LCP crystallization and X-ray diffraction analysis of VcmN, a MATE transporter from Vibrio cholerae.
LCP crystallization and X-ray diffraction analysis of VcmN, a MATE transporter from Vibrio cholerae.
复制标题
DOI：
10.1107/s2053230x16008931
复制
发表时间：
2016-07
期刊：
影响因子：
--
通讯作者：
Nureki O
中科院分区：
文献类型：
--
作者：
Kusakizako T;Tanaka Y;Hipolito CJ;Kuroda T;Ishitani R;Suga H;Nureki O
胆固醇弧菌MATE转运蛋白使用无规立方相（LCP）方法结晶。从在夹层板和坐滴板中获得的单晶收集X射线衍射数据集，分辨率分别为2.5和2.2 μ m。 多药和毒性化合物外排转运蛋白（MATE）是多药转运蛋白家族中的一员，将外源性物质外排至细胞膜的细胞外。由于在细菌病原体中表达的MATE转运蛋白有助于多药耐药性，因此它们是重要的治疗靶点。在这里，从霍乱弧菌，VcmN，MATE-转运蛋白同源物，在大肠杆菌中过表达，纯化和结晶在无水立方相（LCP）。从在夹层板中获得的单晶收集X射线衍射数据至2.5μ m分辨率。晶体属P212121空间群，晶胞参数a = 52.3，B = 93.7，c = 100.2 Ω. 作为进一步的LCP结晶试验的结果，使用坐滴板获得较大尺寸的晶体。从在坐滴板中获得的单晶收集X射线衍射数据至2.2μ m分辨率。晶体属P212121空间群，晶胞参数a = 61.9，B = 91.8，c = 100.9 Ω. 本工作为膜转运蛋白的原子分辨结构测定提供了有价值的见解。
A V. cholerae MATE transporter was crystallized using the lipidic cubic phase (LCP) method. X-ray diffraction data sets were collected from single crystals obtained in a sandwich plate and a sitting-drop plate to resolutions of 2.5 and 2.2 Å, respectively. Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane. Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets. Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP). X-ray diffraction data were collected to 2.5 Å resolution from a single crystal obtained in a sandwich plate. The crystal belonged to space group P212121, with unit-cell parameters a = 52.3, b = 93.7, c = 100.2 Å. As a result of further LCP crystallization trials, crystals of larger size were obtained using sitting-drop plates. X-ray diffraction data were collected to 2.2 Å resolution from a single crystal obtained in a sitting-drop plate. The crystal belonged to space group P212121, with unit-cell parameters a = 61.9, b = 91.8, c = 100.9 Å. The present work provides valuable insights into the atomic resolution structure determination of membrane transporters.