三维电子显微学自动化数据采集的进展

生物三维电子显微学在过去几年取得了巨大的突破,一些具有高对称性的病毒颗粒获得了准原子分辨率的结构,非对称性的生物大分子及其复合体的结构分辨率也有快速的提高。而要获得高分辨率的结构,获取足够多的高质量电子显微照片是其中的一个关键因素。近年来,自动化数据采集技术在电子断层成像术和单颗粒方法中都取得了很大的进展。其广泛应用将使结构测定更加快速并使结构分辨率提高到更高的层次。     

单颗粒电子显微学的研究进展

单颗粒电子显微学是一种新型的结构生物学技术和方法,一方面,其解析生物大分子复合体结构的分辨率日益提高,可以达到近原子分辨率,提供大蛋白分子或复合体的精细结构;另一方面,还可以解析生物大分子在不同功能状态下的结构及变化,对于揭示生物大分子复合体结构的作用机理具有重要作用。本文就单颗粒电子显微学的研究进展作一综述。     

生物三维电子显微学进入全新高速发展时期

生物三维电子显微学主要由三个部分组成——电子晶体学、单颗粒技术和电子断层成像术,其结构解析对象的尺度范围介于x射线晶体学与光学显微镜之间,适合从蛋白质分子结构到细胞和组织结构的解析。以冷冻电镜技术与三维重构技术为基础的低温电子显微学代表了生物电子显微学的前沿。低温单颗粒技术对于高度对称的病毒颗粒的解析最近已达到3．8A分辨率,正在成为解析分子量很大的蛋白质复合体高分辨结构的有效技术手段。低温电子断层成像技术目前对于真核细胞样品的结构解析已达到约40A的分辨率,在今后5年有望达到20A。这样,把x射线晶体学、NMR以及电镜三维重构获得的蛋白质分子及复合体的高分辨率的结构,锚定到较低分辨率的电子断层成像图像中,从而在细胞水平上获得高精确的蛋白质空间定位和原子分辨率的蛋白质相互作用的结构信息。这将成为把分子水平的结构研究与细胞水平的生命活动衔接起来的可行途径。     

使用电子断层技术对IgG1抗体逐个分子的三维重构与结构分析

抗体(antibody)又称免疫球蛋白(immunoglobulin,Ig),是人体免疫反应的重要参与者.了解抗体的结构和结构动态特征,是理解人体免疫作用机理、修复或提高免疫能力、定向设计抗体以治疗各种疾病的基础.本文以人体IgG1抗体为对象,综述了使用透射电子显微学方法研究IgG1抗体结构方向的最新进展.详细介绍了使用逐个分子的电子断层三维重构技术(individual-particle electron tomography,IPET)对抗体进行结构研究的方法,包括样品制备、图像处理和数据分析等.并描述了利用该技术,在研究抗体结合肽分子后的结构形变和通过收集不同构象来研究抗体动态结构特征方面所取得的阶段性成果.最后,对尚待解决的关键问题与该技术未来的发展方向进行了讨论与展望.     

电子显微三维重构技术发展与前沿

本文对电子显微三维重构技术(也称电镜三维重构,electron microscopy 3D reconstruction)进行简要介绍,并在此基础上对该技术当前研究的发展和前沿进行综述,包括高分辨率电镜三维重构、仪器设备性能突破、自动化数据收集和处理、高性能计算技术应用、二/三维图像处理技术的发展和创新、基于三维重构图的模型计算等方面,最后对电子显微三维重构技术的未来进行了展望。     

家蚕传染性软化病病毒的冷冻电子显微镜三维重构

应用冷冻电子显微镜三维重构技术获得了家蚕传染性软化病病毒（Infectious flacherie virus,IFV）衣壳的三维立体结构.重构最终结果使用了5047个病毒粒子的数据.FSC曲线显示该重构结果的分辨率为18？.IFV的衣壳直径为302.4？,遵循拟T=3（P=3）二十面体对称.衣壳为单层,厚度15？,表面光滑致密,无明显突起或凹陷,无孔洞贯穿.将IFV衣壳结构与昆虫的类小RNA病毒-蟋蟀麻痹病毒（Cricket paralysis virus,CrPV）以及人小RNA病毒-人鼻病毒14（human rhinovirus14,HRV14）的衣壳进行比较,发现IFV衣壳结构与CrPV更为接近.与CrPV一样,IFV衣壳表面也缺少＂Rossmann峡谷＂.同时预测了IFV结构蛋白VP2和VP3的肽链折叠拓扑结构,并对亚基在衣壳表面的分布位置进行了推测.     

