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PubMed2004

Release factors eRF1 and RF2: a universal mechanism controls the large conformational changes.

Class I release factors 1 and 2 (RF1 and RF2) terminate protein synthesis by recognizing stop codons on the mRNA via their conserved amino acid motifs (NIKS in eRF1 and SPF in RF2) and by the conserved tripeptide (GGQ) interactions with the ribosomal peptidyltransferase center. Crystal structures of eRF1 and RF2 do not fit their ribosomal binding pocket (approximately 73 angstroms). Cryoelectron microscopy indicates large conformational changes in the ribosome-bound RF2. Here, we investigate the conformational dynamics of the eRF1 and RF2 using molecular dynamics simulation, structural alignment, and electrostatic analysis of domain interactions. We show that relaxed eRF1 has a shape remarkably similar to the ribosome-bound RF2 observed by cryoelectron microscopy. The similarity between the two release factors is as good as between elongation factor G and elongation factor Tu-guanosine-5'(beta,gamma-imido)triphosphate-tRNA. Further, the conformational transitions and dynamics of eRF1 and RF2 between the free and ribosome-bound states are most likely controlled by protonation of conserved histidines. For eRF1, the distance between the NIKS and GGQ motifs shrinks from 97.5 angstroms in the crystal to 70-80 angstroms. For RF2, the separation between SPF and GGQ elongates from 32 angstroms in the crystal to 50 angstroms. Coulombic interaction strongly favors the open conformation of eRF1; however, solvation and histidine protonation modulate the domain interactions, making the closed conformation of eRF1 more accessible. Thus, RF1 and RF2 function like molecular machines, most likely fueled by histidine protonation. The unified conformational control and the shapes of eRF1 and RF2 support the proposition that the termination of protein synthesis involves similar mechanisms across species.

Amino Acid Motifs↗

PubMed2005

Studying the structure of large viruses with multiresolution imaging.

Multiresolution imaging is an extremely useful technique for understanding in detail the structure of large DNA viruses that do not yield to the requirements of protein crystallography. The methodology consists in fitting the atomic structures of capsid components, independently solved, to medium-resolution, 3D maps of the complete virion, obtained by cryoelectron microscopy and image processing. On combining the two kinds of imaging data, one must take into account their intrinsic differences, as they have different resolution, suffer from different imaging artifacts, and are at different scales. These efforts are rewarded by "quasi-atomic" resolution models that provide valuable information about protein-protein interactions in the capsid. Difference maps calculated by subtracting the quasi-atomic model from the cryoelectron microscopy map reveal the molecular envelope of those capsid components whose atomic structure is unknown. A better understanding of the complex interactions involved in capsid assembly, stabilization, and disassembly is thus achieved.

Cryoelectron Microscopy↗

PubMed2002

Electron tomography of frozen-hydrated isolated triad junctions.

Cryoelectron microscopy and tomography have been applied for the first time to isolated, frozen-hydrated skeletal muscle triad junctions (triads) and terminal cisternae (TC) vesicles derived from sarcoplasmic reticulum. Isolated triads were selected on the basis of their appearance as two spherical TC vesicles attached to opposite sides of a flattened vesicle derived from a transverse tubule (TT). Foot structures (ryanodine receptors) were resolved within the gap between the TC vesicles and TT vesicles, and some residual ordering of the receptors into arrays was apparent. Organized dense layers, apparently containing the calcium-binding protein calsequestrin, were found in the lumen of TC vesicles underlying the foot structures. The lamellar regions did not directly contact the sarcoplasmic reticulum membrane, thereby creating an approximately 5-nm-thick zone that potentially constitutes a subcompartment for achieving locally elevated [Ca(2+) ] in the immediate vicinity of the Ca(2+)-conducting ryanodine receptors. The lumen of the TT vesicles contained globular mass densities of unknown origin, some of which form cross-bridges that may be responsible for the flattened appearance of the transverse tubules when viewed in cross-section. The spatial relationships among the TT membrane, ryanodine receptors, and calsequestrin-containing assemblage are revealed under conditions that do not use dehydration, heavy-metal staining, or chemical fixation, thus exemplifying the potential of cryoelectron microscopy and tomography to reveal structural detail of complex subcellular structures.

Animals↗

PubMed1999

Phosphofructokinase-1 from Saccharomyces cerevisiae: analysis of molecular structure and function by electron microscopy and self-catalysed affinity labelling.

