冷冻透射电镜:从分子到系统

单颗粒电镜结合其他方法能够在(近)原子水平提供结构模型,已经成为一种研究大蛋白复合物的有效方法。该文将以两个大的蛋白裂解复合物——tripeptidyl peptidaseII(6MDa)和26S蛋白酶体(2.5MDa)举例说明。低温电子层析能进行非重复的超分子结构分析,如多核糖体和全细胞;能够为超分子组织提供前所未有的信息(可视化蛋白质组学)。     

Cryo-electron microscopy reveals the membrane insertion mechanism of V. cholerae hemolysin

Vibrio cholerae hemolysin (HlyA) is a 65?kDa pore-forming toxin which causes lysis of target eukaryotic cells by forming heptameric channels in the plasma membrane. Deletion of the 15?kDa C-terminus β-prism carbohydrate-binding domain generates a 50?kDa truncated variant (HlyA50) with 1000-fold-reduced pore-forming activity. Previously, we showed by cryo-electron microscopy that the two toxin oligomers have central channels, but the 65?kDa toxin oligomer is a seven-fold symmetric structure with bowl-, ring-, and arm-like domains, whereas the 50?kDa oligomer is an asymmetric jar-like heptamer. In the present study, we determined three-dimensional(3D) structures of HlyA and HlyA50 in presence of erythrocyte stroma and observed that interaction of the 65?kDa toxin with the stroma induced a significant decrease in the height of the β-barrel oligomer with a change in conformation of the ring- and arm-like domains of HlyA. These features were absent in interaction of HlyA50 with stroma. We propose that this conformational transition is critical for membrane-insertion of the toxin.     

Cryo-electron microscopy reveals how acetogenins inhibit mitochondrial respiratory complex I

Mitochondrial complex I (NADH:ubiquinone oxidoreductase), a crucial enzyme in energy metabolism, captures the redox potential energy from NADH oxidation/ubiquinone reduction to create the proton motive force used to drive ATP synthesis in oxidative phosphorylation. High-resolution single-particle electron cryo-EM analyses have provided detailed structural knowledge of the catalytic machinery of complex I, but not of the molecular principles of its energy transduction mechanism. Although ubiquinone is considered to bind in a long channel at the interface of the membrane-embedded and hydrophilic domains, with channel residues likely involved in coupling substrate reduction to proton translocation, no structures with the channel fully occupied have yet been described. Here, we report the structure (determined by cryo-EM) of mouse complex I with a tight-binding natural product acetogenin inhibitor, which resembles the native substrate, bound along the full length of the expected ubiquinone-binding channel. Our structure reveals the mode of acetogenin binding and the molecular basis for structure–activity relationships within the acetogenin family. It also shows that acetogenins are such potent inhibitors because they are highly hydrophobic molecules that contain two specific hydrophilic moieties spaced to lock into two hydrophilic regions of the otherwise hydrophobic channel. The central hydrophilic section of the channel does not favor binding of the isoprenoid chain when the native substrate is fully bound but stabilizes the ubiquinone/ubiquinol headgroup as it transits to/from the active site. Therefore, the amphipathic nature of the channel supports both tight binding of the amphipathic inhibitor and rapid exchange of the ubiquinone/ubiquinol substrate and product.     

Cryo-electron Microscopy Reveals the Structure of the Nuclear Pore Complex

The nuclear pore complex (NPC) is a giant protein assembly that penetrates the double layers of the nuclear membrane. The overall structure of the NPC has approximately eightfold symmetry and is formed by approximately 30 nucleoporins. The great size and complexity of the NPC have hindered the study of its structure for many years until recent breakthroughs were achieved by integrating the latest high-resolution cryo-electron microscopy (cryo-EM), the emerging artificial intelligence-based modeling and all other available structural information from crystallography and mass spectrometry. Here, we review our latest knowledge of the NPC architecture and the history of its structural study from in vitro to in situ with progressively improved resolutions by cryo-EM, with a particular focus on the latest subnanometer-resolution structural studies. The future directions for structural studies of NPCs are also discussed.     