导管三维重构的基本方法

导管是被子植物运输水分和无机盐的管道,它由一串导管分子以穿孔端壁相互衔接而成。关于它在输水过程中的作用,常用的比较方法是通过离析来测定导管分子的长度和直径,或者是通过三切面来观察某一导管分子与邻近细胞的相互关系。由于上述方法所展示的仅仅是某一局部区域中木质部各种组成分子的平面关系或者是某一组成分子的变化模式,所     

冷冻电子显微学在结构生物学研究中的现状与展望

冷冻电子显微学近年来在电子显微镜的硬件设备及结构解析的软件算法等方面取得了多个重要的技术突破,正在成为结构生物学研究的重要技术手段,为越来越多的生物学研究者所重视.冷冻电子显微学的技术特点决定了它所具备的一些独特优势和发展方向,同时作为一个正在迅速发展的科学技术领域,需要多学科的交叉促进.本文主要介绍冷冻电子显微学的研究现状及面临的技术挑战,并提出未来可能实现结构生物学与细胞生物学不同尺度的研究在冷冻电子显微学技术上融合的新方法.     

几种冷冻稀释液与单胺类防冻剂对中缅树鼩精子冷冻存活率的影响

该文实验精子采自昆明地区经笼养驯化的树鼩(Tupaia belangeri),检测其冷冻前后运动度、顶体完整率以及检测部分冷冻精子的受精能力。实验一:选用8种已报道的冷冻稀释液TTE、TCG、TCF、TTG、BWW、BTS、DM、SR稀释鲜精,并添加0.4mol/LDMSO,4℃预冷平衡2h后,TTE、DM和SR稀释液的精子的运动度与鲜精无差别(P>0.05),其余处理组均显著下降(P<0.05)。冷冻复苏后,各组的运动度显著低于预冷平衡处理后的运动度(P<0.05);DM组的复苏运动度显著高于其他稀释液组(P<0.05),BWW组最低(P<0.05)。对于顶体完整率,与鲜精相比,4℃平衡2h后,TTE和DM组精子的顶体完整率显著高于BWW、BTS和SR组(P<0.05)。冷冻复苏后,DM组精子的顶体完整率显著高于其它(除了TTE)冷冻组(P<0.05)。实验二:在DM稀释液基础上分别添加4种浓度0.2、0.4、0.8和1.2mol/L的二甲基甲酰胺(DF)、甲酰胺(F)、二甲乙酰胺(DA)和乙酰胺(A)以及0.4mol/LDMSO,经过预冷平衡处理后,与鲜精相比,各防冻剂组的精子运动度没有下降(P>0.05);冷冻解冻后,各冷冻组精子的运动度显著低于预冷平衡处理后的精子运动度(P<0.05);0.8mol/LDF和0.4mol/LDMSO组精子的运动度显著高于其它冷冻组(P<0.05)。对于顶体完整率,预冷平衡处理后各高浓度组的比率显著下降;冷冻复苏后,0.4mol/LF和0.4mol/LDF组精子的顶体完整率相对较高。实验三,人工授精实验中,DM+0.8mol/LDF冷冻精子的受精率为16.7%,DM+0.4mol/LDMSO的受精率为50.0%。以上实验结果提示,含卵黄的非离子冷冻稀释液对树鼩精子冷冻保护效果好,但单胺类防冻剂的防冻效果还需进一步深入研究。     

单颗粒示踪技术及其在病毒侵染机制研究中的应用

病毒侵染宿主细胞是一个复杂的动态过程,且涉及病毒与细胞多种成分的相互作用,病毒侵染机制研究对研制抗病毒药物以及病毒性疾病的预防和治疗至关重要。单颗粒示踪技术是可视化研究生物动态过程的重要工具,能够研究病毒在活细胞中的侵染行为,现已成为病毒侵染机制研究的有效手段。此文综述了单颗粒示踪技术中病毒标记物、标记方法、活细胞成像及分析进展,以流行性感冒病毒和人免疫缺陷病毒为例阐述了该技术在病毒侵染机制研究中的应用,对该技术的应用前景进行了展望。此文旨在为病毒侵染机制研究介绍新的技术手段、推动病毒学研究。     

