Nanodisc 配合冷凍電鏡提升膜蛋白分辨率

Toxic, hot, and spicy: Nanodiscs improve membrane proteinresolution in cryo-EM
Nanodisc結(jié)合冷凍電鏡應用時，Nanodisc提升了通過冷凍電鏡對膜蛋白的解析率，同時揭示了功能性磷脂所扮演的重要角色。 
 
The last few years have seen a tremendous increase inhigh-resolution protein structures solved by cryo electron microscopy(cryo-EM). Novel electron detecting cameras and sophisticated analysis softwarehave expanded the capacity of cryo-EM to smaller and asymmetric proteins (1).As a true competitor to X-ray crystallography, cryo-EM is particularlyinteresting for hard-to-crystallize targets such as membrane proteins.

在過去的幾年里，使用冷凍電子顯微鏡（冷凍電鏡）對蛋白結(jié)構(gòu)高分辨率結(jié)構(gòu)解析的應用有著很大地增長。新型的電子探測相機和復雜的分析軟件使冷凍電鏡的應用延伸到更小和不對稱的蛋白結(jié)構(gòu)解析
（1）。作為X射線晶體法的真正強勢的替代方法，冷凍電鏡能把如膜蛋白等難以結(jié)晶的蛋白作為應用目標，并引起了各界廣泛的興趣。
 
The importance of sample preparation methods forhigh-resolution cryo-EM data cannot be underestimated. Two recent Naturepublications have shown that nanodiscs are not only excellent tools formembrane protein stabilization, but that they can also improve resolution, inparticular of the transmembrane region, and enable analysis of interactingphospholipids.

在應用的過程中，樣品制備方法對得到高分辨率冷凍電鏡數(shù)據(jù)的重要性是不可低估的。從最近的兩篇發(fā)表到Nature的文章來看，Nanodisc不僅是膜蛋白穩(wěn)定的優(yōu)良工具，而且它也可以提高在電鏡解析的分辨率，特別是膜蛋白的跨膜部分，同時能實現(xiàn)磷脂相互作用的分析。
 
Toxic: Near-atomic detail of a bacterial Tc toxin membraneinsertion (2). StefanRaunser's team at the Max-Planck Institute in Dortmund, Germany unveiled themechanism used by bacterial Tc toxin as it enters the cell. Besides the highmedical relevance of this project - Tc toxins include anthrax, plague, andscarlet-like fever toxins - the conformational changes these toxins undergo aresimply fascinating. Secreted by bacteria as soluble proteins, toxins fold intochannels that perforate the host membrane by a putative entropic springmechanism. In previous attempts with detergent-solubilized protein, it was notpossible to resolve the transmembrane region of the toxin. Now, usingnanodisc-stabilized TcdA1 protein, researchers were able to achieve an overallresolution of 3.5 Angstrom, allowing them to describe this mechanicallyenforced membrane insertion mechanism for the first time.

Toxic: Near-atomic detail of a bacterial Tc toxin membraneinsertion
(2)。德國馬克斯·普朗克研究所的Stefan Raunser團隊闡述了細菌Tc毒素進入細胞的機制。除了這個項目的高度醫(yī)學相關(guān)性價值外( Tc毒素包括炭疽，鼠疫，猩紅熱樣毒素)這些毒素所經(jīng)歷的構(gòu)象變化是有極大吸引力的。由細菌分泌的可溶性蛋白，毒素折疊成通道穿過宿主細胞膜。以前嘗試使用去污劑溶解帶跨膜區(qū)域蛋白進行分析，并不能很好地解析帶跨膜區(qū)域的毒素。而現(xiàn)在使用Nanodisc穩(wěn)定TcdA1蛋白，研究人員能夠獲得到3.5埃的解析度，這讓他們有機會首先發(fā)現(xiàn)并描述了這種機械的強制膜插入的機制。(閱讀Nature原文:http://www.nature.com/nsmb/journal/v23/n10/full/nsmb.3281.html)
 
Hot & spicy: Functional lipids enable detection of heatand hot spices (3). Yifan Cheng's team at UCSFanalyzed the tetrameric transient receptor potential vanilloid 1 (TRPV1) ionchannel at 2.9 Angstrom resolution. TRPV1 reacts to many physical and chemicalstimuli, including heat and capsaicin, an ingredient of chilli peppers. Nanodiscs were crucial to obtain a high resolution structure, as previousattempts with amphipol-stabilized complexes had only yielded a 3.8 Aresolution.

But nanodiscs played another important role in this analysis: By providing aphospholipid bilayer, they enabled the discovery of lipids with a structuralfunction in ligand binding. Similar to the results of the Dortmund group, thetransmembrane regions were those with the highest resolution, stressing thevalue of nanodiscs for cryo-EM analysis.

Hot & spicy: Functional lipids enable detection of heatand hot spices
(3). 加州大學舊金山分校的程亦凡團隊分析了瞬態(tài)電壓感受器陽離子通道1(TRPV1一種在疼痛和熱知覺中起中心作用的蛋白質(zhì)）在2.9埃分辨率離子通道結(jié)構(gòu)。TRPV1對許多物理和化學刺激，包括熱、辣椒素(一種辣椒的成分)都有反應。Nanodisc是此研究中獲得高分辨率的結(jié)構(gòu)十分重要的因素，而以前的嘗試使用雙極性穩(wěn)定復合物只得到了3.8個埃的分辨率。

在這一研究中，Nanodisc還扮演了另一個非常重要的角色：通過提供一個磷脂雙分子層，研究人員得以發(fā)現(xiàn)磷脂具有配基結(jié)合的結(jié)構(gòu)功能。類似于Stefan Raunser團隊的結(jié)果，跨膜區(qū)具有最高的分辨率，大大提升了Nanodisc在結(jié)合冷凍電鏡分析膜蛋白應用中的價值。(閱讀Nature原文:http://www.nature.com/nature/journal/v534/n7607/full/nature17964.html)
 
 
參考文獻：
1.Kühlbrandt,W. Cryo-EM enters a new era. eLIFE (2014) doi:10.7554/eLife.03678
2.Gatsogiannis, C. et al. Membrane insertion of a Tc toxin in near-atomic detail.Nature structural and molecular biology (2016),23,884-890. doi:10.1038/nsmb.3281
3.Gao, Y.et al. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipidaction. Nature (2016) 534(7607):347-351. doi:10.1038/nature17964