Li, XM; Mooney, P; Zheng, S; et al.

Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units

Song, F; Chen, P; Sun, DP; et al.

Beam-induced motion correction for sub-megadalton cryo-EMparticles

Scheres, SHW

Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine

Wong, W; Bai, XC; Brown, A; et al.

結(jié)構(gòu)生物學(xué)研究方法的重大突破——電子直接探測(cè)相機(jī)在冷凍電鏡中的應(yīng)用

柳正，張景強(qiáng)

冷凍電鏡

·編者按·

冷凍電子顯微鏡技術(shù)（cryo-electron microscopy，Cryo-EM）是指運(yùn)用透射電子顯微鏡對(duì)低溫樣品進(jìn)行觀察和成像的顯微技術(shù)，簡(jiǎn)稱冷凍電鏡技術(shù)。冷凍電鏡技術(shù)由低溫制樣、低劑量電鏡成像和計(jì)算機(jī)圖像處理3部分組成。冷凍電鏡是重要的結(jié)構(gòu)生物學(xué)研究方法，與X射線晶體學(xué)和核磁共振一起構(gòu)成了高分辨率結(jié)構(gòu)生物學(xué)研究的基礎(chǔ)，在獲得生物大分子的結(jié)構(gòu)并揭示其功能方面極為重要，在結(jié)構(gòu)生物學(xué)領(lǐng)域引起了研究者的極大關(guān)注。

冷凍電鏡理論在20世紀(jì)70年代提出，但直到21世紀(jì)初，冷凍電鏡的分辨率水平依然沒有得到突破。2013年，由于最新的直接電子探測(cè)器（Direct Electron Detector, DED）的應(yīng)用，以及單顆粒三維重建算法的革新和不斷改進(jìn)，冷凍電鏡技術(shù)取得了飛躍式的發(fā)展，通過該技術(shù)解析得到的生物大分子結(jié)構(gòu)可以達(dá)到近原子分辨率（＜4 ?）水平。

3位冷凍電鏡領(lǐng)域的開拓者Richard Henderson、Joachim Frank和Jacques Dubochet分別在該領(lǐng)域的基本理論、重構(gòu)算法和實(shí)驗(yàn)方面的早期研究中做出了重要貢獻(xiàn)。他們也因?yàn)椤霸陂_發(fā)用于溶液中生物分子高分辨率結(jié)構(gòu)測(cè)定的冷凍電鏡技術(shù)方面的貢獻(xiàn)”，被授予2017年諾貝爾化學(xué)獎(jiǎng)。

本專題得到張景強(qiáng)教授（中山大學(xué)）、朱莉副教授（蘭州大學(xué)）的大力支持。

·熱點(diǎn)數(shù)據(jù)排行·

截至2017年11月9日，中國知網(wǎng)（CNKI）和Web of Science（WOS）的數(shù)據(jù)報(bào)告顯示，以“冷凍電鏡”等為詞條可以檢索到的期刊文獻(xiàn)分別為153、4452條，本專題將相關(guān)數(shù)據(jù)按照：研究機(jī)構(gòu)發(fā)文數(shù)、作者發(fā)文數(shù)、期刊發(fā)文數(shù)、被引用頻次進(jìn)行排行，結(jié)果如下。

研究機(jī)構(gòu)發(fā)文數(shù)量排名（CNKI）

研究機(jī)構(gòu)發(fā)文數(shù)量排名（WOS）

作者發(fā)文數(shù)量排名（CNKI）

作者發(fā)文數(shù)量排名（WOS）

作者發(fā)文數(shù)量排名（CNKI）（續(xù)表）

作者發(fā)文數(shù)量排名（WOS）（續(xù)表）

期刊發(fā)文數(shù)量排名（CNKI）

根據(jù)中國知網(wǎng)（CNKI）數(shù)據(jù)報(bào)告，以“冷凍電鏡”等為詞條可以檢索到的高被引論文排行結(jié)果如下。

國內(nèi)數(shù)據(jù)庫高被引論文排行（續(xù)表）

根據(jù)Web of Science統(tǒng)計(jì)數(shù)據(jù)，以“冷凍電鏡”等為詞條可以檢索到的高被引論文排行結(jié)果如下。

國外數(shù)據(jù)庫高被引論文排行

·經(jīng)典文獻(xiàn)推薦·

基于Web of Science檢索結(jié)果，利用Histcite軟件選取LCS（Local Citation Score，本地引用次數(shù)）TOP 30文獻(xiàn)作為節(jié)點(diǎn)進(jìn)行分析，得到本領(lǐng)域推薦的經(jīng)典文獻(xiàn)如下。

electron microscopy; single-particle analysis;maximum likelihood; image processing; software development

來源出版物：Journal of Structural Biology, 2012, 180(3)：519-530

Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM

Li, XM; Mooney, P; Zheng, S; et al.

Abstract:In recent work with large high-symmetry viruses, single-particle electron cryomicroscopy (cryo-EM)has achieved the determination of near-atomic-resolution structures by allowing direct fitting of atomic models into experimental density maps. However, achieving this goal with smaller particles of lower symmetry remains challenging. Using a newly developed single electroncounting detector, we confirmed that electron beaminduced motion substantially degrades resolution, and we showed that the combination of rapid readout and nearly noiseless electron counting allow image blurring to be corrected to subpixel accuracy, restoring intrinsic image information to high resolution (Thon rings visible to ～3 ?).Using this approach, we determined a 3.3-?-resolution structure of an ～700-kDa protein with D7 symmetry, theThermoplasma acidophilum20S proteasome, showing clear side-chain density. Our method greatly enhances image quality and data acquisition efficiency—key bottlenecks in applying near-atomic-resolution cryo-EM to a broad range of protein samples.

來源出版物：Nature Methods, 2013, 10(6)： 584-590

Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units

Song, F; Chen, P; Sun, DP; et al.

Abstract:The hierarchical packaging of eukaryotic chromatin plays a central role in transcriptional regulation and other DNA-related biological processes. Here, we report the 11-angstrom-resolution cryogenic electron microscopy (cryo-EM) structures of 30-nanometer chromatin fibers reconstituted in the presence of linker histone H1 and with different nucleosome repeat lengths.The structures show a histone H1-dependent left-handed twist of the repeating tetranucleosomal structural units,within which the four nucleosomes zigzag back and forth with a straight linker DNA. The asymmetric binding and the location of histone H1 in chromatin play a role in the formation of the 30-nanometer fiber. Our results provide mechanistic insights into how nucleosomes compact into higher-order chromatin fibers.

來源出版物：Science, 2014, 344(6182)： 376-380

Beam-induced motion correction for sub-megadalton cryo-EMparticles

Scheres, SHW

Abstract:In electron cryo-microscopy (cryo-EM), the electron beam that is used for imaging also causes the sample to move. This motion blurs the images and limits the resolution attainable by single-particle analysis. In a previous Research article we showed that correcting for this motion by processing movies from fast direct-electron detectors allowed structure determination to near-atomic resolution from 35000 ribosome particles. In this Research advance article, we show that an improved movie processing algorithm is applicable to a much wider range of specimens.The new algorithm estimates straight movement tracks by considering multiple particles that are close to each other in the field of view, and models the fall-off of high-resolution information content by radiation damage in a dosedependent manner. Application of the new algorithm to four data sets illustrates its potential for significantly improving cryo-EM structures, even for particles that are smaller than 200 kDa.

來源出版物：Elife, 2014, 3： e03665

Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine

Wong, W; Bai, XC; Brown, A; et al.

Abstract:Malaria inflicts an enormous burden on global human health. The emergence of parasite resistance to front-line drugs has prompted a renewed focus on the repositioning of clinically approved drugs as potential anti-malarial therapies. Antibiotics that inhibit protein translation are promising candidates for repositioning. We have solved the cryo-EM structure of the cytoplasmic ribosome from the human malaria parasite, Plasmodium falciparum, in complex with emetine at 3.2 angstrom resolution. Emetine is an anti-protozoan drug used in the treatment of ameobiasis that also displays potent anti-malarial activity. Emetine interacts with the E-site of the ribosomal small subunit and shares a similar binding site with the antibiotic pactamycin, thereby delivering its therapeutic effect by blocking mRNA/tRNA translocation.As the first cryo-EM structure that visualizes an antibiotic bound to any ribosome at atomic resolution, this establishes cryo-EM as a powerful tool for screening and guiding the design of drugs that target parasite translation machinery.

來源出版物：Elife, 2014, 3： e03080

·推薦綜述·

結(jié)構(gòu)生物學(xué)研究方法的重大突破——電子直接探測(cè)相機(jī)在冷凍電鏡中的應(yīng)用

柳正，張景強(qiáng)

引言

早在20世紀(jì)70年代，材料學(xué)研究中就使用電鏡直接觀察到了原子像。而生物材料由于含水、易受電子?xùn)c損傷及固有的低反差等問題，無法獲得高分辨圖像。后來，人們采用冷凍技術(shù)、低劑量成像及圖像疊加等技術(shù)予以解決，從而在20世紀(jì)80年代出現(xiàn)了—種測(cè)定生物大分子三維結(jié)構(gòu)的新方法——冷凍電鏡技術(shù)。冷凍電鏡技術(shù)由低溫制樣、低劑量電鏡成像和計(jì)算機(jī)圖像處理三部分組成。低溫制樣最先由Taylor和Glaeser成功應(yīng)用，后經(jīng)Dubochet的深入研究而完善，其基本流程是把載有樣品（如緩沖液中病毒、蛋白質(zhì)和DNA組裝的顆粒等）的電鏡載網(wǎng)快速投入經(jīng)液氮/液氦冷卻的液態(tài)乙烷中，在載網(wǎng)孔或支持膜上形成玻璃態(tài)的薄冰，樣品顆粒（粒徑大約十幾到過百nm）分散包埋在其中而形成冷凍樣品。電鏡成像是指保持樣品在液氮或液氦低溫下，使用低電子劑量成像（通常使用20 e/?2左右的低劑量）。計(jì)算機(jī)圖像處理最先是指單顆粒圖像分析，F(xiàn)rank及其同事做了大量的開創(chuàng)性工作，使其成為解析生物大分子三維結(jié)構(gòu)的一個(gè)有力工具。單顆粒圖像分析方法的要點(diǎn)是首先估計(jì)電子顯微像中大量分散的樣品顆粒（如核糖體、病毒顆粒和DNA顆粒等）的取向歐拉角和中心位置，并迭代精修，然后分類疊加平均，再根據(jù)測(cè)定的取向和位置參數(shù)，把二維像在傅里葉空間三維插值重構(gòu)出其三維結(jié)構(gòu)。隨著研究的深入，證明應(yīng)用單顆粒圖像分析方法能夠在原子分辨率尺度上解析生物大分子的結(jié)構(gòu)。

雖然理論上可行，但由于冷凍電鏡所獲得的圖像信噪比很低，在圖像分析中很難準(zhǔn)確地恢復(fù)高頻信號(hào)及測(cè)定每個(gè)顆粒的取向和位置參數(shù)。因此，冷凍電鏡單顆粒技術(shù)理論預(yù)期的能力在實(shí)踐應(yīng)用中很難達(dá)到。過去5年，也有一些高分辨率的結(jié)構(gòu)被冷凍電鏡單顆粒技術(shù)解析出來，但都局限于具有高對(duì)稱性、剛性很好的大分子量球形病毒樣品。近年來，電子顯微鏡開始裝備了電子直接探測(cè)相機(jī)（electron direct detection device，DDD），已有研究組用這一裝置對(duì)非對(duì)稱性核糖體或者低對(duì)稱性膜蛋白的結(jié)構(gòu)進(jìn)行研究，均獲得了高分辨率的結(jié)果。本文將結(jié)合我們以往的冷凍電鏡研究工作，介紹DDD相機(jī)的原理和技術(shù)優(yōu)勢(shì)，并結(jié)合冷凍電鏡中的主要技術(shù)難題，展望DDD相機(jī)可能給冷凍電鏡技術(shù)帶來的突破性進(jìn)展。

