Crystal structure of an eIF4G-like protein from Danio rerio.
Crystal structure of an eIF4G-like protein from Danio rerio.
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DOI：
10.1002/prot.22703
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发表时间：
2010-05-15
影响因子：
2.9
通讯作者：
Phillips, George N., Jr.
中科院分区：
文献类型：
--
作者：
Bae, Euiyoung;Bitto, Eduard;Bingman, Craig A.;McCoy, Jason G.;Wesenberg, Gary E.;Phillips, George N., Jr.
材料和方法选择编码MIF4Gdb的基因作为靶标，作为一组编码蛋白质的基因的一部分，这些基因编码的蛋白质与先前保存在蛋白质数据库中的结构尽可能不同，也与CESG先前选择的靶标尽可能不相似。它被分配了一个项目数据库标识符GO。79294。生产这种蛋白质的完整、详细的方案可以在PepcDB中找到。8简单地，将该基因克隆到pVP33K中，这是我们项目中使用的第一个生产Flexi®载体9，并按照标准的CESG管道方案进行了硒蛋氨酸蛋白的纯化，10个蛋白的表达，11个蛋白的纯化12和整体信息管理。在康宁3775板上，使用名为UW-192的本地筛，在4摄氏度和20摄氏度下进行了13个初始结晶筛。在4℃和20℃下使用Bruker Nonius Crystal Farm监测晶体生长，并使用Crystal Farm Navigator(Nexus BiosSystems，Inc.)对晶体生长进行评分。Tecan Genesis RSP 150机器人组装了沉淀剂解决方案，用于优化实验。在悬滴批量实验中生长出了衍射级晶体。用含7%(w/v)的聚乙二醇4K、0.4 M氯化钠、100 mM MES/醋酸盐pH 5.5的缓冲液(50 mM NaC l、0.3 mM TCEP、5 mM BisTris pH 7.0)混合10 mg/ml蛋白质溶液，在20℃条件下生长晶体。
Materials and MethodsThe gene coding for MIF4Gdb was selected as a target as part of a group of genes chosen to code for proteins that are as dissimilar as possible to structures previously deposited in the Protein Data Bank, and also as dissimilar as possible to targets that CESG had previously selected. It was assigned a project database identifier of GO. 79294. Complete, detailed protocols for the production of this protein can be found in PepcDB. 8 Briefly, the gene was cloned into pVP33K, the first production Flexi® Vector 9 used on our project, and selenomethionyl protein was purified following the standard CESG pipeline protocol for cloning, 10 protein expression, 11 protein purification 12 and overall information management. 13 Initial crystallization screens were conducted at 4 and 20 C, in Corning 3775 plates, using a local screen called UW-192. Crystal growth was monitored using Bruker Nonius Crystal Farms at 4 C and 20 C, and scored using Crystal Farm Navigator (Nexus Biosystems, Inc.). A Tecan Genesis RSP 150 robot assembled precipitant solutions for optimization experiments. Diffraction quality crystals were grown in hanging drop batch experiments. Crystals were grown from 10 mg/ml protein solution in buffer (50 mM NaCl, 3 mM NaN 3, 0.3 mM TCEP, 5 mM BisTris pH 7.0) mixed with an equal amount of reservoir solution containing 7%(w/v) PEG 4K, 0.4 M NaCl, 100 mM MES/acetate pH 5.5 at 20 C. The crystals were cryoprotected in 15%(w/v) ethylene glycol, 10%(w/v) PEG 4K, 100 mM MES/acetate pH 5.5 and were flash-frozen in liquid nitrogen.
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影响因子：
14.9
作者：
UniProt Consortium
通讯作者：
UniProt Consortium
DOI：
10.1007/978-1-59745-196-3_4
发表时间：
2009
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Blommel PG;Martin PA;Seder KD;Wrobel RL;Fox BG
通讯作者：
Fox BG
影响因子：
2.9
作者：
MADEJ, T;GIBRAT, JF;BRYANT, SH
通讯作者：
BRYANT, SH
DOI：
10.1107/s0907444903018043
发表时间：
2003-11-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Grosse-Kunstleve, RW;Adams, PD
通讯作者：
Adams, PD
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K