Visualization of alpha-helices in tobacco mosaic virus by cryo-electron microscopy

We have used tobacco mosaic virus (TMV) as a test specimen, in order to develop techniques for the analysis of high-resolution structural detail in electron micrographs of biological assemblies with helical symmetry. It has previously been shown that internal details of protein structure can be visualized by processing electron micrographs of unstained specimens of extended two-dimensional crystalline arrays. However, the techniques should in principle be applicable to other periodic specimens, such as assemblies with helical symmetry. We show here that data to spacings better than 10 A can be retrieved from electron images of frozen hydrated TMV. The three-dimensional computed map agrees well with that derived from X-ray diffraction and shows the two pairs of alpha-helices forming the core of the coat subunit, the C alpha-helix and the viral RNA. The results demonstrate that it is possible to determine detailed internal structure in helical particles.     

Constructing and validating initial Cα models from subnanometer resolution density maps with pathwalking

A significant number of macromolecular structures solved by electron cryo-microscopy and X-ray crystallography obtain resolutions of 3.5-6?, at which direct atomistic interpretation is difficult. To address this, we developed pathwalking, a semi-automated protocol to enumerate reasonable Cα models from near-atomic resolution density maps without a structural template or sequence-structure correspondence. Pathwalking uses an approach derived from the Traveling Salesman Problem to rapidly generate an ensemble of initial models for individual proteins, which can later be optimized to produce full atomic models. Pathwalking can also be used to validate and identify potential structural ambiguities in models generated from near-atomic resolution density maps. In this work, examples from the EMDB and PDB are used to assess the broad applicability and accuracy of our method. With the growing number of near-atomic resolution density maps from cryo-EM and X-ray crystallography, pathwalking can become an important tool in modeling protein structures.     

Cryo-EM of macromolecular assemblies at near-atomic resolution

With single-particle electron cryomicroscopy (cryo-EM), it is possible to visualize large, macromolecular assemblies in near-native states. Although subnanometer resolutions have been routinely achieved for many specimens, state of the art cryo-EM has pushed to near-atomic (3.3-4.6 ?) resolutions. At these resolutions, it is now possible to construct reliable atomic models directly from the cryo-EM density map. In this study, we describe our recently developed protocols for performing the three-dimensional reconstruction and modeling of Mm-cpn, a group II chaperonin, determined to 4.3 ? resolution. This protocol, utilizing the software tools EMAN, Gorgon and Coot, can be adapted for use with nearly all specimens imaged with cryo-EM that target beyond 5 ? resolution. Additionally, the feature recognition and computational modeling tools can be applied to any near-atomic resolution density maps, including those from X-ray crystallography.     

Effects of phospholipases C and D on ordering of channel proteins in the mitochondrial outer membrane

The effects of phospholipases C and D on the state of order of the channels in the outer membranes of Neurospora mitochondria have been investigated by negative-stain electron microscopy and optical diffraction. Unlike the situation with phospholipase A2, treatment of the isolated membranes with phospholipase C or D does not induce crystallization of the channels in the membrane plane. Furthermore, treatment of already-formed periodic arrays of outer membrane channels with either phospholipase C or D causes loss of long-range order in the arrays. The latter result suggests that zwitterionic phospholipids may play an important role in stabilizing the periodic arrays of the channel-forming protein in this membrane.     

High-resolution electron microscopic investigation of crystalline cholesterol monohydrate in human atheroma at the atomic level

High-resolution electron microscopic investigation of cholesterol monohydrate crystals obtained from human atheroma was carried out for the purpose of characterization of the crystal lattice, demonstration of crystallization processes and identification of crystal disorders. By high-resolution electron microscopy the crystal structures of perfect cholesterol monohydrate crystals were characterized as regular lattice arrays which consisted of stacks of repetitive rod-shaped substructures ca 1.58 nm long and 0.16 nm wide, with the total thickness of bilayered substructures ca 3.36 nm. These substructures were in an end-to-end arrangement of approximately side-to-side parallel packing, with a centre-to-centre spacing ca 0.32 nm. At the atomic level the lattice arrays were made up of regularly spaced rows of dots ca 0.28 nm × 0.16 nm in size. These dots possessed a six-fold ring-like shape, and were arranged in a hexagonal structure with an additional dot in the centre. High-resolution electron microscopic observations of the partially crystallized particles of cholesterol monohydrate showed various stages of cholesterol crystallization, from very small short-ordered segment of lattice arrays to different sized nano- and microcrystallites in the amorphous matrix of the crystals. Furthermore, crystal growth was also demonstrated from detailed examination of the crystal surfaces, the interfaces between the crystals and the boundary structures between the amorphous and crystalline phases. In addition, high-resolution electron microscopy could clearly identify various kinds of crystal defect in the cholesterol monohydrate crystals, including considerable variations of lattice spacings with focal fragmentation of lattice fringes, derangement of atom-sized dots along the lattice fringes and marked alterations of the morphology of atom-sized dots with the vacancies along the lattice arrays. It is hoped that such information obtained from high-resolution electron microscopic observations of the crystalline cholesterol in human atheroma at the atomic or near-atomic level may be helpful by providing a more complete understanding of the pathogenetic mechanisms responsible for the formation, progression and regression of the acellular lipid-rich cores of advanced atherosclerotic plaques.     

