STRUCTURE DETERMINATION OF PTEN TUMOR SUPPRESSOR BY MAD

MAD 测定 PTEN 肿瘤抑制因子的结构

• 批准号: 6667817
• 负责人: NIKOLA P PAVLETICH
• 金额: $ 14.27万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2003-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6667817
• 关键词: bioimaging /biomedical imaging; biological products; biomedical resource; proteins; radiography; structural biology

We determined the structure of the a T cell receptor (TCR) b chain complexed with the superantigen staphylococcal enterotoxin B (SEB) to 2.4
resolution. We also determined the structure, to 2.6
resolution, of the complex between the TCR b chain and a mutant of SEB in which valine at position 26 is replaced by tyrosine (SEB V26Y). The crystals belong to space group P21 with cell dimensions a = 71.2
, b = 83.6
, c = 83.0
for the wild type b-SEB complex and a = 70.9
, b = 83.0
, c = 82.8
for the mutant b-SEB V26Y complex. There are two complex molecules in the asymmetric unit. X-ray diffraction data up to 2.4
(b-SEB) and 2.6
(b-SEB V26Y) were collected at 100 oK from one flash-cooled crystal for each complex using synchrotron radiation at CHESS beamline F-1 with a Princeton 2K CCD detector. The crystals were soaked in 10% PEG 8000, 24% glycerol, and 0.1 M Tris-HCl, pH 8.5, prior to flash-cooling in liquid nitrogen. Data were integrated and merged using HKL/DENZO/SCALEPACK which gives 34,943 unique reflections with Rmerge= 9.2% for b-SEB and 28,757 unique reflections with Rmerge= 8.1% for b-SEB V26Y. The data sets are 91.5% complete to 2.4
for b-SEB (79.1% from 2.5-2.4
) and 97.0% complete to 2.6
for b-SEB V26Y (90.6% from 2.7-2.6
). The structure of the wild type b-SEB complex was solved by the molecular replacement method with the program AMoRe (Navaza, 1994). The search models consisted of the 14.3.d TCR b chain refined at 1.7
resolution (PDB accession code 1bec) and SEB refined at 1.9
resolution (PDB accession code 1SE4). The structure was refined by iterative cycles of simulated annealing and temperature factor (B) refinement using X-PLOR interspersed with model building into Fo- Fc and 2Fo- Fc electron density maps using TURBO-FROD. The final model contains 7,589 protein atoms and 179 water molecule with Rfree= 0.309 and Rwork= 0.228 in the range 6-2.4
. The r.m.s. deviations from ideal bond lengths and bond angles are 0.006
and 1.79o, respectively. The structure determination of the b-SEB V26Y mutant complex was begun from the partially refined structure of the b-SEB wild type complex. The final model of the b-SEB V26Y complex contains 6,915 protein atoms and 31 water molecule with Rfree= 0.326 and Rwork= 0.229 in the range 6-2.6
. The r.m.s. deviations from ideal bond lengths and bond angles are 0.008
and 1.37o, respectively.

我们确定了T细胞受体（TCR）B链的结构 与超抗原葡萄球菌肠毒素B（SEB）络合 2.4分辨率。 我们还确定了结构为2.6 分辨率，TCR B链与SEB突变体之间的复合物 其中26位的Valine被酪氨酸（SEB V26Y）取代。 晶体属于带有细胞尺寸a = 71.2的空间群P21 ，b = 83.6，c = 83.0对于野生型B-seb复合物，a = 70.9 ，突变体B-SEB V26Y复合物的B = 83.0，C = 82.8。 那里 是非对称单元中的两个复杂分子。 X射线衍射 在100处收集最多2.4（B-SEB）和2.6（B-SEB V26Y）的数据 OK使用Synchrotron从每个配合物的一个闪光冷却晶体中 与普林斯顿2K CCD检测器的国际象棋光束线F-1辐射。 这 将晶体浸入10％PEG 8000，24％甘油和0.1 m Tris-HCl，pH 8.5，在液氮中进行闪光冷却之前。 数据 使用hkl/denzo/scalepack集成并合并 34,943个独特的反射，rmerge = 9.2％，为28,757 B-SEB V26Y的Rmerge = 8.1％的唯一反射。 数据集 对于B-SEB（2.5-2.4的79.1％）和97.0％的91.5％已完成至2.4 B-SEB V26Y（2.7-2.6的90.6％）完成至2.6。 这 野生型B-SEB复合物的结构通过分子求解 替代方法与计划（Navaza，1994）。 搜索 模型由14.3.D TCR B链组成，以1.7分辨率精制 （PDB登录代码1BEC）和SEB以1.9分辨率（PDB）精制 登录代码1SE4）。 结构通过迭代循环进行了完善 使用模拟退火和温度因子（b）改进 X-plor散布在模型构建到Fo-fc和2fo-fc 电子密度图使用涡轮豆形。 最终模型包含 7,589种蛋白质原子和179个水分子，RFREE = 0.309，并且 RWORW = 0.228在6-2.4范围内。 R.M.S.与理想的偏差 键长和键角分别为0.006和1.79O。 这 B-SEB V26Y突变体复合物的结构测定开始 从B-SEB野生型复合物的部分精制结构。 B-SEB V26Y复合物的最终模型包含6,915个蛋白质原子 RFREE = 0.326，RWORW = 0.229的31水分子在该范围内 6-2.6。 R.M.S.与理想键长和键角的偏差 分别为0.008和1.37O。