電鏡中的成像載體

一般電鏡中感光并記錄圖像的載體可以分為3類：熒光屏、感光膠片和CCD（charge-couple device）相機(jī)。熒光屏可實(shí)時(shí)顯示所得的電子顯微像，肉眼可直接觀測(cè)但卻無法永久保存。一般使用感光膠片或者CCD相機(jī)永久記錄電子顯微像。

膠片是一種對(duì)電子束敏感、顆粒度很小的溴化物乳膠底片，能很好地記錄保存圖像，但需要經(jīng)常卸換底片（FEI的電鏡通常一次只能裝載56張底片），此過程會(huì)干擾電鏡鏡筒內(nèi)的真空，并可能造成污染。另外，底片須在暗室內(nèi)沖洗，掃描后才能進(jìn)行圖像處理和重構(gòu)。我們?cè)褂酶泄饽z片，應(yīng)用髮夾燈絲200 kV的電鏡，解析出亞納米分辨率（9.8 ?）的戊型肝炎病毒（hepatitis E virus，HEV）的三維結(jié)構(gòu)，在300 kV的場(chǎng)發(fā)射電鏡下，解析了T7噬菌體衣殼的3.5 ?結(jié)構(gòu)。

電鏡CCD相機(jī)首先用閃爍器（scintillator）把入射電子信號(hào)轉(zhuǎn)換成光信號(hào)，接著，用透鏡或者光纖把光信號(hào)傳送到像感應(yīng)器，然后再轉(zhuǎn)換為數(shù)字圖像。入射電子每次進(jìn)入閃爍器，都會(huì)在原位點(diǎn)發(fā)生偏離而形成擴(kuò)散光云（cloud of light），常用點(diǎn)擴(kuò)散函數(shù)（point spread function，PSF）來描述這一過程。點(diǎn)擴(kuò)散函數(shù)隨著入射電子數(shù)量的增加及深入而增大。因此，記錄完整的一次曝光信號(hào)需要比較大的像素尺寸，如常見的CCD像素尺寸為15 μm。

與膠片相比，CCD相機(jī)的優(yōu)點(diǎn)是無需卸換和沖洗，可以直接長(zhǎng)時(shí)間連續(xù)地采集圖像，與其他軟件結(jié)合，還可以實(shí)現(xiàn)全自動(dòng)高通量的數(shù)據(jù)收集。現(xiàn)在有不少實(shí)驗(yàn)室已經(jīng)廣泛使用CCD進(jìn)行實(shí)時(shí)檢測(cè)圖像。我們也曾用CCD相機(jī)，在300 kV場(chǎng)發(fā)射電鏡條件下，解析了昆蟲質(zhì)型多角體病毒（cytoplasmic polyhedrosis virus，CPV）近原子分辨率（3.9 ?）的三維結(jié)構(gòu)。

在CCD相機(jī)成像過程中，從成像電子入射直到最后的像被讀出，要經(jīng)歷閃爍器中電子和光子的散射，以及光纖中的光子散射，還受到透鏡耦合度的影響，這些都可能造成數(shù)據(jù)質(zhì)量的下降。在高分辨率區(qū)域，CCD相機(jī)收集數(shù)據(jù)的檢測(cè)量子效率（detective quantum efficiency，DQE，表示輸入和輸出圖像的信噪比的比率）不如膠片，因此，在使用DDD相機(jī)之前，大多數(shù)的高分辨率病毒結(jié)構(gòu)的電鏡數(shù)據(jù)都是用感光膠片進(jìn)行收集的。

DDD相機(jī)采用的是直接使用電子感光的方式。電子直接探測(cè)技術(shù)在其他領(lǐng)域（如高能粒子物理）早有應(yīng)用，而由于電鏡成像電子的能量很高，容易損傷傳感器，限制了它在電鏡領(lǐng)域的應(yīng)用。經(jīng)過幾年的摸索，研究人員已經(jīng)用DDD相機(jī)采集到的數(shù)據(jù)成功解析了多個(gè)非對(duì)稱性或低對(duì)稱性的高分辨率結(jié)構(gòu)，如病毒、核糖體及其他蛋白等。得到的結(jié)構(gòu)密度圖能夠直接進(jìn)行氨基酸建模，可與解析生物大分子結(jié)構(gòu)的傳統(tǒng)方法（如X射線和NMR技術(shù)）相媲美。

DDD相機(jī)的原理與成像模式

DDD相機(jī)的基本原理是電子直接感光，不需要CCD相機(jī)那樣的中間轉(zhuǎn)換步驟。現(xiàn)代電子直接傳感器主要源于在數(shù)碼相機(jī)中發(fā)展的有源像素傳感器（active pixel sensor，APS）互補(bǔ)金屬氧化物半導(dǎo)體CMOS（complementary metal oxide semiconductor，CMOS）技術(shù)。在相機(jī)的芯片中，每個(gè)像素都有各自的信號(hào)放大器，各自進(jìn)行電荷—電壓的轉(zhuǎn)換，從而提高了探測(cè)入射電子效率和像素讀寫的速度；再結(jié)合特殊線路布局，能夠很好地承受電鏡入射的高能電子。最早一代的DDD相機(jī)還采用背部比較厚的傳感器，隨著入射電子的增加與透入更深，PSF會(huì)增大；當(dāng)前的DDD相機(jī)使用背部薄化（back-thinned）的傳感器，避免了底層對(duì)電子的散射，僅僅在其上層硅質(zhì)的介質(zhì)中直接存貯入射的電子，然后進(jìn)行信號(hào)轉(zhuǎn)換，極大縮小了PSF。因此，這類傳感器可以使用更小的像素尺寸（如K2 summit的為5 μm），從而可以在單位尺寸內(nèi)應(yīng)用更多像素。由于不需要中間的電子、光信號(hào)和圖像的轉(zhuǎn)換，免去了因此而產(chǎn)生的信號(hào)扭曲和失真。

調(diào)制傳遞函數(shù)（modulation transfer function，MTF）和檢測(cè)量子效率（detective quantum efficiency，DQE）可以比較客觀地描述DDD相機(jī)的性能特征。MTF是不同空間頻率下相機(jī)信號(hào)的振幅函數(shù)，從數(shù)學(xué)上可以表征為PSF的傅立葉變換。而DQE是輸入和輸出圖像的信噪比的比率。DDD相機(jī)與其它記錄載體相比具有的特點(diǎn)是MTF和DQE在高頻區(qū)域（高分辨率區(qū)）都很高，因而為電鏡記錄高分辨率的圖像提供了保障。目前，DDD相機(jī)常用的成像模式可分為3種：整合模式（integrating mode）、計(jì)數(shù)模式（counting mode）和超分辨率模式（super-resolution mode）。用戶可以根據(jù)自己的需要和所具備的計(jì)算設(shè)備的圖像處理能力選擇適合的模式。

整合模式與感光膠片和傳統(tǒng)的CCD一樣，DDD可以在曝光時(shí)間內(nèi)把所有成像的入射電子一次性地收集與記錄，并輸出為圖像。一次性的收集往往使得劑量都比較大，成像質(zhì)量的提高來自DDD傳感器自身較強(qiáng)的探測(cè)效率和內(nèi)在較高的轉(zhuǎn)換效率。

計(jì)數(shù)模式是對(duì)入射到探測(cè)器后的單個(gè)成像電子進(jìn)行逐個(gè)記錄，即把入射的電子模擬信號(hào)記錄成離散的單個(gè)輸出，這就要求傳感器具有非常高的讀出速率。一般讀出速率400幀/s，如果相機(jī)的尺寸是4 K×

4 K，讀出速率約為6 700 M·pixel/s，需要的計(jì)算機(jī)處理速率大概為100 Gb/s。

超分辨率模式是計(jì)數(shù)模式的進(jìn)一步改進(jìn)，記錄的單個(gè)信號(hào)突破了物理像素的尺寸限制。其原理在于該模式下的傳感器記錄將PSF進(jìn)一步縮小?？梢园褕D像讀出限制在信號(hào)產(chǎn)生的中心附近，這個(gè)范圍往往都比一個(gè)像素的尺寸還小。因此，可以把信號(hào)局限在亞像素范圍內(nèi)。這種成像模式進(jìn)一步地限制了噪聲并提高了探測(cè)效率。DDD的共同優(yōu)點(diǎn)是：可使用電影模式（movie mode）對(duì)同一區(qū)域進(jìn)行多幀成像，再結(jié)合圖像處理程序矯正研究對(duì)象在成像過程中產(chǎn)生的漂移（電子束相互作用引起的擾動(dòng)，以及樣品臺(tái)的不穩(wěn)定導(dǎo)致的移動(dòng)等）；但是，這樣的數(shù)據(jù)采集方式，大大增加了數(shù)據(jù)量。一般來說，一次曝光存儲(chǔ)25幀的話，對(duì)于4 K×

4 K的DDD，就需要1.6 G來存貯，對(duì)于一個(gè)預(yù)計(jì)重構(gòu)到3點(diǎn)多埃的樣品，如用自動(dòng)采集軟件收集數(shù)據(jù)，通常需要10 000張，這樣一來獲得的數(shù)據(jù)約為15 T。而超分辨模式更是把記錄范圍限制在信號(hào)產(chǎn)生的中心周圍，從而將記錄范圍限制到了亞像素區(qū)域，這樣可以在較低放大倍率下獲得在整合模式或計(jì)數(shù)模式下同樣的目標(biāo)分辨率，從而在同樣的成像區(qū)域內(nèi)采集更多的數(shù)據(jù)，但在成像過程中，圖像輸出花費(fèi)的時(shí)間比其它兩種模式要多，在收集數(shù)據(jù)過程中，單位時(shí)間內(nèi)采集的圖像數(shù)比整合模式和計(jì)數(shù)模式要少。當(dāng)前比較流行的三種DDD相機(jī)分別是K2 base/summit、FalconⅠ/Ⅱ和DE-12/20/64。在200 kV下，K2 base/summit和DE-12相機(jī)的DQE比FalconⅠ/Ⅱ高，而在300 kV，F(xiàn)alconⅡ的DQE在高頻區(qū)比其他兩款相機(jī)要好；另外，DE系列相機(jī)具有視場(chǎng)大的特點(diǎn)，價(jià)格也相對(duì)實(shí)惠。

DDD相機(jī)帶來冷凍電鏡的革新

對(duì)單顆粒技術(shù)的促進(jìn)

