Structure of influenza haemagglutinin at neutral and at fusogenic pH by electron cryo-microscopy

The three-dimensional structures of the complete haemagglutinin (HA) of influenza virus A/Japan/305/57 (H2N2) in its native (neutral pH) and membrane fusion-competent (low pH) form by electron cryo-microscopy at a resolution of 10 A and 14 A, respectively, have been determined. In the fusion-competent form the subunits remain closely associated preserving typical overall features of the trimeric ectodomain at neutral pH. Rearrangements of the tertiary structure in the distal and the stem parts are associated with the formation of a central cavity through the entire ectodomain. We suggest that the cavity is essential for relocation of the so-called fusion sequence of HA towards the target membrane.     

Electron cryo-microscopy and image reconstruction of adeno-associated virus type 2 empty capsids

Adeno-associated virus type 2 empty capsids are composed of three proteins, VP1, VP2 and VP3, which have relative molecular masses of 87, 72 and 62 kDa, respectively, and differ in their N-terminal amino acid sequences. They have a likely molar ratio of 1:1:8 and occupy symmetrical equivalent positions in an icosahedrally arranged protein shell. We have investigated empty capsids of adeno-associated virus type 2 by electron cryo-microscopy and icosahedral image reconstruction. The three-dimensional map at 1.05 nm resolution showed sets of three elongated spikes surrounding the three-fold symmetry axes and narrow empty channels at the five-fold axes. The inside of the capsid superimposed with the previously determined structure of the canine parvovirus (Q. Xie and M.S. Chapman, 1996, J. Mol. Biol., 264, 497–520), whereas the outer surface showed clear discrepancies. Globular structures at the inner surface of the capsid at the two-fold symmetry axes were identified as possible positions for the N-terminal extensions of VP1 and VP2.     

Ultra-stable super-resolution fluorescence cryo-microscopy for correlative light and electron cryo-microscopy

Remarkable progress in correlative light and electron cryo-microscopy(cryo-CLEM) has been made in the past decade. A crucial component for cryo-CLEM is a dedicated cryo-fluorescence microscope(cryo-FM). Here, we describe an ultra-stable superresolution cryo-FM that exhibits excellent thermal and mechanical stability. The temperature fluctuations in 10 h are less than0.06 K, and the mechanical drift over 5 h is less than 200 nm in three dimensions. We have demonstrated the super-resolution imaging capability of this system(average single molecule localization accuracy of ～13.0 nm). The results suggest that our system is particularly suitable for long-term observations, such as single molecule localization microscopy(SMLM) and cryogenic super-resolution correlative light and electron microscopy(csCLEM).     

Analysis of macromolecular structure and dynamics by electron cryo-microscopy.

Electron cryo-microscopy has yielded a wealth of detailed new information on structures of biological macromolecules ranging from alphabeta-tubulin at 3.7 A resolution to hepatitis B virus at 7.4 A resolution, as well as a number of membrane proteins at 6-8 A resolution. Much of this progress was made possible by recent advances in instrumentation and image processing techniques.     

Realizing the potential of electron cryo-microscopy

Structural analysis by electron microscopy of biological macromolecules or macromolecular assemblies embedded in rapidly frozen, vitreous ice has made great advances during the last few years. Electron cryo-microscopy, or cryo-EM, can now be used to analyse the structures of molecules arranged in the form of two-dimensional crystals, helical arrays or as single particles with or without symmetry. Although it has been possible, using crystalline or helical specimens, to reach a resolution adequate to build atomic models (4 A), there is every hope this will soon also be possible with single particles. Small and large single particles present different obstacles to progress.     

Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging

Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads.     

Fine structure of influenza A virus observed by electron cryo-microscopy.

A rapidly frozen vitrified aqueous suspension of influenza A virus was observed by high resolution electron cryomicroscopy. The influenza particles were grouped into small (diameter < 150 nm) spherical particles with well organized interiors, large spherical ones with less internal organization, and filamentous ones. Envelopes of most of the large virus particles were phospholipid bilayers, and the chromatography fraction containing these large particles was largely devoid of viral activity. The envelopes of most of the filamentous and small spherical virus particles, on the other hand, gave a strange contrast which could be ascribed to a combination of a thin outer lipid monolayer and a 7.2 nm thick protein-containing inner layer. These latter particles represented most of the viral activity in the preparation. Densitometric traces of the near in-focus images confirmed these structural differences. Some viral envelope structures apparently intermediate between these two distinct types of membrane were also detected. A structural model of intact biologically active influenza virus particles was formulated from these results, together with computer simulations.     

Low resolution structure of bovine rhodopsin determined by electron cryo-microscopy.

