Three-dimensional structure of GlcNAcalpha1-4Gal releasing endo-beta-galactosidase from Clostridium perfringens.
Three-dimensional structure of GlcNAcalpha1-4Gal releasing endo-beta-galactosidase from Clostridium perfringens.
复制标题
从产气荚膜梭菌中释放内切β-半乳糖苷酶的 GlcNAcalpha1-4Gal 的三维结构。
DOI：
10.1002/prot.20363
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发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
Wang,Bi-Cheng
中科院分区：
文献类型：
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作者：
Tempel,Wolfram;Liu,Zhi-Jie;Horanyi,PeterS;Deng,Lu;Lee,Doowon;Newton,MGary;Rose,JohnP;Ashida,Hisashi;Li,Su-Chen;Li,Yu-Teh;Wang,Bi-Cheng
材料与方法。Endo-β-GalGnGa的结晶已在前面描述过。将晶体转移到1 μL滴的1∶2∶7混合物中，该混合物由2m碘化钾水溶液、甘油和结晶储层溶液[42%硫酸铵、2%聚乙二醇（PEG400）、0.1 mn -2-羟乙基哌嗪- n ！-2-乙磺酸（HEPES-Na） （pH 7.4），并孵育5 s。晶体被安装在纤维环中，并在液氮中快速冷冻。在先进光子源（APS）的17BM （IMCA-CAT）光束线处，使用mar165电荷耦合器件（CCD）探测器，在晶体与探测器之间的距离为110 mm，在1.7 Å波长处采集数据。采用冗余逆波束策略，记录了6组160幅0.5振荡图像。另一个晶体从一个独立的但组成相同的结晶实验中得到，并转移到1 μL的甘油和储层溶液的1:4混合物中。它立即被安装在一个纤维环中，并在液氮中快速冷冻。在APS的光束线22ID处（SER-CAT），在0.98 Å波长处，使用MAR 165 CCD探测器在130 mm的晶体-探测器距离处采集数据。记录了180张0.5振荡图像的单次扫描。使用HKL2000软件套件对衍射数据进行索引、集成和缩放（表1）。使用SOLVE12从单波长异常散射数据确定了11个相，使用1.7 Å x射线测量，从碘化钾浸泡中加入的碘离子中测量。在用RESOLVE进行相位细化后，使用0.98 Å波长数据集和ARP/wARP程序进行了13,14自动跟踪。15最终模型是在使用Xfit对初始模型进行手动调整的几个周期后获得的，16随后使用REFMAC517进行有节制的细化，并使用MOLPROBITY在线工具进行验证。如所示，CCP4套件的其他程序通过其图形用户界面使用。使用DALI服务器进行结构相似性搜索。21模型（表II）保存在蛋白质数据银行22 （PDB）中，访问码为1UPS。
Materials and Methods. The crystallization of Endo-β-GalGnGa was described previously. 7 A crystal was transferred to a 1 μL drop of a 1: 2: 7 mixture of 2 M aqueous potassium iodide solution, glycerol, and crystallization reservoir solution [42% ammonium sulfate, 2% polyethylene glycol (PEG400), 0.1 MN-2-hydroxyethylpiperazine-N!-2-ethanesulfonic acid (HEPES-Na) at pH 7.4] and incubated for 5 s. 8 The crystal was mounted in a fiber loop9 and flash-frozen10 in liquid nitrogen. Data were collected at 1.7 Å wavelength at beamline 17BM (IMCA-CAT) at the Advanced Photon Source (APS) using a MAR 165 chargecoupled device (CCD) detector at a 110 mm crystal-todetector distance. Six sets of 160 0.5 oscillation images were recorded using a redundant inverse beam strategy. Another crystal was obtained from a separate but identically composed crystallization experiment and transferred to a 1 μL drop of a 1: 4 mixture of glycerol and reservoir solution. It was immediately mounted in a fiber loop and flash-frozen in liquid nitrogen. Data were collected at 0.98 Å wavelength at beamline 22ID (SER-CAT) at the APS using a MAR 165 CCD detector at a 130 mm crystal-todetector distance. A single sweep of 180 0.5 oscillation images was recorded.Diffraction data were indexed, integrated and scaled (Table I) using the HKL2000 software suite. 11 Phases were determined using SOLVE12 from single-wavelength anomalous scattering data, measured using 1.7 Å X-rays, from the iodide ions incorporated from the potassium iodide soaking. After phase refinement with RESOLVE, 13, 14 automated tracing was performed using the 0.98 Å wavelength data set and the program ARP/wARP. 15 The final model was obtained after several cycles of manual adjustment of the initial model using Xfit, 16 followed by restrained refinement with REFMAC517 and validation with the MOLPROBITY online tool. 18 Other programs of the CCP4 suite19 were used through its graphical user interface20 as indicated. Structural similarity searches were carried out using the DALI server. 21 The model (Table II) was deposited at the Protein Data Bank22 (PDB) with access code 1UPS.