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Nanodisc配合冷凍電鏡提升膜蛋白的分辨率
Toxic, hot, and spicy: Nanodiscs improve membrane protein resolution in cryo-EM
(作者：Cube Biotech)
Nanodisc結(jié)合冷凍電鏡應(yīng)用時(shí) ，Nanodisc提升了通過冷凍電鏡對(duì)膜蛋白的解析率，同時(shí)表現(xiàn)了功能性磷脂所扮演的重要角色。
The last few years have seen a tremendous increase in high-resolution protein structures solved by cryo electron microscopy (cryo-EM). Novel electron detecting cameras and sophisticated analysis software have expanded the capacity of cryo-EM to smaller and asymmetric proteins (1). As a true competitor to X-ray crystallography, cryo-EM is particularly interesting 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(duì)蛋白結(jié)構(gòu)高分辨率結(jié)構(gòu)解析的應(yīng)用有著很大地增長(zhǎng)。新型的電子探測(cè)相機(jī)和復(fù)雜的分析軟件使冷凍電鏡的應(yīng)用延伸到更小和不對(duì)稱的蛋白結(jié)構(gòu)解析（1）。作為X射線晶體法的真正強(qiáng)勢(shì)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(yīng)用目標(biāo)，并引起了各界廣泛的興趣。
The importance of sample preparation methods for high-resolution cryo-EM data cannot be underestimated. Two recent Nature publications have shown that nanodiscs are not only excellent tools for membrane protein stabilization, but that they can also improve resolution, in particular of the transmembrane region, and enable analysis of interacting phospholipids.
在應(yīng)用的過程中，樣品制備方法對(duì)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(shí)能實(shí)現(xiàn)磷脂相互作用的分析。
Toxic: Near-atomic detail of a bacterial Tc toxin membrane insertion (2). Stefan Raunser's team at the Max-Planck Institute in Dortmund, Germany unveiled the mechanism used by bacterial Tc toxin as it enters the cell. Besides the high medical relevance of this project - Tc toxins include anthrax, plague, and scarlet-like fever toxins - the conformational changes these toxins undergo are simply fascinating. Secreted by bacteria as soluble proteins, toxins fold into channels that perforate the host membrane by a putative entropic spring mechanism. In previous attempts with detergent-solubilized protein, it was not possible to resolve the transmembrane region of the toxin. Now, using nanodisc-stabilized TcdA1 protein, researchers were able to achieve an overall resolution of 3.5 Angstrom, allowing them to describe this mechanically enforced membrane insertion mechanism for the first time.
Toxic: Near-atomic detail of a bacterial Tc toxin membrane insertion (2)。德國(guó)馬克斯·普朗克研究所的Stefan Raunser團(tuán)隊(duì)闡述了細(xì)菌Tc毒素進(jìn)入細(xì)胞的機(jī)制。除了這個(gè)項(xiàng)目的高度醫(yī)學(xué)相關(guān)性價(jià)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(xì)菌分泌的可溶性蛋白，毒素折疊成通道穿過宿主細(xì)胞膜。以前嘗試使用去污劑溶解帶跨膜區(qū)域蛋白進(jìn)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(jī)會(huì)首先發(fā)現(xiàn)并描述了這種機(jī)械的強(qiáng)制膜插入的機(jī)制。
Hot & spicy: Functional lipids enable detection of heat and hot spices (3). Yifan Cheng's team at UCSF analyzed the tetrameric transient receptor potential vanilloid 1 (TRPV1) ion channel at 2.9 Angstrom resolution. TRPV1 reacts to many physical and chemical stimuli, including heat and capsaicin, an ingredient of chilli peppers. Nanodiscs were crucial to obtain a high resolution structure, as previous attempts with amphipol-stabilized complexes had only yielded a 3.8 A resolution.
But nanodiscs played another important role in this analysis: By providing a phospholipid bilayer, they enabled the discovery of lipids with a structural function in ligand binding. Similar to the results of the Dortmund group, the transmembrane regions were those with the highest resolution, stressing the value of nanodiscs for cryo-EM analysis.
Hot & spicy: Functional lipids enable detection of heat and hot spices (3). 加州大學(xué)舊金山分校的Yifan Cheng團(tuán)隊(duì)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(duì)許多物理和化學(xué)刺激，包括熱，辣椒素(一種辣椒的成分)都有反應(yīng)。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(fù)合物只得到了3.8個(gè)埃的分辨率。
在這一研究中，Nanodisc還扮演了另一個(gè)非常重要的角色：通過提供一個(gè)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(tuán)隊(duì)的結(jié)果，跨膜區(qū)具有很高的分辨率，大大提升了Nanodisc在結(jié)合冷凍電鏡分析膜蛋白應(yīng)用中的價(jià)值。
參考文獻(xiàn)：
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 lipid action. Nature (2016) 534(7607):347-351. doi:10.1038/nature17964