對(duì)高對(duì)稱性的樣品來說，應(yīng)用DDD收集數(shù)據(jù)，使獲得高分辨率變得容易：1）同等分辨率下需要的顆粒數(shù)將大大降低，縮短了采集數(shù)據(jù)的時(shí)間。如輪狀病毒，只要807個(gè)顆粒就達(dá)到了4.4 ?；2）達(dá)到同樣分辨率的情況下，對(duì)圖像處理的要求降低。如現(xiàn)在不需要考慮單個(gè)顆粒欠焦值、放大倍率和電子傾斜等多種參數(shù)的精修，就可以得到比較高的分辨率；3）采用同樣的樣品和策略，分辨率將進(jìn)一步提升；4）對(duì)電鏡的要求有所降低，如以往需要300 kV電鏡才能達(dá)到4 ?的分辨率水平，現(xiàn)在用200 kV就可以達(dá)到。

DDD相機(jī)還將進(jìn)一步降低以往電鏡對(duì)研究生物大分子大小的限制（100～200 kD左右）。在傳統(tǒng)的冷凍電鏡技術(shù)中，由于低分子量的生物大分子對(duì)成像電子的散射很弱，圖像輸出信噪比很低，往往很難判斷大分子顆粒的取向和位置相關(guān)參數(shù)，從而限制了其在小分子中的應(yīng)用。對(duì)于某些小生物大分子，有時(shí)還需要借助負(fù)染色的方法來提高反差。DDD相機(jī)對(duì)較低分子量生物大分子成像時(shí)，可以保證產(chǎn)生足夠的識(shí)別信號(hào)以進(jìn)行圖像的取向和位置參數(shù)的配準(zhǔn)。最近已有160 kD的蛋白被解析到4.5 ?，450 kD的蛋白解析到3.2 ?。在膜蛋白結(jié)構(gòu)的測(cè)定中，DDD也可以從去垢劑或者amphipols中的膜蛋白獲得足夠多的信號(hào)，從而滿足圖像處理的要求，重構(gòu)得到高分辨率的結(jié)構(gòu)。

對(duì)DDD相機(jī)采集的數(shù)據(jù)進(jìn)行圖像處理時(shí)，可對(duì)非均一性的樣品實(shí)行有效的計(jì)算機(jī)純化（purification in silico）。在很多執(zhí)行生物功能的大分子樣品中，基于功能因子的結(jié)合與否，構(gòu)成了不同的構(gòu)象狀態(tài)或不同組成的復(fù)合物。目前的生物化學(xué)技術(shù)，如超速離心、柱層析等，不能將它們有效分離。從圖像處理的角度，可用監(jiān)督分類或者無監(jiān)督分類將它們分離開來，尤其最近出現(xiàn)的最大似然估計(jì)（maximum likelihood）的無監(jiān)督分類，給應(yīng)用冷凍電鏡進(jìn)行結(jié)構(gòu)研究帶來了諸多方便，而信噪比的提高使得這些方法更加可靠。因此，冷凍電鏡技術(shù)可以實(shí)時(shí)且在高分辨率下研究處理多種功能狀態(tài)的分子，實(shí)現(xiàn)一次照相獲得多個(gè)高分辨率結(jié)構(gòu)。DDD相機(jī)帶來的信噪比提高，還將會(huì)使得一些特殊的圖像處理方法更加可靠。在研究具有區(qū)域?qū)ΨQ性的樣品時(shí)，如T7噬菌體，其具有二十面體的衣殼，還有不同對(duì)稱性的尾部（參與構(gòu)造的蛋白對(duì)稱性各不相同），應(yīng)用區(qū)域性重構(gòu)的方法，可以避免其主要對(duì)稱性對(duì)尾部的干擾而將其單獨(dú)解析出來。

對(duì)冷凍電子斷層技術(shù)的促進(jìn)

與單顆粒技術(shù)采集大量散落的單個(gè)顆粒相比，電子斷層成像只對(duì)單個(gè)對(duì)象（病毒、細(xì)菌、復(fù)合物和細(xì)胞等）從多個(gè)角度進(jìn)行二維成像，然后將多個(gè)二維圖像整合成三維結(jié)構(gòu)。這種方法在研究非定形、不對(duì)稱和不具全同性的生物樣品的三維結(jié)構(gòu)和功能中，有著不可替代的作用。

但是，電子斷層成像需對(duì)同一樣品區(qū)域多次曝光，增加了曝光的電子劑量，可能會(huì)造成電子?xùn)c損傷。由于生物樣品對(duì)電子輻射比較敏感，能承受的總電子劑量有限。如果將多次曝光累加的總電子劑量控制在稍高于單顆粒技術(shù)成像的電子劑量，在單張圖像上的劑量就會(huì)很低，則圖像的信噪比也很低?，F(xiàn)在有研究者采用相位板或在圖像處理時(shí)進(jìn)行圖像降噪以提高反差。顯而易見，DDD相機(jī)的引入是很好的解決方法。首先，可在低劑量的情況下獲得高信噪比；其次，能夠在高頻區(qū)域保存可以提取的高分辨率信息。如再結(jié)合其他已有的方法和技術(shù)，如多幀成像、配準(zhǔn)疊加算法等，可以更進(jìn)一步提高信噪比，使后續(xù)的圖像處理變得更加可靠，分辨率也因?yàn)閳D像質(zhì)量的提高和算法的可靠性而提高。結(jié)合已有的sub-volume平均，在某些樣品上實(shí)現(xiàn)高分辨率或許值得期待。

對(duì)二維高分辨成像的促進(jìn)

細(xì)胞超微結(jié)構(gòu)的電鏡研究是細(xì)胞生物學(xué)的重要基礎(chǔ)，它著重對(duì)細(xì)胞內(nèi)環(huán)境或者特定功能狀態(tài)下的生物大分子或標(biāo)記了的生物大分子復(fù)合物進(jìn)行研究，以便從中獲得重要的信息——細(xì)胞亞結(jié)構(gòu)的病理變化、生理變化，以及生物大分子在細(xì)胞內(nèi)的遷移和變化情況等。這類研究最關(guān)心的是如何保持樣品處于活的狀態(tài)和獲得高分辨，以便觀察到更小結(jié)構(gòu)及其變化，而高分辨二維成像技術(shù)可滿足這些要求。以往，普通超薄切片技術(shù)采用固定、脫水、包埋等一系列化學(xué)處理，使用化學(xué)染色幫助提高圖像中的反差，但這些化學(xué)處理會(huì)損傷觀察對(duì)象，在電鏡下形成假象，且分辨率只達(dá)到幾個(gè)nm水平。近年來，冷凍超薄切片技術(shù)的出現(xiàn)，使生物材料快速冰凍法固定和在-185～-15℃環(huán)境下進(jìn)行超薄切片成為可能。應(yīng)用這一方法，能制備水合樣品，使生物結(jié)構(gòu)保持或接近活體狀態(tài)，防止可溶性物質(zhì)的抽取、流失和移位，保持了生物大分子的活性，并大大縮短了樣品的制備時(shí)間。

有了好樣品，還要使用冷凍電鏡技術(shù)提高分辨率。如果使用DDD相機(jī)電影模式的多幀成像并結(jié)合相應(yīng)的圖像處理方法，一來可以在低劑量的照相條件下保持樣品的完整性，二來可以除去顆粒移動(dòng)引起的圖像模糊。在圖像處理的過程中，還可進(jìn)行配準(zhǔn)和疊加平均以提高圖像的反差，可以在高頻區(qū)看到更多的細(xì)節(jié)，從而提高圖像的分辨率。如區(qū)域性配準(zhǔn)算法，可選擇多幀配準(zhǔn)算法和多參照相對(duì)位置配準(zhǔn)，從而大大提高對(duì)圖像的分辨能力，進(jìn)而獲得高分辨率（＜1 nm）的結(jié)構(gòu)，這將對(duì)細(xì)胞生物學(xué)研究產(chǎn)生很大的促進(jìn)作用。

結(jié)論

冷凍電鏡可以解析生物大分子的高分辨率結(jié)構(gòu)，這一理論已提出了近20年，但在使用中，由于各種局限而未能發(fā)揮預(yù)期能力。這些局限主要有高通量收集數(shù)據(jù)、所研究的生物分子的異質(zhì)性、電子輻射損傷、樣品漂移和顯微像信噪比低等。隨著DDD相機(jī)的成熟使用，這些局限性可以在不同程度上被直接消除，或通過與其他方法（如圖像處理新算法）結(jié)合來加以消除。因此，結(jié)合DDD相機(jī)，應(yīng)用冷凍電鏡技術(shù)可以比以往更容易解析到高分辨率的結(jié)構(gòu)，冷凍電鏡的高分辨率研究可以從高對(duì)稱性拓寬到無對(duì)稱性樣品，進(jìn)一步降低研究對(duì)象的分子量下限，順利解析難以結(jié)晶的蛋白（如在生物體內(nèi)非常重要的膜蛋白等）結(jié)構(gòu)。最近，著名結(jié)構(gòu)生物學(xué)家Rossmann也指出，由于成熟的DDD相機(jī)的應(yīng)用，冷凍電鏡技術(shù)將會(huì)成為結(jié)構(gòu)生物學(xué)研究的主要工具。因而，我們完全有理由相信，這一結(jié)構(gòu)解析的方法將在生物學(xué)中發(fā)揮更大的作用。

致謝 在本文寫作過程中，諶東華博士閱讀文稿并提供了寶貴意見，特此感謝！

冷凍電子斷層成像可以在納米級(jí)尺度上研究那些結(jié)構(gòu)不具有均一性的分子、病毒、細(xì)胞器以及它們之間組成的復(fù)合體的三維結(jié)構(gòu)。在過去的10年中，電子顯微鏡硬件、冷凍制樣設(shè)備和技術(shù)，以及自動(dòng)化斷層數(shù)據(jù)收集方法的進(jìn)步使得本研究領(lǐng)域得到快速發(fā)展。本文對(duì)冷凍電子斷層成像的方法，包括基本原理、樣品制備、斷層數(shù)據(jù)采集和圖像處理、三維重構(gòu)以及重建信息的理解和展示、近年來在生物樣品領(lǐng)域的一些典型應(yīng)用以及前景作一簡(jiǎn)單介紹。

關(guān)鍵詞：冷凍電鏡；電子斷層成像

來源出版物：生物物理學(xué)報(bào), 2010 (7)： 570-578

被引頻次：7

結(jié)構(gòu)生物學(xué)的新進(jìn)展

張景強(qiáng)，盧炘英，張勤奮

摘要：文章概括地介紹了結(jié)構(gòu)生物學(xué)在整個(gè)生命科學(xué)中的地位，結(jié)構(gòu)生物學(xué)的研究?jī)?nèi)容和結(jié)構(gòu)生物學(xué)的3種主要的研究方法：X射線單晶衍射方法、核磁共振（NMR）方法和電子顯微方法，介紹了這3種方法在近年來所取得的新成果。重點(diǎn)介紹了冷凍電子顯微技術(shù)結(jié)合計(jì)算機(jī)三維重構(gòu)技術(shù)研究病毒三維結(jié)構(gòu)的方法、特點(diǎn)以及所取得的成果。并簡(jiǎn)單介紹了物理學(xué)家在結(jié)構(gòu)生物學(xué)研究中的作用。

關(guān)鍵詞：結(jié)構(gòu)生物學(xué)；三維結(jié)構(gòu)；冷凍電鏡；計(jì)算機(jī)重構(gòu)方法

來源出版物：物理, 2001, 30(7)： 407-412

被引頻次：6

第一類肽鏈釋放因子結(jié)構(gòu)與功能研究的新進(jìn)展

陳潔，柴寶峰，梁愛華

摘要：蛋白質(zhì)生物合成過程的終止是由于第一類肽鏈釋放因子識(shí)別終止密碼子，并導(dǎo)致肽酰-tRNA酯鍵水解，釋放出新合成的多肽鏈。近期，通過冷凍電鏡、結(jié)晶學(xué)、核磁共振、分子動(dòng)力學(xué)和生物化學(xué)等方面的研究，使第一類肽鏈釋放因子的結(jié)構(gòu)與功能逐漸清晰。對(duì)近期的研究進(jìn)行了分析和整理。

