridine and 0.95 Å from crystals of the free form grown under microgravity. All crystals belong to the space groupP21and have similar unit-cell parameters. The crystal of uridine phosphorylase grown under microgravity diffracted to ultra-high resolution and gave high-quality X-ray diffraction data.

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 Crystallization and preliminary X-ray crystallographic studies of dehydroascorbate reductase (DHAR) fromOryza sativaL.japonica

2014 ◽  
Vol 70 (6) ◽  
pp. 781-785 ◽  
Author(s):  
Hackwon Do ◽  
Il-Sup Kim ◽  
Young-Saeng Kim ◽  
Sun-Young Shin ◽  
Jin-Ju Kim ◽  
...  
Keyword(s):  
Crop Production ◽  
Dehydroascorbate Reductase ◽  
Diffusion Method ◽  
Oxygen Species ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Key Enzyme ◽  
Important Enzyme

Dehydroascorbate reductase fromOryza sativaL.japonica(OsDHAR), a key enzyme in the regeneration of vitamin C, maintains reduced pools of ascorbic acid to detoxify reactive oxygen species. In previous studies, the overexpression of OsDHAR in transgenic rice increased grain yield and biomass as well as the amount of ascorbate, suggesting that ascorbate levels are directly associated with crop production in rice. Hence, it has been speculated that the increased level of antioxidants generated by OsDHAR protects rice from oxidative damage and increases the yield of rice grains. However, the crystal structure and detailed mechanisms of this important enzyme need to be further elucidated. In this study, recombinant OsDHAR protein was purified and crystallized using the sitting-drop vapour-diffusion method at pH 8.0 and 298 K. Plate-shaped crystals were obtained using 0.15 Mpotassium bromide, 30%(w/v) PEG MME 2000 as a precipitant, and the crystals diffracted to a resolution of 1.9 Å on beamline 5C at the Pohang Accelerator Laboratory. The X-ray diffraction data indicated that the crystal contained one OsDHAR molecule in the asymmetric unit and belonged to space groupP21with unit-cell parametersa= 47.03,b= 48.38,c= 51.83 Å, β = 107.41°.

Technology of high quality protein crystals’ obtaining aboard the ISS at execution of joint Japanese–Russian experiments

2020 ◽  
pp. 5-25
Author(s):  
Koji INAKA ◽  
Saori ICHIMIZU ◽  
Izumi YOSHIZAKI ◽  
Kiyohito KIHIRA ◽  
Elena G. LAVRENKO ◽  
...  
Keyword(s):  
Drug Design ◽  
International Space Station ◽  
Space Station ◽  
Protein Crystals ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
High Quality ◽  
International Space ◽  
X Ray ◽  
High Quality Protein

A series of space experiments aboard the International Space Station (ISS) associated with high-quality Protein Crystal Growth (PCG) in microgravity conditions can be considered as a unique and one of the best examples of fruitful collaboration between Japanese and Russian scientists and engineers in space, which includes also other ISS International Partners. X-ray diffraction is still the most powerful tool to determine the protein three dimensional structure necessary for Structure based drug design (SBDD). The major purpose of the experiment is to grow high quality protein crystals in microgravity for X-ray diffraction on Earth. Within one and a half decade, Japan and Russia have established an efficient process over PCG in space to support latest developments over drug design and structural biology. One of the keys for success of the experiment lies in how precisely pre-launch preparations are made. Japanese party provides flight equipment for crystallization and ensures the required environment to support the experiment aboard of the ISS’s Kibo module, and also mainly takes part of the experiment ground support such as protein sample characterization, purification, crystallization screening, and solution optimization for microgravity experiment. Russian party is responsible for integration of the flight items equipped with proteins and precipitants on board Russian transportation space vehicles (Soyuz or Progress), for delivery them at the ISS, transfer to Kibo module, and returning the experiments’ results back on Earth aboard Soyuz manned capsule. Due to close cooperation of the parties and solid organizational structure, samples can be launched at the ISS every half a year if the ground preparation goes smoothly. The samples are crystallized using counter diffusion method at 20 degree C for 1–2.5 months. After samples return, the crystals are carefully taken out from the capillary, and frozen for X-ray diffraction at SPring8 facility in Japan. Extensive support of researchers from both countries is also a part of this process. The paper analyses details of the PCG experiment scheme, unique and reliable technology of its execution, and contains examples of the application. Key words: International Space Station, Protein crystals, Microgravity, International collaboration.

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.

scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of a hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) fromCoffea canephorainvolved in chlorogenic acid biosynthesis

2012 ◽  
Vol 68 (7) ◽  
pp. 824-828 ◽  
Author(s):  
Laura A. Lallemand ◽  
James G. McCarthy ◽  
Sean McSweeney ◽  
Andrew A. McCarthy
Keyword(s):  
Structural Data ◽  
Coffea Canephora ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Key Enzyme ◽  
Substrate Specifities ◽  
Drop Technique ◽  
Better Than

Chlorogenic acids (CGAs) are a group of soluble phenolic compounds that are produced by a variety of plants, includingCoffea canephora(robusta coffee). The last step in CGA biosynthesis is generally catalysed by a specific hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferase (HQT), but it can also be catalysed by the more widely distributed hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT). Here, the cloning and overexpression of HCT fromC. canephorainEscherichia colias well as its purification and crystallization are presented. Crystals were obtained by the sitting-drop technique at 293 K and X-ray diffraction data were collected on the microfocus beamline ID23-2 at the ESRF. The HCT crystals diffracted to better than 3.0 Å resolution, belonged to space groupP42212 with unit-cell parametersa=b= 116.1,c= 158.9 Å and contained two molecules in the asymmetric unit. The structure was solved by molecular replacement and is currently under refinement. Such structural data are needed to decipher the molecular basis of the substrate specifities of this key enzyme, which belongs to the large plant acyl-CoA-dependent BAHD acyltransferase superfamily.