Cryo-electron tomography of clathrin-coated vesicles: structural implications for coat assembly

Clathrin-coated vesicles mediate vesicular traffic in cells. Three-dimensional image reconstructions of homogenous populations of in vitro assembled clathrin coats have yielded a molecular model for clathrin and its interactions with some of its partners. The intrinsic averaging required for those calculations has precluded detailed analysis of heterogeneous populations of clathrin-coated vesicles isolated from cells. We have therefore used cryo-electron tomography to study the lattice organization of individual clathrin-coated vesicles and the disposition of the captured vesicle with respect to the surrounding coat. We find a wide range of designs for the clathrin lattice, with different patterns of pentagonal, hexagonal, and occasionally heptagonal facets. Many coats, even smaller ones, enclose membrane vesicles, which are generally offset from the center of the clathrin shell. The electron density distribution between the coat and the underlying vesicle is not uniform, and the number of apparent contacts that anchor the clathrin lattice to the vesicle membrane is significantly less than the number of clathrin heavy chains in the assembly. We suggest that the eccentric position of the vesicle reflects the polarity of assembly, from initiation of coat formation to membrane pinching.     

Cryo-electron microscopy of hepatitis B virions reveals variability in envelope capsid interactions

Hepatitis B virus (HBV) is a major human pathogen causing about 750,000 deaths per year. The virion consists of a nucleocapsid and an envelope formed by lipids, and three integral membrane proteins. Although we have detailed structural insights into the organization of the HBV core, the arrangement of the envelope in virions and its interaction with the nucleocapsid is elusive. Here we show the ultrastructure of hepatitis B virions purified from patient serum. We identified two morphological phenotypes, which appear as compact and gapped particles with nucleocapsids in distinguishable conformations. The overall structures of these nucleocapsids resemble recombinant cores with two alpha-helical spikes per asymmetric unit. At the charged tips the spikes are contacted by defined protrusions of the envelope proteins, probably via electrostatic interactions. The HBV envelope in the two morphotypes is to some extent variable, but the surface proteins follow a general packing scheme with up to three surface protein dimers per asymmetric unit. The variability in the structure of the envelope indicates that the nucleocapsid does not firmly constrain the arrangement of the surface proteins, but provides a general template for the packing.     

Cryo-electron microscopy of artificial biological membranes

Vitrified synthetic phosphatidycholine liposome suspensions were studied by cryo-electron microscopy. The bilayer structure is resolved on vitrified liposome images. The packing of the aliphatic chains of the lipid within vitrified liposomes can be determined by the analysis of electron diffraction patterns. Images and electron diffraction patterns show that the structure of vitrified liposomes is related to the structure that liposomes have before vitrification. In fact, vitrified liposomes have a different structure, depending whether they are maintained before cooling at a temperature higher or lower than that corresponding to the ‘melting’ of the hydrocarbon chain of the lipids. Below the melting temperature, liposomes are formed by small domains.     

Cryo-electron microscopy structure of the intact photosynthetic light-harvesting antenna-reaction center complex from a green sulfur bacterium

The photosynthetic reaction center complex (RCC) of green sulfur bacteria (GSB) consists of the membrane-imbedded RC core and the peripheric energy transmitting proteins called Fenna–Matthews–Olson (FMO). Functionally, FMO transfers the absorbed energy from a huge peripheral light-harvesting antenna named chlorosome to the RC core where charge separation occurs. In vivo, one RC was found to bind two FMOs, however, the intact structure of RCC as well as the energy transfer mechanism within RCC remain to be clarified. Here we report a structure of intact RCC which contains a RC core and two FMO trimers from a thermophilic green sulfur bacterium Chlorobaculum tepidum at 2.9 Å resolution by cryo-electron microscopy. The second FMO trimer is attached at the cytoplasmic side asymmetrically relative to the first FMO trimer reported previously. We also observed two new subunits (PscE and PscF) and the N-terminal transmembrane domain of a cytochrome-containing subunit (PscC) in the structure. These two novel subunits possibly function to facilitate the binding of FMOs to RC core and to stabilize the whole complex. A new bacteriochlorophyll (numbered as 816) was identified at the interspace between PscF and PscA-1, causing an asymmetrical energy transfer from the two FMO trimers to RC core. Based on the structure, we propose an energy transfer network within this photosynthetic apparatus.     