Conventional and cryoelectron microscopy portray native octameric yeast phosphofructokinase-1 (PFK) as consisting of two identical heterotetrameric tetrahedron-like moieties being rotated relative to each other. Immunoelectron microscopy employing subunit-specific IgG identifies alpha-type subunits in the contact zone of the two tetrahedrons, while beta-chains are recognized exclusively at the tips of the octamer. The chemical reaction of phosphofructokinase with analogues of fructose 6-phosphate followed by autocatalytic phosphoryl transfer from [gamma-32P]-ATP results in a specific labelling of the alpha-subunit. AMP and fructose 2,6-bisphosphate affect labelling by stimulating the binding of substrate analogue; AMP additionally promotes phosphoryl transfer. No stimulation of labelling is observed with proteolytically modified tetrameric 12-S phosphofructokinase.

Adenosine Monophosphate↗

PubMed2005

Localization of a disease-associated mutation site in the three-dimensional structure of the cardiac muscle ryanodine receptor.

The cardiac muscle ryanodine receptor (RyR2) functions as a calcium release channel in the heart. Up to 40 mutations in RyR2 have been linked to genetic forms of sudden cardiac death. These mutations are largely clustered in three regions of the sequence of the polypeptide: one near the N terminus, one in the central region, and the third in the C-terminal region. The central region includes 11 mutations, and an isoleucine-proline motif (positions 2427 and 2428) in the same region is predicted to contribute to the binding of FKBP12.6 protein. We have mapped the central mutation site in the three-dimensional structure of RyR2 by green fluorescent protein insertion, cryoelectron microscopy, and single-particle image processing. The central mutation site was mapped to a "bridge" of density that connects cytoplasmic domains 5 and 6, which have been suggested to undergo conformational changes during channel gating. Moreover, the location of this central mutation site is not close to that of the FKBP12.6-binding site mapped previously by cryoelectron microscopy.

Animals↗

PubMed1997

The ATP-binding site of Ca(2+)-ATPase revealed by electron image analysis.

The location of the ATP-binding site of a P-type ion pump, Ca(2+)-ATPase from rabbit sarcoplasmic reticulum, was examined by cryoelectron microscopy. A nonhydrolyzable analog of ATP, beta, gamma-bidentate chromium (III) complex of ATP (CrATP), was used to stabilize the enzyme in the Ca(2+)-occluded state. Tubular crystals were then induced by vanadate in the presence of EGTA, keeping CrATP bound to the enzyme. The three-dimensional structures of the crystals were determined at 14 A resolution by cryoelectron microscopy and helical image analysis. Statistical comparison of the structures with and without CrATP showed clear and significant differences at the groove proposed previously as the ATP-binding pocket.

Adenosine Triphosphate↗

PubMed2004

Amylopectin: a major component of the residual body in Cryptosporidium parvum oocysts.

Amylopectin is used for carbohydrate storage in different life-stages of a number of apicomplexan parasites. We have performed an ultrastructural analysis of amylopectin granules from the oocyst residual body and sporozoites of Cryptosporidium parvum. Amylopectin granules were studied in situ and after isolation from 'French' press disrupted parasites, by conventional transmission electron microscopy (TEM) of sectioned oocysts and various negative staining and cryoelectron microscopy techniques. Within the membrane-enclosed oocyst residuum large amylopectin granules (0.1-0.3 microm) can be found besides a characteristic large lipid body and a crystalline protein inclusion. Smaller granules were detected in sectioned sporozoites. Negative staining of isolated amylopectin granules revealed some ultrastructural features not readily visible in sectioned material. The large amylopectin granules had a smooth surface with a 'ball of string'-like inner structure. Granules isolated from sporozoites were more irregularly shaped and showed a rod-like particulate composition. With the exception of alpha-amylase, which led to some degree of damage of the surface of the particles, treatment of amylopectin granules with other glycohydrolases had little effect on the overall structure. However, granules adhered to one another. Only when the granules were boiled did the 'ball of string' structure gradually dissolve.

Amylopectin↗

PubMed2001

Molecular biology of astroviruses: selected highlights.

Human astrovirus, the prototype of the Astroviridae family, is a non-enveloped positive-strand RNA virus with distinctive morphology. Initially named for a characteristic 5-6 point star evident on the surface of faecally shed viral particles by direct electron microscopy, a recent study using cryoelectron microscopy and image reconstruction indicates that viral particles consist of a smoothly rippled, solid capsid decorated with short spikes. Mechanisms underlying the assembly of these viral particles have not been fully elucidated. However, studies of two full-length cDNA clones of human astrovirus serotype 1 suggest that capsid residue Thr227 plays a critical role in the assembly of infectious viral progeny. The development of a full-length clone (pAVIC) from which infectious RNA can be transcribed has also facilitated studies of the viral 3C-like serine protease, encoded in ORF1a. These studies demonstrate that the full-length ORF1a product (101 kDa) is processed in vitro to an N-terminal 64 kDa fragment and a C-terminal 38 kDa fragment. Mutation of the predicted catalytic triad inhibits proteolysis. In other studies based on modifications of pAVIC, preliminary evidence supports the feasibility of developing a reporter cell line to facilitate astrovirus detection.

