Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins.
Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins.
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DOI：
10.3389/fmolb.2022.882288
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发表时间：
2022
影响因子：
5
通讯作者：
中科院分区：
文献类型：
--
作者：
成功的样品制备是任何结构生物学技术的基础。膜蛋白尤其令人感兴趣，因为它们是药物设计的重要目标，但也是出了名的难以处理。对于电子冷冻显微镜(Cryo-EM)，样品纯度、均一性、完整性以及生化活性的生物物理表征是制备高质量冷冻-EM网格的先决条件，因为这些因素影响计算重建的结果。在这里，我们提出了在单颗粒冷冻-EM网格制备之前的质量控制管道，使用生物物理技术的组合来解决膜蛋白及其复合体的完整性、纯度和低聚状态，从而为样品玻璃化提供可重复的条件。差示扫描荧光法(NDSF)用于优化缓冲液和洗涤剂条件，而质量光度法和动态光散射法用于评估聚集行为、重组效率和齐聚。在NDSF和MASS光度仪器上收集的数据可以通过spc.embl-hamburg.de上公开提供的网络服务器进行分析。在冷冻-EM膜蛋白样品制备前优化条件的案例研究提供了一个质量评估实例，证实了我们管道的有效性。
Successful sample preparation is the foundation to any structural biology technique. Membrane proteins are of particular interest as these are important targets for drug design, but also notoriously difficult to work with. For electron cryo-microscopy (cryo-EM), the biophysical characterization of sample purity, homogeneity, and integrity as well as biochemical activity is the prerequisite for the preparation of good quality cryo-EM grids as these factors impact the result of the computational reconstruction. Here, we present a quality control pipeline prior to single particle cryo-EM grid preparation using a combination of biophysical techniques to address the integrity, purity, and oligomeric states of membrane proteins and its complexes to enable reproducible conditions for sample vitrification. Differential scanning fluorimetry following the intrinsic protein fluorescence (nDSF) is used for optimizing buffer and detergent conditions, whereas mass photometry and dynamic light scattering are used to assess aggregation behavior, reconstitution efficiency, and oligomerization. The data collected on nDSF and mass photometry instruments can be analyzed with web servers publicly available at spc.embl-hamburg.de. Case studies to optimize conditions prior to cryo-EM sample preparation of membrane proteins present an example quality assessment to corroborate the usefulness of our pipeline.
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影响因子：
64.8
作者：
DEROSIER, DJ;KLUG, A
通讯作者：
KLUG, A
影响因子：
4.4
作者：
KOPPEL, DE
通讯作者：
KOPPEL, DE
影响因子：
64.8
作者：
Ford, MGJ;Mills, IG;McMahon, HT
通讯作者：
McMahon, HT
影响因子：
14.8
作者：
通讯作者：
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DOI：
10.1016/0739-6260(91)90054-4
发表时间：
1991-01-01
期刊：
MICRON AND MICROSCOPICA ACTA
影响因子：
--
作者：
BOEKEMA, EJ
通讯作者：
BOEKEMA, EJ