Cryo-electron microscopy reconstructions of two types of wild rabbit hemorrhagic disease viruses characterized the structural features of Lagovirus

Rabbit hemorrhagic disease was described in China in 1984 and can cause hemorrhagic necrosis of the liver within two or three days after infection. The etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the Caliciviridae family. Compared to other calicivirus, such as rNV and SMSV, the structure of Lagovirus members is not well characterized. In this report, structures of two types of wild RHDV particles, the intact virion and the core-like particle (CLP), were reconstructed by cryo-electron microscopy at 11Å and 17Å, respectively. This is the first time the 3D structure of wild caliciviruses CLP has been provided, and the 3D structure of intact RHDV virion is the highest resolution structure in Lagovirus. Comparison of the intact virion and CLP structures clearly indicated that CLP was produced from the intact virion with the protrusion dissociated. In contrast with the crystal structures of recombinant Norovirus and San Miguel sea lion virus, the capsomers of RHDV virion exhibited unique structural features and assembly modes. Both P1 and P2 subdomains have interactions inside the AB capsomer, while only P2 subdomains have interaction inside CC capsomer. The pseudo atomic models of RHDV capsomers were constructed by homology modeling and density map fitting, and the rotation of RHDV VP60 P domain with respect to its S domain, compared with SMSV, was observed. Collectively, our cryo-electron microscopic studies of RHDV provide close insight into the structure of Lagovirus, which is important for functional analysis and better vaccine development in the future.     

冷冻电镜技术的突破导致结构生物学发生革命性变化

最近,冷冻电镜技术的突破引起结构生物学发生了革命。这一革命导致2017年诺贝尔化学奖授予对冷冻电镜技术发展做出开创性贡献的3位科学家Jacques Dubochet、Joachim Frank和Richard Henderson。本文将综述冷冻电镜的发展历程,导致结构生物学革命的冷冻电镜关键技术,包括电镜、图像记录装置和图像处理算法方面的突破,以及中国科学家应用冷冻电镜取得的重要科学成就,涵盖基因表达/调控、蛋白质合成/降解、膜蛋白、免疫、病毒等相关蛋白复合体。最后,对冷冻电镜的未来发展方向进行展望。     

低温电子显微技术在膜蛋白结构研究中的应用和展望

介绍了蛋白质电子晶体学和单颗粒分析技术这两种低温电子显微技术在膜蛋白和膜蛋白复合体结构研究中的具体方法和近10~20年来的实际应用,并分别分析了这两种方法的优势和瓶颈。此外,还介绍了Amphipol替代、Streptavidin二维晶体锚定脂质体和纳米球包被脂质体等近两年来出现的新的用于低温电镜成像的膜蛋白样品制备方法。最后对膜蛋白的低温电子显微研究的未来发展做了展望。     

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.     

Cryo-electron tomography provides novel insights into nuclear pore architecture: implications for nucleocytoplasmic transport

To go beyond the current structural consensus model of the nuclear pore complex (NPC), we performed cryo-electron tomography of fully native NPCs from Xenopus oocyte nuclear envelopes (NEs). The cytoplasmic face of the NPC revealed distinct anchoring sites for the cytoplasmic filaments, whereas the nuclear face was topped with a massive distal ring positioned above the central pore with indications of the anchoring sites for the nuclear basket filaments and putative intranuclear filaments. The rather "spongy" central framework of the NPC was perforated by an elaborate channel and void system, and at the membrane pore interface it exhibited distinct "handles" protruding into the lumen of the NE. The most variable structural moiety of the NPC was a rather tenuous central plug partially obstructing the central pore. Its mobile character was documented by time-lapse atomic force microscopy. Taken together, the new insights we gained into NPC structure support the notion that the NPC acts as a constrained diffusion pore for molecules and particles without retention signal and as an affinity gate for signal-bearing cargoes.     