關(guān)鍵詞：第一類肽鏈釋放因子；冷凍電鏡；晶體結(jié)構(gòu)；核磁共振；分子動(dòng)力學(xué)

來源出版物：生物化學(xué)與生物物理進(jìn)展, 2009, 36(7)：817-822

被引頻次：5

用含水冷凍電鏡技術(shù)測(cè)定的水稻矮縮病毒的三維結(jié)構(gòu)

盧光瑩，蔡德友，陳聲祥，等

摘要：用含水冷凍電鏡技術(shù)和計(jì)算機(jī)數(shù)據(jù)處理方法分別測(cè)定了水稻矮縮病毒（RDV）完整顆粒和只含內(nèi)殼層的顆粒的三維結(jié)構(gòu)，其分辨率分別為2.6 nm和3.3 nm。從完整顆粒的結(jié)構(gòu)中可以清晰地看到它的外殼層和內(nèi)殼層的雙層結(jié)構(gòu)及其內(nèi)部的RNA或非結(jié)構(gòu)蛋白質(zhì)的電子密度。完整顆粒和內(nèi)殼層的直徑分別為69.8 nm和54.0 nm，外殼層和內(nèi)殼層的厚度分別為6.9 nm和2.5 nm。外殼層表面的三角形剖分?jǐn)?shù)T=13。外殼層由260個(gè)衣粒（三體）組成，因此共有780個(gè)蛋白質(zhì)亞基。外殼層和內(nèi)殼層上均有許多通道。內(nèi)外殼層之間以及外殼層的衣粒之間是以一種十分新穎的聯(lián)鎖方式相互連接的。

關(guān)鍵詞：863計(jì)劃項(xiàng)目；美國W. M. keck基金；國家研究資源中心；國家普通醫(yī)學(xué)科學(xué)研究所

來源出版物：高技術(shù)通訊, 1995(1)： 1-4

被引頻次：5

單顆粒電子顯微學(xué)的研究進(jìn)展

蔡剛

摘要：?jiǎn)晤w粒電子顯微學(xué)是一種新型的結(jié)構(gòu)生物學(xué)技術(shù)和方法，一方面，其解析生物大分子復(fù)合體結(jié)構(gòu)的分辨率日益提高，可以達(dá)到近原子分辨率，提供大蛋白分子或復(fù)合體的精細(xì)結(jié)構(gòu)；另一方面，還可以解析生物大分子在不同功能狀態(tài)下的結(jié)構(gòu)及變化，對(duì)于揭示生物大分子復(fù)合體結(jié)構(gòu)的作用機(jī)理具有重要作用。本文就單顆粒電子顯微學(xué)的研究進(jìn)展作一綜述。

關(guān)鍵詞：?jiǎn)晤w粒電子顯微學(xué)；生物大分子復(fù)合體；自然生理?xiàng)l件；冷凍電鏡；近原子分辨率；動(dòng)態(tài)過程

來源出版物：生物物理學(xué)報(bào), 2010, 26(7)： 560-569

被引頻次：3

冷凍電鏡單顆粒重構(gòu)中的病毒三維顯示

李晶，李鯤鵬，柳正，等

摘要：利用冷凍電鏡顯微技術(shù)和單顆粒三維重建方法獲得BmCPV（家蠶質(zhì)多角體）；CSBV（中蜂囊狀幼蟲病病毒）；C6/36DNV（C6/36濃核病毒）的三維結(jié)構(gòu)體數(shù)據(jù)，用空間低通濾波對(duì)體數(shù)據(jù)矢量場(chǎng)進(jìn)行處理并進(jìn)行三維顯示，較之原來的顯示，提高了信噪比，增強(qiáng)了顯示穩(wěn)定性與顯示質(zhì)量。病毒每個(gè)表面細(xì)節(jié)和軸上突起更加清晰可見。

關(guān)鍵詞：冷凍電子顯微技術(shù)；三維重構(gòu)；低通濾波；三維顯示；病毒

來源出版物：北京郵電大學(xué)學(xué)報(bào), 2005, 28(s1)： 96-98

被引頻次：3

結(jié)構(gòu)生物學(xué)研究方法的重大突破——電子直接探測(cè)相機(jī)在冷凍電鏡中的應(yīng)用

柳正，張景強(qiáng)

摘要：近年來，科學(xué)家應(yīng)用冷凍電鏡技術(shù)（cryo-EM）解析出了低對(duì)稱性生物大分子的高分辨率（3～5 ?）三維結(jié)構(gòu)，并用其密度圖直接進(jìn)行了分子建模。與傳統(tǒng)的X-射線和NMR方法相比，冷凍電鏡技術(shù)具有適用于分子量較大的生物分子、樣品不需結(jié)晶且用量很少等優(yōu)勢(shì)。尤其是電子直接探測(cè)相機(jī)（electron direct detection device，DDD）在冷凍電鏡技術(shù)中的應(yīng)用，使高分辨率的結(jié)構(gòu)研究變得更加簡(jiǎn)單、應(yīng)用更為廣泛，是一個(gè)重大突破。文章介紹DDD相機(jī)的原理和技術(shù)優(yōu)勢(shì)，及其在解決冷凍電鏡技術(shù)困難中的一些應(yīng)用，進(jìn)而展望了DDD相機(jī)可能給冷凍電鏡技術(shù)應(yīng)用帶來的突破性進(jìn)展。

關(guān)鍵詞：冷凍電鏡；電子直接探測(cè)相機(jī)；低對(duì)稱生物大分子；高分辨率

來源出版物：生物物理學(xué)報(bào), 2014, 30(6)： 405-415

被引頻次：2

含水納米材料冷凍電鏡直接成像研究

李茵茵，李鯤鵬，李向輝，等

摘要：對(duì)細(xì)菌、聚合物膠束、載藥脂質(zhì)體和脂質(zhì)體立方晶等含水納米材料進(jìn)行冷凍電鏡直接成像研究。結(jié)果表明，與負(fù)染色技術(shù)相比，文中利用冷凍電鏡技術(shù)觀察到了保存更完整的細(xì)菌膜結(jié)構(gòu)和細(xì)菌的部分內(nèi)部結(jié)構(gòu)、精細(xì)的多層聚合物膠束結(jié)構(gòu)、包裹在脂質(zhì)體內(nèi)的納米藥物以及脂質(zhì)體立方晶的晶格結(jié)構(gòu)。與負(fù)染色技術(shù)相比，冷凍電鏡技術(shù)能避免染色假象、真空變形等缺陷，更真實(shí)地反映了有關(guān)納米材料的原有結(jié)構(gòu)特點(diǎn)。

關(guān)鍵詞：冷凍電鏡；聚合物膠束；脂質(zhì)體

來源出版物：電子顯微學(xué)報(bào), 2012, 31(4)： 346-349.

被引頻次：2

基于小波變換和高斯差分冷凍電鏡生物大分子圖像的自動(dòng)分割

巫小蓉，吳效明

摘要：冷凍電鏡生物大分子圖像分割是進(jìn)行冷凍電鏡生物大分子顆粒識(shí)別的基礎(chǔ)。文章分析了冷凍電鏡生物大分子圖像的主要特點(diǎn)，提出了基于小波變換和高斯差分的冷凍電鏡生物大分子圖像自動(dòng)分割方法。該方法利用小波變換得到原圖像的低分辨率圖像，抑制了噪聲，提高了圖像的對(duì)比度；同時(shí)采用高斯差分算子解決了圖像亮度不均勻的問題，并對(duì)高斯差分圖像采用基于灰度梯度信息融合的分割方法。實(shí)驗(yàn)結(jié)果表明，該算法能有效的減少噪聲對(duì)邊緣提取的影響，分割效果良好，是一種全新的冷凍電鏡生物大分子圖像自動(dòng)分割算法。

關(guān)鍵詞：顆粒識(shí)別；小波變換；高斯差分；最大類間方差；冷凍電鏡生物大分子圖像分割

來源出版物：中國組織工程研究, 2009, 13(48)： 9479-9482

被引頻次：2

基于MDL的病毒冷凍電鏡圖像邊緣檢測(cè)

楊磊，吳效明，巫曉蓉

摘要：信息技術(shù)在生物分子病毒微觀研究上有著越來越多的應(yīng)用。本文介紹了應(yīng)用MDL小波軟閾值去噪結(jié)合盲目反卷積來實(shí)現(xiàn)病毒原圖像的恢復(fù)，以達(dá)到對(duì)比度提高的效果。首先簡(jiǎn)單介紹了小波軟閾值去噪和盲目反卷積原理。然后結(jié)合這兩種算法，有效的恢復(fù)了冷凍電鏡病毒圖像。實(shí)驗(yàn)表明用該算法得到的病毒恢復(fù)圖像來做邊緣檢測(cè)，在同一些經(jīng)典圖像復(fù)原算法比較中，襯度上有明顯的改善。

關(guān)鍵詞：小波軟閾值去噪；盲目反卷積；冷凍電鏡；圖像恢復(fù)；邊緣檢測(cè)

來源出版物：中國醫(yī)學(xué)物理學(xué)雜志, 2006, 23(6)： 408-411

被引頻次：2488

Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy

Henderson, R; Baldwin, JM; Ceska, TA; et al.

Abstract:The light-driven proton pump-bacteriorhodopsin occurs naturally as two-dimensional crystals. A threedimensional density map of the structure, at near-atomic resolution, has been obtained by studying the crystals using electron cryo-microscopy to obtain electron diffraction patterns and high-resolution micrographs. New methods were developed for analysing micrographs from tilted specimens, incorporating methods previously developed for untilted specimens that enable large areas to be analysed and corrected for distortions. Data from 72 images, from both tilted and untilted specimens, were analysed to produce the phases of 2700 independent Fourier components of the structure. The amplitudes of these components were accurately measured from 150 diffraction patterns. Together, these data represent about half of the full three-dimensional transform to 3.5 ?. The map of the structure has a resolution of 3.5 ? in a direction parallel to the membrane plane but lower than this in the perpendicular direction. It shows many features in the density that are resolved from the main density of the seven α-helices. We interpret these features as the bulky aromatic side-chains of phenylalanine, tyrosine and tryptophan residues. There is also a very dense feature, which is the β-ionone ring of the retinal chromophore. Using these bulky side-chains as guide points and taking account of bulges in the helices that indicate smaller side-chains such as leucine, a complete atomic model for bacteriorhodopsin between amino acid residues 8 and 225 has been built.There are 21 amino acid residues, contributed by all seven helices, surrounding the retinal and 26 residues,contributed by five helices, forming the proton pathway or channel. Ten of the amino acid residues in the middle of the proton channel are also part of the retinal binding site.The model also provides a useful basis for consideration of the mechanism of proton pumping and allows a consistent interpretation of a great deal of other experimental data. In particular, the structure suggests that pKchanges in the Schiff base must act as the means by which light energy is converted into proton pumping pressure in the channel.Asp96 is on the pathway from the cytoplasm to the Schiff base and Asp85 is on the pathway from the Schiff base to the extracellular surface.