Protein interactions in the murine cytomegalovirus capsid revealed by cryoEM

Cytomegalovirus (CMV) is distinct among members of the Herpesviridae family for having the largest dsDNA genome (230 kb). Packaging of large dsDNA genome is known to give rise to a highly pressurized viral capsid, but molecular interactions conducive to the formation of CMV capsid resistant to pressurization have not been described. Here, we report a cryo electron microscopy (cryoEM) structure of the murine cytomegalovirus (MCMV) capsid at a 9.1 ? resolution and describe the molecular interactions among the ~3000 protein molecules in the CMV capsid at the secondary structure level. Secondary structural elements are resolved to provide landmarks for correlating with results from sequence-based prediction and for structure-based homology modeling. The major capsid protein (MCP) upper domain (MCPud) contains α-helices and β-sheets conserved with those in MCPud of herpes simplex virus type 1 (HSV-1), with the largest differences identified as a “saddle loop” region, located at the tip of MCPud and involved in interaction with the smallest capsid protein (SCP). Interactions among the bacteriophage HK97-like floor domain of MCP, the middle domain of MCP, the hook and clamp domains of the triplex proteins (hoop and clamp domains of TRI-1 and clamp domain of TRI-2) contribute to the formation of a mature capsid. These results offer a framework for understanding how cytomegalovirus uses various secondary structural elements of its capsid proteins to build a robust capsid for packaging its large dsDNA genome inside and for attaching unique functional tegument proteins outside.     

Rapid Three-Dimensional Reconstruction at the Light Microscopic Level and a Technique for Re-Embedding the Same Semithin Sections for Electron Microscopic Examination

Rapid three-dimensional reconstruction of serial sections at the light microscopic and ultrastructural levels was accomplished using a two-step technique. Fixed specimens were embedded in Epon and 1 μm sections were cut and placed on glass slides. One of every four sections was drawn onto transparency film for rapid three-dimensional reconstruction. The semi-thin sections were re-embedded in Epon and sectioned at 90 nm for examination in the electron microscopy.     

Structural basis for the slow dynamics of the actin filament pointed end

The actin filament has clear polarity where one end, the pointed end, has a much slower polymerization and depolymerization rate than the other end, the barbed end. This intrinsic difference of the ends significantly affects all actin dynamics in the cell, which has central roles in a wide spectrum of cellular functions. The detailed mechanism underlying this difference has remained elusive, because high-resolution structures of the filament ends have not been available. Here, we present the structure of the actin filament pointed end obtained using a single particle analysis of cryo-electron micrographs. We determined that the terminal pointed end subunit is tilted towards the penultimate subunit, allowing specific and extra loop-to-loop inter-strand contacts between the two end subunits, which is not possible in other parts of the filament. These specific contacts prevent the end subunit from dissociating. For elongation, the loop-to-loop contacts also inhibit the incorporation of another actin monomer at the pointed end. These observations are likely to account for the less dynamic pointed end.     

The integrative role of cryo electron microscopy in molecular and cellular structural biology

After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo‐EM) is now heading off at unprecedented speed towards high‐resolution analysis of biological objects of various sizes. This ‘revolution in resolution’ is happening largely thanks to new developments of new‐generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo‐EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell. These advances reinforce the integrative role of cryo‐EM in synergy with other methods such as X‐ray crystallography, fluorescence imaging or focussed‐ion beam milling as exemplified here by some recent studies from our laboratory on ribosomes, viruses, chromatin and nuclear receptors. Such multi‐scale and multi‐resolution approaches allow integrating molecular and cellular levels when applied to purified or in situ macromolecular complexes, thus illustrating the trend of the field towards cellular structural biology.     

Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1

The respiratory chain in the inner mitochondrial membrane contains three large multi‐enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19‐Å 3D map of the 1.7‐MDa amphipol‐solubilized supercomplex I₁III₂IV₁ from bovine heart obtained by single‐particle electron cryo‐microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X‐ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol‐binding sites of complexes I and III, and of 10–11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred.     

Cryoelectron microscopy reveals new features in the three-dimensional structure of phosphorylase kinase

Phosphorylase kinase (PhK), a regulatory enzyme in the cascade activation of glycogenolysis, is a 1.3-MDa hexadecameric complex, (alphabetagammadelta)(4). PhK comprises two arched octameric (alphabetagammadelta)(2) lobes that are oriented back-to-back with overall D(2) symmetry and connected by small bridges. These interlobal bridges, arguably the most questionable structural component of PhK, are one of several structural features that potentially are artifactually generated or altered by conventional sample preparation techniques for electron microscopy (EM). To minimize such artifacts, we have solved by cryoEM the first three-dimensional (3D) structure of nonactivated PhK from images of frozen hydrated molecules of the kinase. Minimal dose electron micrographs of PhK in vitreous ice revealed particles in a multitude of orientations. A simple model was used to orient the individual images for 3D reconstruction, followed by multiple rounds of refinement. Three-dimensional reconstruction of nonactivated PhK from approximately 5000 particles revealed a bridged, bilobal molecule with a resolution estimated by Fourier shell correlation analysis at 25 A. This new structure suggests that several prominent features observed in the structure of PhK derived from negatively stained particles arise as artifacts of specimen preparation. In comparison to the structure from negative staining, the cryoEM structure shows three important differences: (1) a dihedral angle between the two lobes of approximately 90 degrees instead of 68 degrees, (2) a compact rather than extended structure for the lobes, and (3) the presence of four, rather than two, connecting bridges, which provides the first direct evidence for these components as authentic elements of the kinase solution structure.     