The visual pigment rhodopsin is a member of the G protein-coupled receptor family. Electron cryo-microscopy was used to determine the three-dimensional structure of bovine rhodopsin from tilted two-dimensional crystals embedded in vitrified water. The effective resolution in a map obtained from the 23 best crystals was about 9.5 A horizontally and approximately 47 A normal to the plane of the membrane. Four clearly resolved tracks of density in the map correspond to four alpha-helices oriented nearly perpendicular to the plane of the membrane. One of these helices appears to be more tilted than anticipated from the projection structure published previously. The remaining three helices are presumably more highly tilted, given that they form a continuous "arc-shaped" feature and could not be resolved to the same extent. The overall density distribution in the low resolution map shows an arrangement of the helices in which the "arc-shaped" feature is extended by a fourth, less tilted helix. The band of these four tilted helices is flanked by a straight helix on the outer side and a pair of straight helices on its inner side.     

Structural characterization of components of protein assemblies by comparative modeling and electron cryo-microscopy

We explore structural characterization of protein assemblies by a combination of electron cryo-microscopy (cryoEM) and comparative protein structure modeling. Specifically, our method finds an optimal atomic model of a given assembly subunit and its position within an assembly by fitting alternative comparative models into a cryoEM map. The alternative models are calculated by MODELLER [J. Mol. Biol. 234 (1993) 313] from different sequence alignments between the modeled protein and its template structures. The fitting of these models into a cryoEM density map is performed either by FOLDHUNTER [J. Mol. Biol. 308 (2001) 1033] or by a new density fitting module of MODELLER (Mod-EM). Identification of the most accurate model is based on the correlation between the model accuracy and the quality of fit into the cryoEM density map. To quantify this correlation, we created a benchmark consisting of eight proteins of different structural folds with corresponding density maps simulated at five resolutions from 5 to 15 angstroms, with three noise levels each. Each of the proteins in the set was modeled based on 300 different alignments to their remotely related templates (12-32% sequence identity), spanning the range from entirely inaccurate to essentially accurate alignments. The benchmark revealed that one of the most accurate models can usually be identified by the quality of its fit into the cryoEM density map, even for noisy maps at 15 angstroms resolution. Therefore, a cryoEM density map can be helpful in improving the accuracy of a comparative model. Moreover, a pseudo-atomic model of a component in an assembly may be built better with comparative models of the native subunit sequences than with experimentally determined structures of their homologs.     

Structural glycobiology in the age of electron cryo-microscopy

1. Download : Download high-res image (122KB)
2. Download : Download full-size image

     

Near-atomic resolution reconstructions of icosahedral viruses from electron cryo-microscopy

Nine different near-atomic resolution structures of icosahedral viruses, determined by electron cryo-microscopy and published between early 2008 and late 2010, fulfil predictions made 15 years ago that single-particle cryo-EM techniques could visualize molecular detail at 3-4? resolution. This review summarizes technical developments, both in instrumentation and in computation, that have led to the new structures, which advance our understanding of virus assembly and cell entry.     

Structure of native oligomeric Sprouty2 by electron microscopy and its property of electroconductivity

Receptor tyrosine kinases (RTKs) regulate many cellular processes, and Sprouty2 (Spry2) is known as an important regulator of RTK signaling pathways. Therefore, it is worth investigating the properties of Spry2 in more detail. In this study, we found that Spry2 is able to self-assemble into oligomers with a high-affinity KD value of approximately 16 nM, as determined through BIAcore surface plasmon resonance analysis. The three-dimensional (3D) structure of Spry2 was resolved using an electron microscopy (EM) single-particle reconstruction approach, which revealed that Spry2 is donut-shaped with two lip-cover domains. Furthermore, the method of energy dispersive spectrum obtained through EM was analyzed to determine the elements carried by Spry2, and the results demonstrated that Spry2 is a silicon- and iron-containing protein. The silicon may contribute to the electroconductivity of Spry2, and this property exhibits a concentration-dependent feature. This study provides the first report of a silicon- and iron-containing protein, and its 3D structure may allow us (1) to study the potential mechanism through the signal transduction is controlled by switching the electronic transfer on or off and (2) to develop a new type of conductor or even semiconductor using biological or half-biological hybrid materials in the future.     

Structure of ribosomes from Thermomyces lanuginosus by electron microscopy and image processing

Multivariate statistical analysis and hierarchical ascendant classification techniques have been used to sort electron images of ribosomes from the thermophilic fungus Thermomyces lanuginosus into their characteristic views. Three predominant views were elucidated, called here overlap, non-overlap and top, showing reproducible detail approaching 1.8 nm resolution. The overlap and non-overlap forms of the fungal ribosomes appeared to be similar to those from the eubacterium Escherichia coli, despite differences in rRNA composition. The non-overlap projection predominated for the fungal complexes, suggesting different adsorption properties for ribosomes from the two species. Additionally, the top view has not been previously described for eubacteria. No major morphological differences could be detected between the fungal and eubacterial ribosomes at the resolution achieved in this study, suggesting a strong conservation of tertiary structure of this macromolecular complex despite the evolutionary gap between these two organisms.     