Spermatogenesis in animals as revealed by electron microscopy

Summary The development of nuclei and cytoplasmic microtubules was studied in the maturing spermatids of the grasshopper, Acrida lata, fixed with glutaraldehyde-potassium bichromate-osmium tetroxide and embedded in epoxy Epon-resin. Utilization of microkaryosomes for the formation of paracrystalline nucleoprotein is suggested by the fact that they are no longer visible in the advanced spermatid nuclei showing the paracrystalline structure. The cytoplasmic microtubules approximately 220 Å in diameter develop in close association with a linear material similar in density to the nuclear envelope. Only a single layer of the double-layered nuclear envelope is visible during the development of microtubules. Although cytoplasmic microtubules are assumed to have several physiological functions, such apparatus seem to be related to the polymerization of nucleoproteins as well, since the depolymerization of nucleoproteins occurs simultaneously along with the disappearance of cytoplasmic microtubules.     

Cryo-electron microscopy of coagulation Factor VIII bound to lipid nanotubes

Factor VIII (FVIII) is a key protein in blood coagulation, deficiency or malfunction of which causes Haemophilia A. The sole cure for this condition is intravenous administration of FVIII, whose membrane-bound structure we have studied by Cryo-electron microscopy and image analysis. Self-assembled lipid nanotubes were optimised to bind FVIII at close to native conditions. The tubes diameter was constant at 30 nm and the lipid bilayer resolved. The FVIII molecules were well defined, forming an 8.5 nm thick outer layer, and appeared to reach the hydrophobic core of the bilayer. The two known FVIII atomic models were superimposed with the averaged 2D protein densities. The insertion of the FVIII within the membrane was evaluated, reaffirming that the membrane-binding C2 or C1-C2 domain(s) fully penetrate the outer leaflet of the lipid layer. The presented results lay the basis for new models of the FVIII overall orientation and membrane-binding mechanism.     

Cryo-electron microscopy of vitreous sections

Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction.     

Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy

The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.     

Cryo-electron microscopy targets in CASP13: Overview and evaluation of results

Structures of seven CASP13 targets were determined using cryo-electron microscopy (cryo-EM) technique with resolution between 3.0 and 4.0 Å. We provide an overview of the experimentally derived structures and describe results of the numerical evaluation of the submitted models. The evaluation is carried out by comparing coordinates of models to those of reference structures (CASP-style evaluation), as well as checking goodness-of-fit of modeled structures to the cryo-EM density maps. The performance of contributing research groups in the CASP-style evaluation is measured in terms of backbone accuracy, all-atom local geometry and similarity of inter-subunit interfaces. The results on the cryo-EM targets are compared with those on the whole set of eighty CASP13 targets. A posteriori refinement of the best models in their corresponding cryo-EM density maps resulted in structures that are very close to the reference structure, including some regions with better fit to the density.     

Cryo-electron microscopy reveals macromolecular organization within biological liquid cyrstals seen in the polarizing microscope

A method is described for examining water dispersible biopolymers in the frozen, hydrated state by electron microscopy using the filamentous bacterial viruses Pf1 and fd as examples. The technique reveals liquid-crystalline textures that correlate well with polarizing microscopy of magnetically oriented specimens. At higher magnification the packing of the virus particle is revealed to a spatial resolution of better than 30 Å, thus linking directly with data from X-ray diffraction and optical microscopy. Electron diffraction confirms that the structure is preserved to high resolution (4 Å). The technique permits a detailed understanding of the processes involved in the orientation of these samples in a strong magnetic field and clarifies the long-range bi-axial properties of some fibres as seen by X-ray diffraction and optical microscopy.