A measure for the angle between projections based on the extent of correlation between corresponding central sections

A pre-condition for the ab initio assignment of Euler angles to a set of projections from an asymmetric object is that at least three of the available projections correspond to rotations about different axes. For symmetric objects this condition may be relaxed. There are some applications of single-particle electron microscopy, such as the reconstruction of filamentous macromolecular assemblies, where all available projections more-or-less correspond to rotations about a common rotation axis making it difficult to satisfy this condition. Here, a method has been developed to overcome this problem, based on the fact that the correlation between two central sections of the Fourier transform of a compact object will not be limited to an infinitesimal central line but will have a finite extent, which is related to the angle between the corresponding projections. Projections from model filaments, with different degrees of rotational symmetry about the long axis, have been used to test the methodology. The results show that angle determination is robust down to signal-to-noise ratios as low as 2 and that, in general, the error decreases as the degree of symmetry increases. The method has been used to assign angles to a set of negatively stained muscle thick filament projections to obtain an initial 3D reconstruction. The main features of the projections are seen to be faithfully reproduced in the reprojections from the reconstruction. A real-space adaptation of this method is also discussed.     

The location of ubiquitin in Lethocerus arthrin

Arthrin is a ubiquitinated actin that is present in flight muscles of some insects. In addition, it has been found in the malaria parasite Plasmodium falciparum. The role of this monoubiquitylation is not clear, and it does not appear to be associated with proteolytic degradation. The stoichiometry of arthrin to actin in Lethocerus indirect flight muscle, 1:6, suggests that there would be one arthrin molecule for each Tm-Tn (tropomyosin-troponin) complex. The appearance of arthrin after tropomyosin and troponin in Drosophila development is consistent with the Tm-Tn complex determining which actin subunit is targeted for conjugation with ubiquitin. We have used a new approach of three-dimensional reconstruction of helical filaments, the iterative helical real space reconstruction method, to extract segments of homogeneous arthrin out of long filaments where the conformation of the ubiquitin is more heterogeneous. Surprisingly, the location of the ubiquitin is on the face of actin subdomain 1, opposite to where tropomyosin binds in the “off” state, suggesting that there could not be a direct interaction between the ubiquitin and the tropomyosin. It is possible that the troponin complex in the “on” state that is bound to one actin strand makes an unfavorable contact with a ubiquitin molecule attached to the opposite actin strand. This might be the basis for a destabilization of the on state at rest length. Lys118 is the most likely residue to which the ubiquitin is conjugated, based upon fitting atomic structures of actin and ubiquitin into the reconstruction.     

MonoRes: Automatic and Accurate Estimation of Local Resolution for Electron Microscopy Maps

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The N-terminal domains of myosin binding protein C can bind polymorphically to F-actin

The regulation of vertebrate striated muscle contraction involves a number of different molecules, including the thin-filament accessory proteins tropomyosin and troponin that provide Ca²⁺-dependent regulation by controlling access to myosin binding sites on actin. Cardiac myosin binding protein C (cMyBP-C) appears to modulate this Ca²⁺-dependent regulation and has attracted increasing interest due to links with inherited cardiac diseases. A number of single amino acid mutations linked to clinical diseases occur in the N-terminal region of cMyBP-C, including domains C0 and C1, which previously have been shown to bind to F-actin. This N-terminal region also has been shown to both inhibit and activate actomyosin interactions in vitro. Using electron microscopy and three-dimensional reconstruction, we show that C0 and C1 can each bind to the same two distinctly different positions on F-actin. One position aligns well with the previously reported binding site that clashes with the binding of myosin to actin, but would force tropomyosin into an “on” position that exposes myosin binding sites along the filament. The second position identified here would not interfere with either myosin binding or tropomyosin positioning. It thus appears that the ability to bind to at least two distinctly different positions on F-actin, as observed for tropomyosin, may be more common than previously considered for other actin binding proteins. These observations help to explain many of the seemingly contradictory results obtained with cMyBP-C and show how cMyBP-C can provide an additional layer of regulation to actin-myosin interactions. They also suggest a redundancy of C0 and C1 that may explain the absence of C0 in skeletal muscle.