Structure of an influenza neuraminidase-diabody complex by electron cryomicroscopy and image analysis

The structure of a complex between a bivalent diabody and its antigen, influenza neuraminidase, has been determined by electron cryomicroscopy of single particles and image analysis. A three-dimensional reconstruction has been interpreted in terms of high-resolution X-ray models of the component proteins. The complex consists of two neuraminidase tetramers cross-linked by four diabodies with 422 point symmetry. The structure and symmetry of the complex is determined uniquely by packing constraints consistent with the maximum possible number of diabody cross-links. Diabodies may provide a useful approach to the structure determination of small proteins by incorporating the proteins into large symmetric complexes followed by single-particle electron microscopy.     

Three-dimensional reconstruction of maltoporin from electron microscopy and image processing.

Two dimensional crystals of maltoporin (or phage lambda receptor) were obtained by reconstitution of purified maltoporin trimers and Escherichia coli phospholipids by detergent dialysis. Two different trimer packing forms were observed. One was hexagonal (a = 7.8 nm) and one rectangular (a = 7.8 nm, b = 13.6 nm). In this paper we describe the three-dimensional structure of maltoporin, deduced from the study of the rectangular form by electron microscopy and image processing. At a resolution of approximately 2.5 nm, maltoporin trimers form aqueous channel triplets which appear to merge into a single outlet at the periplasmic surface of the outer membrane. The pore defined by maltoporin has a similar structure to that outlined by the matrix protein. From the results of functional studies by conductance measurement, it is concluded that the three channels defined by maltoporin act, contrary to those formed by the porin (OmpF protein), as a single conducting unit. A tentative outline of the maltoporin promoter is given. Maltoporin appears to be constituted by three different domains: a major rod-like domain spanning the membrane, a minor domain located near the periplasmic surface of the membrane and finally a central domain responsible for the splitting of the channel.     

Structure of mitochondrial F1-ATPase studied by electron microscopy and image processing

The structure of soluble F₁-ATPase (EC 3.6.1.3) has been investigated by computer analysis of individual molecular images extracted from electron micrographs of negatively stained particles. A total of 1241 images was interactively selected from several digitized micrographs and these images were subsequently aligned relative to different reference images. They were then submitted to a multivariate statistical classification procedure. We have focussed our attention on the main ‘hexagonal’ view which represents some 40% of our population of images. In this view, six masses are located on the outer region of the projection which are associated with the alpha and the beta subunits of the protein. A seventh mass is located close to the centre of the hexagon, but slightly off its exact midpoint. It has the shape of the letter V and its two legs point to two of the outer protein masses, or one alpha-beta subunit pair. The corner of the V has a density as high as those of the large subunits. Possible subunit arrangements and their consequences for the mechanism of ATP synthesis are discussed.     

Time-resolved molecular dynamics of bacteriophage HK97 capsid maturation interpreted by electron cryo-microscopy and X-ray crystallography

The bacteriophage HK97 capsid is a molecular machine that exhibits large-scale conformational rearrangements of its 420 identical protein subunits during capsid maturation. Immature empty capsids, termed Prohead II, assemble in vivo in an Escherichia coli expression system. Maturation of these particles may be induced in vitro, converting them into Head II capsids that are indistinguishable in conformation from the capsid of an infectious phage particle. One method of in vitro maturation requires acidification to drive the reaction through two expansion intermediates (EI-I, EI-II) to its penultimate particle state (EI-III), which has 86% more internal volume than Prohead II. Neutralization of EI-III produces the fully mature capsid, Head II. The three expansion intermediates and the acid expansion pathway were characterized by cryo-EM analysis and 3D reconstruction. We now report that, although large-scale structural changes are involved, the electron density maps for these intermediate states are readily interpreted in terms of quasi-atomic models based on subunit structures determined by prior crystallographic analysis of Head II. Progression through the expansion intermediate states primarily represents rigid-body rotations and translations of the subunits, accompanied by refolding of two small regions, the N-terminal arm and a beta-hairpin called the E-loop. Movies made with these pseudo-atomic coordinates and the Head II X-ray coordinates illuminate various aspects of the maturation pathway in the course of which the pattern of inter-subunit interactions is sequentially transformed while the integrity of the capsid is maintained.     

Conformational change in the mitochondrial channel, VDAC, detected by electron cryo-microscopy.

Crystalline arrays of the voltage-dependent channel, VDAC, can be produced by treatment of Neurospora mitochondrial outer membranes with phospholipase A2. The membrane crystals undergo a lateral phase transition (lattice contraction) that can be induced by an amphipathic polyanion, which also reduces the channel's gating potential. Electron cryo-microscopy of frozen-hydrated crystals indicates that the mean projected diameters of the channels do not decrease with lattice contraction. Instead, contraction is associated with the disappearance of lateral protein "arms" that normally extend between the channels. A model is presented that explains the changes in channel packing and gating potential in terms of a conformational change involving the movement of a protein "arm" between the bilayer and the channel.