Two states of a light-sensitive membrane protein captured at room temperature using thin-film sample mounts.
Two states of a light-sensitive membrane protein captured at room temperature using thin-film sample mounts.
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DOI：
10.1107/s2059798321011220
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发表时间：
2022-01-01
期刊：
影响因子：
--
通讯作者：
Moraes I
中科院分区：
文献类型：
--
作者：
Axford D;Judge PJ;Bada Juarez JF;Kwan TOC;Birch J;Vinals J;Watts A;Moraes I
高分辨率的X射线衍射数据收集在室温下的光和暗适应状态的古紫质-3感光体使用透明和不透明的聚合物膜样品安装。室温衍射方法是非常理想的生物大分子的动态研究，因为它们允许高分辨率的结构数据被收集的蛋白质经历构象变化。对于在无规立方相（LCP）中生长的晶体，挤出机通常用于使微晶流通过X射线束;然而，对于许多膜蛋白，这种方法所需的样品量可能难以产生。使用两层低X射线透射率的聚合物膜来安装古视紫红质-3（AR 3）感光体的晶体，并获得室温衍射数据，从而创建了更有效的样品环境。通过使用透明和不透明的聚合物薄膜，两种结构，一个对应于脱敏，暗适应（DA）状态和其他的地面或光适应（LA）状态，解决了优于1.9 μ m的分辨率。 AR 3的所有关键结构特征都得到了解析，包括视网膜发色团，其在DA状态下以13-顺式异构体存在，在LA状态下以全反式异构体存在。薄膜夹层样品环境使衍射数据能够在室温下在照明和黑暗条件下记录，这更接近体内的情况。这种简单的方法适用于广泛的膜蛋白结晶在LCP和光敏样品一般在同步加速器和实验室X射线源。
High-resolution X-ray diffraction data were collected at room temperature for light- and dark-adapted states of the archaerhodopsin-3 photoreceptor using transparent and opaque polymer-film sample mounts. Room-temperature diffraction methods are highly desirable for dynamic studies of biological macromolecules, since they allow high-resolution structural data to be collected as proteins undergo conformational changes. For crystals grown in lipidic cubic phase (LCP), an extruder is commonly used to pass a stream of microcrystals through the X-ray beam; however, the sample quantities required for this method may be difficult to produce for many membrane proteins. A more sample-efficient environment was created using two layers of low X-ray transmittance polymer films to mount crystals of the archaerhodopsin-3 (AR3) photoreceptor and room-temperature diffraction data were acquired. By using transparent and opaque polymer films, two structures, one corresponding to the desensitized, dark-adapted (DA) state and the other to the ground or light-adapted (LA) state, were solved to better than 1.9 Å resolution. All of the key structural features of AR3 were resolved, including the retinal chromophore, which is present as the 13-cis isomer in the DA state and as the all-trans isomer in the LA state. The film-sandwich sample environment enables diffraction data to be recorded at room temperature in both illuminated and dark conditions, which more closely approximate those in vivo. This simple approach is applicable to a wide range of membrane proteins crystallized in LCP and light-sensitive samples in general at synchrotron and laboratory X-ray sources.