Imaging of organelles by electron microscopy reveals protein-protein interactions in mitochondria and chloroplasts

Ongoing progress in electron microscopy (EM) offers now an opening to visualize cells at the nanoscale by cryo-electron tomography (ET). Large protein complexes can be resolved at near-atomic resolution by single particle averaging. Some examples from mitochondria and chloroplasts illustrate the possibilities with an emphasis on the membrane organization. Cryo-ET performed on non-chemically fixed, unstained, ice-embedded material can visualize specific large membrane protein complexes. In combination with averaging methods, 3D structures were calculated of mitochondrial ATP synthase at 6 nm resolution and of chloroplast photosystem II at 3.5 nm.     

Single particle refinement in electron crystallography: a pilot study

Electron crystallography can be used to determine the structures of membrane proteins at near-atomic resolution in some cases. However, most electron crystallography projects remain at a resolution around 10 Å. This might be partly due to lack of flatness of many two-dimensional crystals. We have investigated this problem and suggest single particle processing of locally averaged unit cells to improve the quality and possibly the resolution of three-dimensional maps. Applying this method to the secondary transporter melibiose permease we have calculated a three-dimensional map that is clearer and easier to interpret than the map derived using purely electron-crystallographic methods.     

Refinement of Protein Structures into Low-Resolution Density Maps Using Rosetta

We describe a method based on Rosetta structure refinement for generating high-resolution, all-atom protein models from electron cryomicroscopy density maps. A local measure of the fit of a model to the density is used to directly guide structure refinement and to identify regions incompatible with the density that are then targeted for extensive rebuilding. Over a range of test cases using both simulated and experimentally generated data, the method consistently increases the accuracy of starting models generated either by comparative modeling or by hand-tracing the density. The method can achieve near-atomic resolution starting from density maps at 4-6 Å resolution.     

Structure of paracrystalline arrays on outer membranes of rat-liver and rat-heart mitochondria.

Crystalline arrays are induced in outer membranes of rat-liver and rat-heart mitochondria by phosphotungstate and silicotungstate. The basic structure of the arrays has been determined by correlation averaging of electron microscopic images of side views of tubular arrays and en face views of planar arrays. The arrays consist of rows of bilobed projecting subunits and are similar (in lattice parameters and projected subunit dimensions) to periodic arrays of ion transport ATPases, e.g., arrays of Ca(2+)-ATPase induced by vanadate in sarcoplasmic reticulum. Hexokinase-labeled colloidal gold particles do not specifically decorate the arrays, suggesting that the hexokinase receptor (VDAC channel) is not a component of the arrays.     

Self-assembly of dendrimer-encapsulated nanoparticle arrays using 2-D microbial S-layer protein biotemplates

We investigated the formation of self-assembled two-dimensional (2-D) arrays of dendrimer-encapsulated platinum nanoparticles (Pt-DENs) using prokaryotic surface-layer (S-layer) proteins as biomacromolecular templates. The Pt-DENs (mean core diameter 1.8 +/- 0.5 nm) were synthesized by chemical reduction of metal ion species complexed within the interior of fourth-generation, hydroxyl-terminated, starburst poly(amidoamine) dendrimers (G4 PAMAM-OH). Detailed structural and elemental composition analyses performed using high-resolution transmission electron microscopy, energy-dispersive X-ray spectroscopy, electron energy loss spectroscopy, and X-ray photoelectron spectroscopy indicated that the dendrimer-metal nanocomposite particles were crystalline in nature rather than amorphous and that at least some quantity of the platinum found within the particles is present in the expected zerovalent state. By using the S-layer lattices from the acidothermophilic archaeon Sulfolobus acidocaldarius and the Gram-positive bacterium Deinococcus radiodurans as a biotemplate, hexagonal- and honeycomb-ordered arrays of the Pt-DENs were successfully fabricated under a range of different pH conditions via noncovalent nanoparticle-protein interactions. Fast Fourier transform analyses of transmission electron microscopy images verified that the fabricated Pt-DEN assemblies displayed mean periodicities that corresponded well with the lattice constants of the native protein templates (i.e., 22 and 18 nm for S. acidocaldarius and D. radiodurans S layers, respectively). Our results demonstrate that utilizing pre-synthesized Pt-DENs in conjunction with microbial S-layer proteins displaying highly periodic topochemical properties can be an effective, novel route for creating patterned arrays of Pt nanoparticles with potential technological applications.     