來源出版物：Journal of Molecular Biology, 1990, 213(4)：899-929

被引頻次：784

Cryo-electron microscopy of viruses

Adrian, M; Dubochet, J; Lepault, J; et al.

Abstract:Thin vitrified layers of unfixed, unstained and unsupported virus suspensions can be prepared for observation by cryo-electron microscopy in easily controlled conditions. The viral particles appear free from the kind of damage caused by dehydration, freezing or adsorption to a support that is encountered in preparing biological samples for conventional electron microscopy. Cryo-electron microscopy of vitrified specimens offers possibilities for high resolution observations that compare favourably with any other electron microscopical method.

來源出版物：Nature, 1984, 308(5954)： 32-36

被引頻次：704

RELION: Implementation of a Bayesian approach to cryo-EMstructure determination

Scheres, SHW

Abstract:RELION, for REgularized LIkelihood OptimizatioN, is an open-source computer program for the refinement of macromolecular structures by single-particle analysis of electron cryo-microscopy (cryo-EM) data.Whereas alternative approaches often rely on user expertise for the tuning of parameters, RELION uses a Bayesian approach to infer parameters of a statistical model from the data. This paper describes developments that reduce the computational costs of the underlying maximum a posteriori (MAP) algorithm, as well as statistical considerations that yield new insights into the accuracy with which the relative orientations of individual particles may be determined. A so-called gold-standard Fourier shell correlation (FSC) procedure to prevent overfitting is also described. The resulting implementation yields highquality reconstructions and reliable resolution estimates with minimal user intervention and at acceptable computational costs.

關(guān)鍵詞：electron microscopy; single-particle analysis;maximum likelihood; image processing; software development

來源出版物：Journal of Structural Biology, 2012, 180(3)：519-530

被引頻次：573

Determination of the fold of the core protein of hepatitis B virus ky electron cryomicroscopy

Bottcher, B; Wynne, SA; Crowther, R

Abstract:Hepatitis B virus, a major human pathogen with an estimated 300 million carriers worldwide, can lead to cirrhosis and liver cancer in cases of chronic infection. The virus consists of an inner nucleocapsid or core, surrounded by a lipid envelope containing virally encoded surface proteins. The core protein, when expressed in bacteria,assembles into core shell particles, closely resembling the native core of the virus. Here we use electron cryomicroscopy to solve the structure of the core protein to 7.4 ? resolution. Images of about 6400 individual particles from 34 micrographs at different levels of defocus were combined, imposing icosahedral symmetry. The threedimensional map reveals the complete fold of the polypeptide chain, which is quite unlike previously solved viral capsid proteins and is largely α-helical. The dimer clustering of subunits produces spikes on the surface of the shell, which consist of radial bundles of four long α-helices. Our model implies that the sequence corresponding to the immunodominant region of the core protein lies at the tip of the spike and also explains other properties of the core protein.

來源出版物：Nature, 1997, 386(6620)： 88-91

被引頻次：571

Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM

Li, XM; Mooney, P; Zheng, S; et al.

Abstract:In recent work with large high-symmetry viruses, single-particle electron cryomicroscopy (cryo-EM)has achieved the determination of near-atomic-resolution structures by allowing direct fitting of atomic models into experimental density maps. However, achieving this goal with smaller particles of lower symmetry remains challenging. Using a newly developed single electroncounting detector, we confirmed that electron beaminduced motion substantially degrades resolution, and we showed that the combination of rapid readout and nearly noiseless electron counting allow image blurring to be corrected to subpixel accuracy, restoring intrinsic image information to high resolution (Thon rings visible to ～3 ?).Using this approach, we determined a 3.3-?-resolution structure of an ～700 kDa protein with D7 symmetry, theThermoplasma acidophilum20 S proteasome, showing clear side-chain density. Our method greatly enhances image quality and data acquisition efficiency—key bottlenecks in applying near-atomic-resolution cryo-EM to a broad range of protein samples.

來源出版物：Nature Methods, 2013, 10(6)： 584-590

被引頻次：538

Optimal determination of particle orientation,absolute hand, and contrast loss in single-particle electron cryomicroscopy

Rosenthal, PB; Henderson, R

Abstract:A computational procedure is described for assigning the absolute hand of the structure of a protein or assembly determined by single-particle electron microscopy.The procedure requires a pair of micrographs of the same particle field recorded at two tilt angles of a single tilt-axis specimen holder together with the three-dimensional map whose hand is being determined. For orientations determined from particles on one micrograph using the map, the agreement (average phase residual) between particle images on the second micrograph and map projections is determined for all possible choices of tilt angle and axis. Whether the agreement is better at the known tilt angle and axis of the microscope or its inverse indicates whether the map is of correct or incorrect hand.An increased discrimination of correct from incorrect hand(free hand difference), as well as accurate identification of the known values for the tilt angle and axis, can be used as targets for rapidly optimizing the search or refinement procedures used to determine particle orientations.Optimized refinement reduces the tendency for the model to match noise in a single image, thus improving the accuracy of the orientation determination and therefore the quality of the resulting map. The hand determination and refinement optimization procedure is applied to image pairs of the dihydrolipoyl acetyltransferase (E2) catalytic core of the pyruvate dehydrogenase complex fromBacillus stearothermophilustaken by low-dose electron cryomicroscopy. Structure factor amplitudes of a three-dimensional map of the E2 catalytic core obtained by averaging untilted images of 3667 icosahedral particles are compared to a scattering reference using a Guinier plot. A noisedependent structure factor weight is derived and used in conjunction with a temperature factor (B=-1 000 ?2) to restore high-resolution contrast without amplifying noise and to visualize molecular features to 8.7 ? resolution,according to a new objective criterion for resolution assessment proposed here.

關(guān)鍵詞：electron cryomicroscopy; single particle reconstruction; absolute hand; pyruvate dehydrogenase; tilt pairs

來源出版物：Journal of Molecular Biology, 2003, 333(4)：721-745

被引頻次：409

Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy

Yonekura, K; Maki-Yonekura, S; Namba, K; et al.

Abstract:The bacterial flagellar filament is a helical propeller for bacterial locomotion. It is a helical assembly of a single protein, flagellin, and its tubular structure is formed by 11 protofilaments in two distinct conformations,L- and R-type, for supercoiling. The X-ray crystal structure of a flagellin fragment lacking about 100 terminal residues revealed the protofilament structure, but the full filament structure is still essential for understanding the mechanism of supercoiling and polymerization. Here we report a complete atomic model of the R-type filament by electron cryomicroscopy. A density map obtained from image data up to 4 ? resolution shows the feature of α-helical backbone and some large side chains. The atomic model built on the map reveals intricate molecular packing and an-helical coiled coil formed by the terminal chains in the inner core of the filament, with its intersubunit hydrophobic interactions having an important role in stabilizing the filament.

來源出版物：Nature, 2003, 424(6949)： 643-650

被引頻次：387

Cryo-electron microscopy structure of an SH3 amyloid fibril and model of the molecular packing

Jimenez, JL; Guijarro, JL; Orlova, E; et al.

Abstract:Amyloid fibrils are assemblies of misfolded proteins and are associated with pathological conditions such as Alzheimer’s disease and the spongiform encephalopathies. In the amyloid diseases, a diverse group of normally soluble proteins self-assemble to form insoluble fibrils. X-ray fibre diffraction studies have shown that the protofilament cores of fibrils formed from the various proteins all contain a cross-β-scaffold, with β-strands perpendicular and β-sheets parallel to the fibre axis. We have determined the threedimensional structure of an amyloid fibril, formed by the SH3 domain of phosphatidylinositol-3′-kinase, using cryo-electron microscopy and image processing at 25 ? resolution. The structure is a double helix of two protofilament pairs wound around a hollow core, with a helical crossover repeat of ～600 ? and an axial subunit repeat of ～27 ?.The native SH3 domain is too compact to fit into the fibril density, and must unfold to adopt a longer, thinner shape in the amyloid form. The 20×40-? protofilaments can only accommodate one pair of flat β-sheets stacked against each other, with very little inter-strand twist. We propose a model for the polypeptide packing as a basis for understanding the structure of amyloid fibrils in general.

關(guān)鍵詞：amyloid fibrils; cryo-electron microscopy; protein misfolding; SH3 domain; single particle analysis

來源出版物：The EMBO Journal, 1999, 18(4)： 815-821

被引頻次：345

Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy

Crowther, RA; Kiselev, NA; Bottcher, B; et al.

Abstract:Human hepatitis B virus core protein expressed in E. coli assembles into two sizes of particle. We have determined their three-dimensional structures by electron cryomicroscopy and image processing. The large and small particles correspond to triangulation number T = 4 and T =3 dimer clustered packings, containing 240 and 180 protein subunits, respectively. The local packing of subunits is very similar in the two sizes of particle and shows holes or channels through the shell. The native viral core particle packages RNA and is active in reverse transciption to DNA. The holes we observe may provide access for the necessary small molecules. Shells assembled from the intact core protein contain additional material, probably RNA, which appears as an icosahedrally ordered inner shell in the three-dimensional map.

來源出版物：Cell, 1994, 77(6)： 943-950

被引頻次：327

Visualization of a 4-helix bundle in the hepatitis B virus capsid by cryo-electron microscopy

Conway, JF; Cheng, N; Zlotnick, A; et al.

Abstract:Despite the development of vaccines, the hepatitis B virus remains a major cause of human liver disease. The virion consists of a lipoprotein envelope surrounding an icosahedral capsid composed of dimers of a 183-residue protein, ‘core antigen’ (HBcAg)2. Knowledge of its structure is important for the design of antiviral drugs, but it has yet to be determined. Residues 150-183 are known to form a protamine-like domain required for packaging RNA, and residues 1-149 form the ‘a(chǎn)ssembly domain’ that polymerizes into capsids and, unusually for a capsid protein, is highly α-helical. Density maps calculated from cryo-electron micrographs show that the assembly domain dimer is T-shaped： its stem constitutes the dimer interface and the tips of its arms make the polymerization contacts. By refining the procedures used to calculate the map, we have extended the resolution to 9 ?,revealing major elements of secondary structure. In particular, the stem, which protrudes as a spike on the capsid’s outer surface, is a 4-helix bundle, formed by the pairing of α-helical hairpins from both subunits.

