Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography.
Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography.
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DOI：
10.3791/54463
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发表时间：
2016-09-20
期刊：
影响因子：
--
通讯作者：
Liu W
中科院分区：
文献类型：
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作者：
Ishchenko A;Cherezov V;Liu W
膜蛋白（MPs）是细胞膜的重要组成部分，也是主要的药物靶点。合理的药物设计依赖于精确的结构信息，通常是通过晶体学获得的；然而MPs很难结晶。最近MP结构测定的进展很大程度上得益于脂质立方相（LCP）结晶方法的发展，这种方法通常产生良好的衍射，但通常是小晶体，在传统的同步加速器晶体学数据收集过程中受到辐射损伤。新一代x射线自由电子激光（XFEL）源的发展，产生极其明亮的飞秒脉冲，使室温数据从微晶体收集没有或可以忽略不计的辐射损伤。我们最近将LCP技术与序列飞秒晶体学（LCP- sfx）相结合的努力已经产生了几种人类G蛋白偶联受体的高分辨率结构，这是一个众所周知的难以确定结构的目标。在LCP- sfx技术中，LCP被用作基质，用于MP微晶体的生长和输送到注入流与XFEL光束的交叉处，以收集晶体学数据。已经证明，当只有低于10µm的晶体可用时，或者当在室温下使用较小的晶体可以克服与较大的冷冻晶体相关的各种问题（如缺陷积累，高镶嵌性和冷冻伪像）时，LCP-SFX可以显着提高衍射分辨率。x射线源和探测器技术的未来发展将使串行晶体学不仅在XFELs上实现，而且在更容易获得的同步加速器光束线上实现，具有很高的吸引力和实用性。在这里，我们提出了详细的视觉协议的制备，表征和传递微晶体的LCP系列晶体学实验。这些方案包括在注射器中进行结晶实验，检测和表征晶体样品，优化晶体密度，将微晶负载的LCP加载到注射器装置中，并将样品输送到光束中进行数据收集。
Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 µm crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.
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DOI：
10.1126/science.1244142
发表时间：
2013-12-20
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Liu W;Wacker D;Gati C;Han GW;James D;Wang D;Nelson G;Weierstall U;Katritch V;Barty A;Zatsepin NA;Li D;Messerschmidt M;Boutet S;Williams GJ;Koglin JE;Seibert MM;Wang C;Shah ST;Basu S;Fromme R;Kupitz C;Rendek KN;Grotjohann I;Fromme P;Kirian RA;Beyerlein KR;White TA;Chapman HN;Caffrey M;Spence JC;Stevens RC;Cherezov V
通讯作者：
Cherezov V
影响因子：
3.3
作者：
Kulkarni, Chandrashekhar V.;Wachter, Wolfgang;Ahualli, Silvia
通讯作者：
Ahualli, Silvia
影响因子：
7.4
作者：
Kissick, David J.;Gualtieri, Ellen J.;Cherezov, Vadim
通讯作者：
Cherezov, Vadim
影响因子：
1.2
作者：
Liu, Wei;Cherezov, Vadim
通讯作者：
Cherezov, Vadim
影响因子：
14.8
作者：
通讯作者：
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