Two-dimensional crystals of Escherichia coli maltoporin and their interaction with the maltose-binding protein.

We have reconstituted Escherichia coli maltoporin into phospholipid membranes at low lipid-to-protein ratios to produce two-dimensional crystals of this membrane protein. Electron microscopy of negatively stained membranes showed three different types of arrays, two of them hexagonal and the third rectangular, all diffracting to approximately (2 nm)-1. Furthermore, we have core-constituted maltoporin with the maltose-binding protein from E. coli, a soluble periplasmic protein that has been proposed to interact with maltoporin. One of the hexagonal arrays was found to bind maltose-binding protein molecules in a regular way, while the maltose-binding protein binding sites were not accessible in the other crystal forms. Difference maps from averaged decorated arrays and undecorated controls showed three symmetry-related maltose-binding protein binding sites per maltoporin trimer, of which not more than one is likely to be occupied at a given time. Using multivariate statistical analysis to select similar unit cells of the decorated maltoporin array, we have obtained a map showing the rough outline of a maltose-binding protein molecule interacting with the pore formed by a maltoporin trimer.     

Structure of light-harvesting chlorophyll a/b protein complex from plant photosynthetic membranes at 7 A resolution in projection

The structure of thin three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex, an integral membrane protein from the photosynthetic membrane of chloroplasts, has been determined at 7 A (1 A = 0.1 nm) resolution in projection. The structure analysis was carried out by image processing of low-dose electron micrographs, and electron diffraction of thin three-dimensional crystals preserved in tannin. The three-dimensional crystals appeared to be stacks of two-dimensional crystals having p321 symmetry. Results of the image analysis indicated that the crystals were disordered, due to random translational displacement of stacked layers. This was established by a translation search routine that used the low-resolution projection of a single layer as a reference. The reference map was derived from the symmetrized average of two images that showed features consistent with the projected structure of negatively stained two-dimensional crystals. The phase shift resulting from the displacement of each layer was corrected. Phase shifts were then refined by minimizing the phase residual, bringing all layers to the same phase origin. Refined phases from different images were in agreement and reliable to 7 A resolution. A projection map was generated from the averaged phases and electron diffraction amplitudes. The map showed that the complex was a trimer composed of three protein monomers related by 3-fold symmetry. The projected density within the protein monomer suggested membrane-spanning alpha-helices roughly perpendicular to the crystal plane. The density in the centre and on the periphery of the trimeric complex was lower than that of the protein, indicating that this region contained low-density matter, such as lipids and antenna chlorophylls.     

7A projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals

Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7A resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.     

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

The light-driven proton pump bacteriorhodopsin occurs naturally as two-dimensional crystals. A three-dimensional 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 A. The map of the structure has a resolution of 3.5 A 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 alpha-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 beta-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 pK changes 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.     

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。     

Structure of actin-containing filaments from two types of non-muscle cells

Bundles of actin-containing filaments from the acrosomal process of horseshoe crab sperm and from sea urchin egg contain a second protein having a molecular weight of about 55,000. Electron micrographs of these filamentous bundles show features reminiscent of paracrystalline arrays of actin except that bundles from the sea urchin egg have distinctive transverse bands every 110 Å. From optical diffraction patterns of the micrographs, we deduced very similar models for both structures. The models consist of hexagonal arrays of actin filaments cross-linked by the second protein. The pattern of transverse bands in bundles derived from the sea urchin eggs is accounted for by postulating that the second protein is bonded to actin only at positions where cross-linking can occur, rather than being bonded to every actin. The helical symmetry of the actin requires that the bonding contacts involved in the cross-linking be slightly different at different positions along the length of the bundle. The technique of image reconstruction was used to obtain a three-dimensional map of the bundles from the acrosomal process.     

7 Å projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals

Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7 Å resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.     

Ultrastructural chemistry of isolated molecules. Application to biological macromolecules. Study of metachromasia

The ultrastructural chemistry on isolated macromolecules enables to localize chemical function on spread molecules by means of reactions whose product is visible in electron microscopy (metal colloids, lectins, etc.). The reaction of linear polyanions with metachromatic dyes (bifunctional ligands) results in the formation of metachromatic complexes with a periodic structure : ligand molecules are intercalated between the polyanions arranged in parallel arrays; this structure is correlated with the metachromasy.     

In vitro assembly of gap junctions.

Gap junction structures were assembled in vitro from octyl-beta-D-glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = b = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg2+ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.