來源出版物：Nature, 1997, 386(6620)： 91-94

·推薦論文摘要·

冷凍電鏡單顆粒技術(shù)解析生物大分子結(jié)構(gòu)綜述

張世超，歐陽燕，劉善輝，等

摘要：冷凍電鏡單顆粒技術(shù)是從20世紀(jì)80年代發(fā)展起來的結(jié)構(gòu)生物學(xué)新技術(shù)，特別是最近幾年，由于高分辨成像設(shè)備的應(yīng)用以及圖像處理方法的改進(jìn)，該領(lǐng)域取得了革命性的技術(shù)突破，分辨率達(dá)到原子水平，迅速發(fā)展成為結(jié)構(gòu)生物學(xué)新的主流研究技術(shù)，尤其適合于研究生物大分子復(fù)合物的結(jié)構(gòu)。簡(jiǎn)述了冷凍電鏡單顆粒技術(shù)解析生物大分子結(jié)構(gòu)的技術(shù)流程、最新技術(shù)進(jìn)步以及未來發(fā)展方向。

關(guān)鍵詞：冷凍電鏡；單顆粒；三維重構(gòu)；蛋白質(zhì)結(jié)構(gòu)

來源出版物：生物學(xué)雜志, 2017, 34(3)： 74-77

聯(lián)系郵箱：朱莉，zhuli@lzu.edu.cn

cryoSPARC軟件：生物大分子冷凍電子顯微鏡三維重構(gòu)技術(shù)的最新進(jìn)展

蘇曉東，高寧

摘要：2017年2月6日，Nature Methods雜志在線發(fā)表了加拿大多倫多大學(xué)和約克大學(xué)的電子工程與計(jì)算機(jī)科學(xué)系、生物化學(xué)系、醫(yī)學(xué)生物物理系等多學(xué)科人員聯(lián)合組成的研究小組開發(fā)的應(yīng)用于冷凍電子顯微鏡（cryo-electron microscopy，cryo-EM）生物大分子結(jié)構(gòu)解析的軟件，cryoSPARC（cryo-EM single-particle ab initio reconstruction and classification），可以進(jìn)行由低到高分辨率的快速、自動(dòng)化的生物大分子結(jié)構(gòu)解析。

來源出版物：中國科學(xué)：生命科學(xué), 2017, 47(3)： 345-346

聯(lián)系郵箱：蘇曉東，xdsu@pku.edu.cn

冷凍電鏡技術(shù)的發(fā)展推動(dòng)染色質(zhì)高級(jí)結(jié)構(gòu)的研究

朱平

摘要：染色質(zhì)的結(jié)構(gòu)及動(dòng)態(tài)變化在基因轉(zhuǎn)錄及表觀遺傳調(diào)控中起了關(guān)鍵作用，但對(duì)于30 nm染色質(zhì)纖維（通常認(rèn)為是基因組DNA的二級(jí)結(jié)構(gòu)）的高級(jí)結(jié)構(gòu)組成以及細(xì)胞體內(nèi)是否存在30 nm染色質(zhì)的組織形式一直存在較大爭(zhēng)議。近年來，冷凍電鏡三維重構(gòu)技術(shù)發(fā)展迅速，為研究30 nm染色質(zhì)纖維高級(jí)結(jié)構(gòu)提供了一個(gè)良好的工具，并起了較大的推動(dòng)作用。該文介紹了本領(lǐng)域相關(guān)的一些研究進(jìn)展。

關(guān)鍵詞：染色質(zhì)高級(jí)結(jié)構(gòu)；冷凍電鏡；表觀遺傳調(diào)控

來源出版物：中國細(xì)胞生物學(xué)學(xué)報(bào), 2015, 37(11)：1465-1471

聯(lián)系郵箱：朱平，zhup@ibp.ac.cn

結(jié)構(gòu)生物學(xué)研究方法的重大突破——電子直接探測(cè)相機(jī)在冷凍電鏡中的應(yīng)用

柳正，張景強(qiáng)

摘要：近年來，科學(xué)家應(yīng)用冷凍電鏡技術(shù)（cryo-EM）解析出了低對(duì)稱性生物大分子的高分辨率（3～5 ?）三維結(jié)構(gòu)，并用其密度圖直接進(jìn)行了分子建模。與傳統(tǒng)的X-射線和NMR方法相比，冷凍電鏡技術(shù)具有適用于分子量較大的生物分子、樣品不需結(jié)晶且用量很少等優(yōu)勢(shì)。尤其是電子直接探測(cè)相機(jī)（electron direct detection device，DDD）在冷凍電鏡技術(shù)中的應(yīng)用，使高分辨率的結(jié)構(gòu)研究變得更加簡(jiǎn)單、應(yīng)用更為廣泛，是一個(gè)重大突破。文章介紹DDD相機(jī)的原理和技術(shù)優(yōu)勢(shì)，及其在解決冷凍電鏡技術(shù)困難中的一些應(yīng)用，進(jìn)而展望了DDD相機(jī)可能給冷凍電鏡技術(shù)應(yīng)用帶來的突破性進(jìn)展。

關(guān)鍵詞：冷凍電鏡；電子直接探測(cè)相機(jī)；低對(duì)稱生物大分子；高分辨率

來源出版物：生物物理學(xué)報(bào), 2014, 30(6)： 405-415

聯(lián)系郵箱：張景強(qiáng)，lsszhjq@mail.sysu.edu.cn

三維冷凍電鏡成像技術(shù)成功運(yùn)用于解析30nm染色質(zhì)纖維的高分辨率結(jié)構(gòu)

劉駿

摘要：經(jīng)過多年來的不懈努力，中國科學(xué)院生物物理研究所的科學(xué)家，在破譯“生命信息”的分子機(jī)理研究中，取得了重大成果。在這項(xiàng)突破性成果里，朱平研究員和李國紅研究員領(lǐng)導(dǎo)的科研團(tuán)隊(duì)，利用先進(jìn)的三維冷凍電子顯微鏡成像技術(shù)，首次解析了30 nm染色質(zhì)纖維的高分辨率結(jié)構(gòu)，并提出了一種全新的染色質(zhì)纖維的雙螺旋結(jié)構(gòu)模型。

來源出版物：中國科學(xué)：生命科學(xué), 2014(6)： 636-636

聯(lián)系郵箱：劉駿，Jun.Liu.1@uth.tmc.edu

26S蛋白酶體的結(jié)構(gòu)生物學(xué)研究進(jìn)展

王豐，施一公

摘要：26S蛋白酶體是真核細(xì)胞內(nèi)負(fù)責(zé)蛋白質(zhì)降解的主要分子機(jī)器，通過特異性降解目的蛋白質(zhì)，幾乎參與了生物體的絕大多數(shù)生命活動(dòng)。26S蛋白酶體在結(jié)構(gòu)上可分為19S調(diào)節(jié)顆粒和20S核心顆粒兩部分。19S調(diào)節(jié)顆粒負(fù)責(zé)識(shí)別帶有泛素鏈標(biāo)記的蛋白質(zhì)底物及對(duì)其進(jìn)行去折疊，并最終將去折疊的蛋白質(zhì)底物傳送至20S核心顆粒中進(jìn)行降解。由于26S蛋白酶體的結(jié)構(gòu)組成復(fù)雜，分子量十分巨大，現(xiàn)有的X-ray技術(shù)和NMR技術(shù)對(duì)其完整結(jié)構(gòu)的解析都無能為力，僅能解析出部分單個(gè)蛋白成員或分子量較低的亞復(fù)合物晶體結(jié)構(gòu)。而冷凍電鏡技術(shù)在相當(dāng)一段時(shí)間內(nèi)處于發(fā)展的初級(jí)階段，導(dǎo)致其三維結(jié)構(gòu)的研究進(jìn)展曾經(jīng)十分緩慢，嚴(yán)重阻礙了人們對(duì)其結(jié)構(gòu)和功能的了解。近年來，隨著在X-ray技術(shù)領(lǐng)域?qū)Υ蠓肿訌?fù)合物結(jié)構(gòu)解析的經(jīng)驗(yàn)積累和冷凍電鏡技術(shù)領(lǐng)域的技術(shù)革命，完整的26S蛋白酶體三維結(jié)構(gòu)解析取得了飛速的發(fā)展。本文回顧了近幾年在26S蛋白酶體結(jié)構(gòu)生物學(xué)領(lǐng)域的重要進(jìn)展，并展望了該領(lǐng)域未來的發(fā)展及面臨的挑戰(zhàn)。

關(guān)鍵詞：26S蛋白酶體；結(jié)構(gòu)生物學(xué)；冷凍電鏡；X-ray晶體學(xué)

來源出版物：中國科學(xué)：生命科學(xué), 2014, 44(10)： 965-974

聯(lián)系郵箱：施一公，shi-lab@tsinghua.edu.cn

電子顯微學(xué)在結(jié)構(gòu)生物學(xué)研究中的新進(jìn)展

王大能，陳勇，隋森芳

摘要：電子顯微學(xué)是結(jié)構(gòu)生物學(xué)的重要分支，已經(jīng)成為一種公認(rèn)的研究生物大分子、超分子復(fù)合體及亞細(xì)胞結(jié)構(gòu)的有力手段。本文先回顧了近期電子顯微學(xué)在技術(shù)上的新進(jìn)展，后概述了生物電子顯微學(xué)的3個(gè)組成部分——電子晶體學(xué)，單顆粒技術(shù)，電子斷層成像術(shù)的基本原理，技術(shù)方法與研究現(xiàn)狀。最后，對(duì)電子顯微學(xué)與其他結(jié)構(gòu)生物學(xué)研究手段的結(jié)合以及電子顯微學(xué)的未來發(fā)展做了簡(jiǎn)單的展望。

關(guān)鍵詞：結(jié)構(gòu)生物學(xué)；電子顯微學(xué)；電子晶體學(xué)；單顆粒技術(shù)；電子斷層成像術(shù)

來源出版物：電子顯微學(xué)報(bào), 2003, 22(5)： 449-456

Phase-plate cryo-EM structure of a class B GPCR-G-protein complex

Liang, YL; Khoshouei, M ; Radjainia, M; et al.

Abstract:Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gαsβγ protein determined by Volta phase-plate singleparticle cryo-electron microscopy. The peptide agonist engages the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7.This conformation is accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G-protein coupling via an interaction with the Gβ subunit. This structure provides a new framework for understanding G-protein-coupled receptor function.

來源出版物：Nature, 2017, 546(7656)： 118-123

聯(lián)系郵箱：Wootten, D; denise.wootten@monash.edu

Using the Volta phase plate with defocus for cryo-EM single particle analysis

Danev, R; Tegunov, D; Baumeister, W

Abstract:Previously, we reported an in-focus data acquisition method for cryo-EM single-particle analysis with the Volta phase plate (Danev and Baumeister, 2016).Here, we extend the technique to include a small amount of defocus which enables contrast transfer function measurement and correction. This hybrid approach simplifies the experiment and increases the data acquisition speed. It also removes the resolution limit inherent to the in-focus method thus allowing 3D reconstructions with resolutions better than 3 angstrom.

來源出版物：Elife, 2017, 6： e23006

聯(lián)系郵箱：Danev, R; danev@biochem.mpg.de

Cryo-EM structure of a native, fully glycosylated,cleaved HIV-1 envelope trimer

Lee, JH; Ozorowski, G; Ward, AB

Abstract:The envelope glycoprotein trimer (Env) on the surface of HIV-1 recognizes CD4+T cells and mediates viral entry. During this process, Env undergoes substantial conformational rearrangements, making it difficult to study in its native state. Soluble stabilized trimers have provided valuable insights into the Env structure, but they lack the hydrophobic membrane proximal external region (MPER,an important target of broadly neutralizing antibodies), the transmembrane domain, and the cytoplasmic tail. Here we present (i) a cryogenic electron microscopy (cryo-EM)structure of a clade B virus Env, which lacks only the cytoplasmic tail and is stabilized by the broadly neutralizing antibody PGT151, at a resolution of 4.2 angstroms and (ii) a reconstruction of this form of Env in complex with PGT151 and MPER-targeting antibody 10E8 at a resolution of 8.8 angstroms. These structures provide new insights into the wild-type Env structure.

來源出版物：Science, 2016, 351(6277)： 1043-10486

聯(lián)系郵箱：Ward, AB; abward@scripps.edu

Breaking cryo-EM resolution barriers to facilitate drug discovery

Merk, A; Bartesaghi, A; Banerjee, S; et al.