High-synconformation of uridine and asymmetry of the hexameric molecule revealed in the high-resolution structures ofShewanella oneidensisMR-1 uridine phosphorylase in the free form and in complex with uridine

2014 ◽  
Vol 70 (12) ◽  
pp. 3310-3319 ◽  
Author(s):  
Tatyana N. Safonova ◽  
Sergey N. Mikhailov ◽  
Vladimir P. Veiko ◽  
Nadezhda N. Mordkovich ◽  
Valentin A. Manuvera ◽  
...  
Keyword(s):  
High Resolution ◽  
Active Sites ◽  
Shewanella Oneidensis ◽  
Free Form ◽  
Uridine Phosphorylase ◽  
Glycerol Molecule ◽  
Closed Conformation ◽  
Pyrimidine Salvage ◽  
Key Enzyme ◽  
Crystallization Solution

Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Expression of UP fromShewanella oneidensisMR-1 (SoUP) was performed inEscherichia coli. The high-resolution X-ray structure of SoUP was solved in the free form and in complex with uridine. A crystal of SoUP in the free form was grown under microgravity and diffracted to ultrahigh resolution. Both forms of SoUP contained sulfate instead of phosphate in the active site owing to the presence of ammonium sulfate in the crystallization solution. The latter can be considered as a good mimic of phosphate. In the complex, uridine adopts a high-synconformation with a nearly planar ribose ring and is present only in one subunit of the hexamer. A comparison of the structures of SoUP in the free form and in complex with the natural substrate uridine showed that the subunits of the hexamer are not identical, with the active sites having either an open or a closed conformation. In the monomers with the closed conformation, the active sites in which uridine is absent contain a glycerol molecule mimicking the ribose moiety of uridine.

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

2014 ◽  
Vol 70 (9) ◽  
pp. 1272-1275 ◽  
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.

scholarly journals Preliminary X-ray diffraction analysis of a thermophilic β-1,3–1,4-glucanase fromClostridium thermocellum

2014 ◽  
Vol 70 (7) ◽  
pp. 946-948 ◽  
Author(s):  
Lilan Zhang ◽  
Puya Zhao ◽  
Chun-Chi Chen ◽  
Chun-Hsiang Huang ◽  
Tzu-Ping Ko ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
High Specific Activity ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Glycosidic Bonds ◽  
Hydrolysis Of

β-1,3–1,4-Glucanases catalyze the specific hydrolysis of internal β-1,4-glycosidic bonds adjacent to the 3-O-substituted glucose residues in mixed-linked β-glucans. The thermophilic glycoside hydrolase CtGlu16A fromClostridium thermocellumexhibits superior thermal profiles, high specific activity and broad pH adaptability. Here, the catalytic domain of CtGlu16A was expressed inEscherichia coli, purified and crystallized in the trigonal space groupP3121, with unit-cell parametersa=b= 74.5,c= 182.9 Å, by the sitting-drop vapour-diffusion method and diffracted to 1.95 Å resolution. The crystal contains two protein molecules in an asymmetric unit. Further structural determination and refinement are in progress.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of AntE, a crotonyl-CoA carboxylase/reductase fromStreptomycessp. NRRL 2288

2014 ◽  
Vol 70 (6) ◽  
pp. 734-737 ◽  
Author(s):  
Lihan Zhang ◽  
Jing Chen ◽  
Takahiro Mori ◽  
Yan Yan ◽  
Wen Liu ◽  
...  
Keyword(s):  
Diffusion Method ◽  
Antimycin A ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion ◽  
Unsaturated Acyl ◽  
Reductive Carboxylation

AntE fromStreptomycessp. NRRL 2288 is a crotonyl-CoA carboxylase/reductase that catalyzes the reductive carboxylation of various α,β-unsaturated acyl-CoAs to provide the building block at the C7 position for antimycin A biosynthesis. Recombinant AntE expressed inEscherichia coliwas crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space groupI222 orI212121, with unit-cell parametersa= 76.4,b= 96.7,c= 129.6 Å, α = β = γ = 90.0°. A diffraction data set was collected at the KEK Photon Factory to 2.29 Å resolution.

Crystallization and preliminary X-ray diffraction studies on the N-utilizing substance-B (NusB) from Mycobacterium tuberculosis

2000 ◽  
Vol 56 (1) ◽  
pp. 64-66 ◽  
Author(s):  
B. Gopal ◽  
R. A. Cox ◽  
M. J. Colston ◽  
G. G. Dodson ◽  
S. J. Smerdon ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ribosomal Rna ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Core Complex ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters

N-utilizing substance B (NusB) is a protein which forms part of a complex assembly in transcriptional antitermination in Mycobacterium tuberculosis. It forms a heterodimer with the product of the NusE gene (identical to the ribosomal protein S10) and mediates the process of transcriptional antitermination by forming the core complex with the nut site of the ribosomal RNA along with other protein factors. NusB has been cloned and overexpressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. The space group is P212121, with unit-cell parameters a = 46.6, b = 64.2, c = 90.1 Å. A native data set complete to 1.6 Å resolution has been collected from a single crystal.

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