Abstract:Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for wellordered protein complexes with sizes ≥～200 kDa.Whether cryo-EM methods are equally useful for highresolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 ? resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allostericsmall-molecule inhibitor ML309. We also report 2.8-?- and 1.8-?-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase(334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome： (1)crossing 2 ? resolution and (2) obtaining structures of proteins with sizes ＜ 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.

來源出版物：Cell, 2016, 165(7)： 1698-1707

聯(lián)系郵箱：Subramaniam, S; ss1@nih.gov

Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 ? resolution

Nguyen, THD; Galej, WP; Bai, XC; et al.

Abstract:WU4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure ofSaccharomyces cerevisiaeU4/U6.U5 tri-snRNP at 3.7 ? resolution led to an essentially complete atomic model comprising 30 proteins plus U4/U6 and U5 small nuclear RNAs (snRNAs). The structure reveals striking interweaving interactions of the protein and RNA components, including extended polypeptides penetrating into subunit interfaces. The invariant ACAGAGA sequence of U6 snRNA, which base-pairs with the 5′-splice site during catalytic activation,forms a hairpin stabilized by Dib1 and Prp8 while the adjacent nucleotides interact with the exon binding loop 1 of U5 snRNA. Snu114 harbours GTP, but its putative catalytic histidine is held away from the γ-phosphate by hydrogen bonding to a tyrosine in the amino-terminal domain of Prp8. Mutation of this histidine to alanine has no detectable effect on yeast growth. The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre.

來源出版物：Nature, 2016, 530(7590)： 298-302

聯(lián)系郵箱：Nguyen, THD; knguyen@mrc-lmb.cam.ac.uk

2.2 angstrom resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor

Bartesaghi, A; Merk, A; Banerjee, S; et al.

Abstract:Cryo-electron microscopy (cryo-EM) is rapidly emerging as a powerful tool for protein structure determination at high resolution. Here we report the structure of a complex between Escherichia coli betagalactosidase and the cell-permeant inhibitor phenylethyl beta-D-thiogalactopyranoside (PETG), determined by cryo-EM at an average resolution of similar to 2.2 angstroms (angstrom). Besides the PETG ligand, we identified densities in the map for similar to 800 water molecules and for magnesium and sodium ions. Although it is likely that continued advances in detector technology may further enhance resolution, our findings demonstrate that preparation of specimens of adequate quality and intrinsic protein flexibility, rather than imaging or image-processing technologies, now represent the major bottlenecks to routinely achieving resolutions close to 2 angstrom using single-particle cryo-EM.

來源出版物：Science, 2015, 348(6239)： 1147-1151

聯(lián)系郵箱：Subramaniam, S; ss1@nih.gov

Measuring the optimal exposure for single particle cryo-EMusing a 2.6 angstrom reconstruction of rotavirus VP6

Grant, T; Grigorieff, N

Abstract:Biological specimens suffer radiation damage when imaged in an electron microscope, ultimately limiting the attainable resolution. At a given resolution, an optimal exposure can be defined that maximizes the signal-to-noise ratio in the image. Using a 2.6 ? resolution single particle cryo-EM reconstruction of rotavirus VP6, determined from movies recorded with a total exposure of 100 electrons/?2,we obtained accurate measurements of optimal exposure values over a wide range of resolutions. At low and intermediate resolutions, our measured values are considerably higher than obtained previously for crystalline specimens, indicating that both images and movies should be collected with higher exposures than are generally used. We demonstrate a method of using our optimal exposure values to filter movie frames, yielding images with improved contrast that lead to higher resolution reconstructions. This ‘high-exposure’ technique should benefit cryo-EM work on all types of samples,especially those of relatively low-molecular mass.

來源出版物：Elife, 2015, 4： e06980

聯(lián)系郵箱：Grigorieff, N; niko@grigorieff.org

Structure of theE. coliribosome-EF-Tu complex at ＜3 ? resolution by Cs-corrected cryo-EM

Fischer, N; Neumann, P; Konevega, A; et al.

Abstract:Single particle electron cryomicroscopy(cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis.However, cryo-EM structures can be highly non-uniform in local resolution and all structures available to date have been limited to resolutions above 3 ?. Here we present the cryo-EM structure of the 70S ribosome fromEscherichiacoliin complex with elongation factor Tu, aminoacyltRNA and the antibiotic kirromycin at 2.65-2.9 ? resolution using spherical aberration (Cs)-corrected cryo-EM. Overall,the cryo-EM reconstruction at 2.9 ? resolution is comparable to the best-resolved X-ray structure of theE. coli70S ribosome (2.8 ?), but provides more detailed information (2.65 ?) at the functionally important ribosomal core. The cryo-EM map elucidates for the first time the structure of all 35 rRNA modifications in the bacterial ribosome, explaining their roles in fine-tuning ribosome structure and function and modulating the action of antibiotics. We also obtained atomic models for flexible parts of the ribosome such as ribosomal proteins L9 and L31. The refined cryo-EM-based model presents the currently most complete high-resolution structure of theE. coliribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes.

來源出版物：Nature, 2015, 520(7548)： 567-570

聯(lián)系郵箱：Fischer, N; niels.fischer@mpibpc.mpg.de

Semi-automated selection of cryo-EM particles in RELION-1.3

Scheres, SHW

Abstract:The selection of particles suitable for highresolution cryo-EM structure determination from noisy micrographs may represent a tedious and time-consuming step. Here, a semi-automated particle selection procedure is presented that has been implemented within the opensource software RELION. At the heart of the procedure lies a fully CTF-corrected template-based picking algorithm,which is supplemented by a fast sorting algorithm and reference-free 2D class averaging to remove false positives. With only limited user-interaction, the proposed procedure yields results that are comparable to manual particle selection. Together with an improved graphical user interface, these developments further contribute to turning RELION from a stand-alone refinement program into a convenient image processing pipeline for the entire single-particle approach.

關(guān)鍵詞：electron cryo-microscopy; single-particle analysis;automated particle picking

來源出版物：Journal of Structural Biology, 2015, 189(2)：114-122

聯(lián)系郵箱：Scheres, SHW; scheres@mrc-lmb.cam.ac.uk

Structure of the TRPV1 ion channel determined by electron cryo-microscopy

Maofu, L; Erhu, C; David, J; et al.

Abstract:Transient receptor potential (TRP) channels are sensors for a wide range of cellular and environmental signals, but elucidating how these channels respond to physical and chemical stimuli has been hampered by a lack of detailed structural information. Here we exploit advances in electron cryo-microscopy to determine the structure of a mammalian TRP channel, TRPV1, at 3.4 ? resolution, breaking the side-chain resolution barrier for membrane proteins without crystallization. Like voltagegated channels, TRPV1 exhibits four-fold symmetry around a central ion pathway formed by transmembrane segments 5-6 (S5-S6) and the intervening pore loop,which is flanked by S1-S4 voltage-sensor-like domains.TRPV1 has a wide extracellular ‘mouth’ with a short selectivity filter. The conserved ‘TRP domain’ interacts with the S4-S5 linker, consistent with its contribution to allosteric modulation. Subunit organization is facilitated by interactions among cytoplasmic domains, including aminoterminal ankyrin repeats. These observations provide a structural blueprint for understanding unique aspects of TRP channel function.

關(guān)鍵詞：electron cryo-microscopy; single-particle analysis;automated particle picking

來源出版物：Nature, 2013, 504(7478)： 107-12.

聯(lián)系郵箱：David, J; david.julius@ucsf.edu

2.9 ? resolution Cryo-EM 3D reconstruction of close-packed virus particles

Liu, Z; Guo, F; Wang, F; et al.

Abstract:Single-particle cryoelectron microscopy typically discards close-packed particle images as unusable data. Here, we report an image processing strategy and case study of obtaining near-atomic resolution 3D reconstructions from close-packed particles. Multiple independent de novo initial models were constructed to determine and cross-validate the particle parameters. The particles with consistent views were further refined including not only Euler angles and center positions but also defocus, astigmatism, beam tilt, and overall and anisotropic magnification. We demonstrated this strategy with a 2.9 ? resolution reconstruction of a 1.67 MDa virus-like particle of a circovirus, PCV2, recorded on 86 photographic films. The map resolution was further validated with a phase-randomization test and local resolution assessment, and the atomic model was validated with MolProbity and EMRinger. Close-packed virus particles were thus shown not only to be useful for high-resolution 3D reconstructions but also to allow data collection at significantly improved throughput for nearatomic resolution reconstructions.

來源出版物：Structure, 2016, 24(2)： 319-328

聯(lián)系郵箱：Wen, J; jiang12@purdue.edu

Cryo-EM shows the polymerase structures and a nonspooled genome within a dsRNA virus

Liu, H; Cheng, L

Abstract:Double-stranded RNA (dsRNA) viruses possess a segmented dsRNA genome and a number of RNA-dependent RNA polymerases (RdRps) enclosed in a capsid.Until now, the precise structures of genomes and RdRps within the capsids have been unknown. Here we report the structures of RdRps and associated RNAs within nontranscribing and transcribing cypoviruses (NCPV and TCPV, respectively), using a combination of cryo-electron microscopy (cryo-EM) and a symmetry-mismatch reconstruction method. The RdRps and associated RNAs appear to exhibit a pseudo-D3 symmetric organization in both NCPV and TCPV. However, the molecular interactions between RdRps and the genomic RNA were found to differ in these states. Our work provides insight into the mechanisms of the replication and transcription in dsRNA viruses and paves a way for structural determination of lower-symmetry complexes enclosed in higher-symmetry structures.

來源出版物：Science, 2015, 349(6254)： 1347-50.

聯(lián)系郵箱：Liu, H; hrliu@hunnu.edu.cn

3.88 ? structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy

Yu, X; Jin, L; Zhou, ZH

Abstract:Cytoplasmic polyhedrosis virus (CPV) is unique within the Reoviridae family in having a turreted singlelayer capsid contained within polyhedrin inclusion bodies,yet being fully capable of cell entry and endogenous RNA transcription. Biochemical data have shown that the amino-terminal 79 residues of the CPV turret protein (TP)is sufficient to bring CPV or engineered proteins into the polyhedrin matrix for micro-encapsulation. Here we report the three-dimensional structure of CPV at 3.88 A resolution using single-particle cryo-electron microscopy. Our map clearly shows the turns and deep grooves of alpha-helices,the strand separation in beta-sheets, and densities for loops and many bulky side chains; thus permitting atomic model-building effort from cryo-electron microscopy maps. We observed a helix-to-beta-hairpin conformational change between the two conformational states of the capsid shell protein in the region directly interacting with genomic RNA. We have also discovered a messenger RNA release hole coupled with the mRNA capping machinery unique to CPV. Furthermore, we have identified the polyhedrinbinding domain, a structure that has potential in nanobiotechnology applications.

來源出版物：Nature, 2008, 453(7193)： 415

聯(lián)系郵箱：Zhou, ZH; Hong.Zhou@ucla.edu

Cryo-EM: Beyond the microscope

Earl, LA, Falconieri, V, Milne, JL; et al.

Abstract:The pace at which cryo-EM is being adopted as a mainstream tool in structural biology has continued unabated over the past year. Initial successes in obtaining near-atomic resolution structures with cryo-EM were enabled to a large extent by advances in microscope and detector technology. Here, we review some of the complementary technical improvements that are helping sustain the cryo-EM revolution. We highlight advances in image processing that permit high resolution structure determination even in the presence of structural and conformational heterogeneity. We also review selected examples where biochemical strategies for membrane protein stabilization facilitate cryo-EM structure determination,and discuss emerging approaches for further improving the preparation of reliable plunge-frozen specimens.

來源出版物：Current Opinion in Structural Biology, 2017,46： 71-78

聯(lián)系郵箱：Subramaniam, S; ss1@nih.gov

Unravelling biological macromolecules with cryo-electron microscopy

Fernandez-Leiro, R; Scheres, SH

Abstract:Knowledge of the three-dimensional structures of proteins and other biological macromolecules often aids understanding of how they perform complicated tasks in the cell. Because many such tasks involve the cleavage or formation of chemical bonds, structural characterization at the atomic level is most useful. Developments in the electron microscopy of frozen hydrated samples (cryoelectron microscopy) are providing unprecedented opportunities for the structural characterization of biological macromolecules. This is resulting in a wave of information about processes in the cell that were impossible to characterize with existing techniques in structural biology.

來源出版物：Nature, 2016, 537(7620)： 339

Achieving better-than-3-? resolution by single-particle cryo-EM at 200 keV

Herzik, MA Jr; Wu, M; Lander, GC

Abstract:Nearly all single-particle cryo-EM structures resolved to better than 4-? resolution have been determined using 300-keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain reconstructions of macromolecular complexes of different sizes to better than 3-? resolution using a 200 keV TEM.These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules.

來源出版物：Nature Methods, 2017, 10.1038/nmeth.4461

Cryo-EM structures of human γ-secretase

Yang, G; Zhou, R; Shi, Y

Abstract:γ-secretase, a membrane-embedded aspartate protease, catalyzes peptide bond hydrolysis of a large variety of type I integral membrane proteins exemplified by amyloid precursor protein (APP). Cleavage of APP leads to formation of β-amyloid plaque, which is a hallmark of Alzheimer’s disease (AD). Over 200 AD-associated mutations are mapped to presenilin 1 (PS1), the catalytic component of γ-secretase. In the past three years,several cryo-electron microscopy (cryo-EM) structures of human γ-secretase have been determined at near atomic resolutions. Here we summarize the methods involved and discuss structural features of γ-secretase and the associated functional insights.

來源出版物：Current Opinion in Structural Biology,2017, 46： 55

聯(lián)系郵箱：Yang, G; guanghui_yang@163.com

Structure of phycobilisome from the red algaGriffithsia pacifica

Jun, Z; Jianfei, M; Desheng, L; et al.

Abstract:Life on Earth depends on photosynthesis for its conversion of solar energy to chemical energy.Photosynthetic organisms have developed a variety of light-harvesting systems to capture sunlight. The largest light-harvesting complex is the phycobilisome (PBS), the main light-harvesting antenna in cyanobacteria and red algae. It is composed of phycobiliproteins and linker proteins but the assembly mechanisms and energy transfer pathways of the PBS are not well understood. Here we report the structure of a 16.8-megadalton PBS from a red alga at 3.5 ? resolution obtained by single-particle cryoelectron microscopy. We modelled 862 protein subunits,including 4 linkers in the core, 16 rod-core linkers and 52 rod linkers, and located a total of 2048 chromophores.This structure reveals the mechanisms underlying specific interactions between linkers and phycobiliproteins, and the formation of linker skeletons. These results provide a firm structural basis for our understanding of complex assembly and the mechanisms of energy transfer within the PBS.

來源出版物：Nature, 2017, 10.1038/nature24278

Near-atomic resolution structure determination in over-focus with volta phase plate by Cs-corrected cryo-EM

Xiao, F; Lingyun, Z; Chuan, L; et al.

Abstract:Volta phase plate (VPP) is a recently developed transmission electron microscope (TEM) apparatus that can significantly enhance the image contrast of biological samples in cryoelectron microscopy, and therefore provide the possibility to solve structures of relatively small macromolecules at high-resolution. In this work, we performed theoretical analysis and found that using phase plate on objective lens spherical aberration (Cs)-corrected TEM may gain some interesting optical properties,including the over-focus imaging of macromolecules. We subsequently evaluated the imaging strategy of frozenhydrated apo-ferritin with VPP on a Cs-corrected TEM and obtained the structure of apo-ferritin at near-atomic resolution from both under- and over-focused dataset,illustrating the feasibility and new potential of combining VPP with Cs-corrected TEM for high-resolution cryo-EM.

關(guān)鍵詞：cryo-EM; volta phase plate; Cs-corrector;over-focus; apo-ferritin; CTF; graphene grid

來源出版物：Structure, 2017, 25(10)： 1623-1630

Structure of an intron lariat spliceosome fromSaccharomyces cerevisiae

Ruixue, W; Chuangye, Y; Rui, B; et al.

Abstract:The disassembly of the intron lariat spliceosome(ILS) marks the end of a splicing cycle. Here we report a cryoelectron microscopy structure of the ILS complex fromSaccharomyces cerevisiaeat an average resolution of 3.5 ?. The intron lariat remains bound in the spliceosome whereas the ligated exon is already dissociated. The step II splicing factors Prp17 and Prp18, along with Cwc21 and Cwc22 that stabilize the 5′ exon binding to loop I of U5 small nuclear RNA (snRNA), have been released from the active site assembly. The DEAH family ATPase/helicase Prp43 binds Syf1 at the periphery of the spliceosome, with its RNA-binding site close to the 3′ end of U6 snRNA. The C-terminal domain of Ntr1/Spp382 associates with the GTPase Snu114, and Ntr2 is anchored to Prp8 while interacting with the superhelical domain of Ntr1. These structural features suggest a plausible mechanism for the disassembly of the ILS complex.

關(guān)鍵詞：pre-mRNA splicing; intron lariat spliceosome; ILS complex; cryo-EM; spliceosome disassembly; Prp43;DEAH-box ATPase/helicase; Ntr complex; Ntr1; Ntr2

來源出版物：Cell, 2017, 171(1)： 120-132

Structure of the Nav1.4-β1 complex from electric eel

Zhen, Y; Qiang, Z; Lin, W; et al.

Abstract:Voltage-gated sodium (Nav) channels initiate and propagate action potentials. Here, we present the cryo-EM structure of EeNav1.4, the Navchannel from electric eel, in complex with the β1 subunit at 4.0 ? resolution. The immunoglobulin domain of β1 docks onto the extracellular L5Iand L6IVloops of EeNav1.4 via extensive polar interactions, and the single transmembrane helix interacts with the third voltage-sensing domain (VSDIII). The VSDs exhibit “up” conformations, while the intracellular gate of the pore domain is kept open by a digitonin-like molecule.Structural comparison with closed NavPaS shows that the outward transfer of gating charges is coupled to the iris-like pore domain dilation through intricate force transmissions involving multiple channel segments. The IFM fast inactivation motif on the III-IV linker is plugged into the corner enclosed by the outer S4-S5 and inner S6 segments in repeats III and IV, suggesting a potential allosteric blocking mechanism for fast inactivation.

關(guān)鍵詞：voltage-gated sodium channels; Nav channels;Nav1.4; electromechanical coupling; fast inactivation;structural biology; cryo-EM; the beta-1 subunit

來源出版物：Cell, 2017, 170(3)： 470-482

Particle segmentation algorithm for flexible single particle reconstruction

Qiang, Z; Niyun, Z; Hongwei, W; et al.

Abstract:As single particle cryo-electron microscopy has evolved to a new era of atomic resolution, sample heterogeneity still imposes a major limit to the resolution of many macromolecular complexes, especially those with continuous conformational flexibility. Here, we describe a particle segmentation algorithm towards solving structures of molecules composed of several parts that are relatively flexible with each other. In this algorithm, the different parts of a target molecule are segmented from raw images according to their alignment information obtained from a preliminary 3D reconstruction and are subjected to single particle processing in an iterative manner. This algorithm was tested on both simulated and experimental data and showed improvement of 3D reconstruction resolution of each segmented part of the molecule than that of the entire molecule.

關(guān)鍵詞：single particle reconstruction; Cryo-EM; particle segmentation; local reconstruction

來源出版物：Biophysics Reports, 2017, 3(1)： 43-55

聯(lián)系郵箱：Qiang, Z; zhouqiang00@tsinghua.org.cn

Structural insights into Ca2+-activated long-range allosteric channel gating of RyR1

Risheng, W; Xue, W; Yan, Z; et al.

Abstract:Ryanodine receptors (RyRs) are a class of giant ion channels with molecular mass over 2.2 mega-Daltons.These channels mediate calcium signaling in a variety of cells. Since more than 80% of the RyR protein is folded into the cytoplasmic assembly and the remaining residues form the transmembrane domain, it has been hypothesized that the activation and regulation of RyR channels occur through an as yet uncharacterized long-range allosteric mechanism. Here we report the characterization of a Ca2+-activated open-state RyR1 structure by cryo-electron microscopy. The structure has an overall resolution of 4.9 ? and a resolution of 4.2 ? for the core region. In comparison with the previously determined apo/closedstate structure, we observed long-range allosteric gating of the channel upon Ca2+activation. In-depth structural analyses elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism of RyR1. Our work not only provides structural insights into the molecular mechanisms of channel gating and regulation of RyRs, but also sheds light on structural basis for channel-gating and ion selectivity mechanisms for the six-transmembranehelix cation channel family.

來源出版物：Cell Research, 2016, 26(9)： 977-994

RELION: Implementation of a Bayesian approach to cryo-EMstructure determination

Scheres, SHW

RELION, for REgularized LIkelihood OptimizatioN, is an open-source computer program for the refinement of macromolecular structures by single-particle analysis of electron cryo-microscopy (cryo-EM) data.Whereas alternative approaches often rely on user expertise for the tuning of parameters, RELION uses a Bayesian approach to infer parameters of a statistical model from the data. This paper describes developments that reduce the computational costs of the underlying maximum a posteriori(MAP) algorithm, as well as statistical considerations that yield new insights into the accuracy with which the relative orientations of individual particles may be determined. A so-called gold-standard Fourier shell correlation (FSC)procedure to prevent overfitting is also described. The resulting implementation yields high-quality reconstructions and reliable resolution estimates with minimal user intervention and at acceptable computational costs.

文章題目第一作者來源出版物1 RELION： Implementation of a Bayesian approach to Scheres, SHW Journal of Structural Biology, 2012,cryo-EMstructure determination 180(3)： 519-530 2 Electron counting and beam-induced motion correction Li, XM Nature Methods, 2013, 10(6)： 584-590 enable near-atomic-resolution single-particle cryo-EM 3 Cryo-EM study of the chromatin fiber reveals a double Song, F Science, 2014, 344(6182)： 376-380 helix twisted by tetranucleosomal units 4 Beam-induced motion correction for sub-megadalton Scheres, SHW Elife, 2014, 3： e03665 cryo-EMparticles 5 Cryo-EM structure of the Plasmodium falciparum 80S Wong, W Elife, 2014, 3： e03080 ribosome bound to the anti-protozoan drug emetine

哥倫比亞大學(xué)生物化學(xué)與分子生物物理學(xué)系；中山大學(xué)生命科學(xué)學(xué)院】

（摘自《生物物理學(xué)報(bào)》2014年6期）

·高被引論文摘要·

被引頻次：9

冷凍電子斷層成像技術(shù)及其在生物研究領(lǐng)域的應(yīng)用

黃曉星，宋曉偉，朱平

責(zé)任編輯：王微