Amino Acid Sequence↗

PubMed2006

Deleted in cancer 1 (DICE1) is an essential protein controlling the topology of the inner mitochondrial membrane in C. elegans.

DICE1 (deleted in cancer 1), first identified in human lung carcinoma cell lines, is a candidate tumor suppressor, but the details of its activity remain largely unknown. We have found that RNA interference of its C. elegans homolog (DIC-1) produced inviable embryos with increased apoptosis, cavities in cells and abnormal morphogenesis. In the dic-1(RNAi) germ line, ced-3-dependent apoptosis increased, and cell cavities appeared at the late-pachytene/oogenic stage, leading to defective oogenesis. Immunofluorescence microscopy of DIC-1 revealed its ubiquitous expression in the form of cytoplasmic foci, and cryoelectron microscopy narrowed down the location of the foci to the inner membrane of mitochondria. After dic-1 RNAi, mitochondria had an irregular morphology and contained numerous internal vesicles. Homozygous embryos from a heterozygous dic-1 mother arrested at the L3 larval stage, in agreement with the essential role of DIC-1 in mitochondria. In summary, C. elegans DIC-1 plays a crucial role in the formation of normal morphology of the mitochondrial cristae/inner membrane. Our results suggest that human DICE1 may have several functions in multiple intracellular locations.

Animals↗

PubMed2001

Characterization of Rous sarcoma virus Gag particles assembled in vitro.

Purified retrovirus Gag proteins or Gag protein fragments are able to assemble into virus-like particles (VLPs) in vitro in the presence of RNA. We have examined the role of nucleic acid and of the NC domain in assembly of VLPs from a Rous sarcoma virus (RSV) Gag protein and have characterized these VLPs using transmission electron microscopy (TEM), scanning TEM (STEM), and cryoelectron microscopy (cryo-EM). RNAs of diverse sizes, single-stranded DNA oligonucleotides as small as 22 nucleotides, double-stranded DNA, and heparin all promoted efficient assembly. The percentages of nucleic acid by mass, in the VLPs varied from 5 to 8%. The mean mass of VLPs, as determined by STEM, was 6.5 x 10(7) Da for both RNA-containing and DNA oligonucleotide-containing particles, corresponding to a stoichiometry of about 1,200 protein molecules per VLP, slightly lower than the 1,500 Gag molecules estimated previously for infectious RSV. By cryo-EM, the VLPs showed the characteristic morphology of immature retroviruses, with discernible regions of high density corresponding to the two domains of the CA protein. In spherically averaged density distributions, the mean radial distance to the density corresponding to the C-terminal domain of CA was 33 nm, considerably smaller than that of equivalent human immunodeficiency virus type 1 particles. Deletions of the distal portion of NC, including the second Zn-binding motif, had little effect on assembly, but deletions including the charged residues between the two Zn-binding motifs abrogated assembly. Mutation of the cysteine and histidine residues in the first Zn-binding motif to alanine did not affect assembly, but mutation of the basic residues between the two Zn-binding motifs, or of the basic residues in the N-terminal portion of NC, abrogated assembly. Together, these findings establish VLPs as a good model for immature virions and establish a foundation for dissection of the interactions that lead to assembly.

Avian Sarcoma Viruses↗

PubMed2006

Nuclear pore complex structure and plasticity revealed by electron and atomic force microscopy.

To study the ultrastructure of nuclear pore complexes (NPCs), a wide spectrum of different electron microscopy (EM) or atomic force microscopy (AFM) techniques can be employed. The combination of these methods can reveal new insights into the structural and functional organization of this important supramolecular machine through which nucleocytoplasmic transport occurs. Negative staining, quick freezing/freeze-drying/rotary metal shadowing, embedding and thin sectioning, cryoelectron microscopy and tomography, scanning electron microscopy, or combination with immunolabeling techniques are tools for collecting data and information about the three-dimensional structure and architecture of the NPCs. AFM enables investigation of the functional dynamics of native NPCs under physiological conditions.

Animals↗

PubMed2004

Electron and atomic force microscopy of the trimeric ammonium transporter AmtB.

Escherichia coli AmtB is an archetypal member of the ammonium transporter (Amt) family, a family of proteins that are conserved in all domains of life. Reconstitution of AmtB in the presence of lipids produced large, ordered two-dimensional crystals. From these, a 12 A resolution projection map was determined by cryoelectron microscopy, and high-resolution topographs were acquired using atomic force microscopy. Both techniques showed the trimeric structure of AmtB in which each monomer seems to have a pseudo-two-fold symmetry. This arrangement is likely to represent the in vivo structure. This work provides the first views of the structure of any member of the Amt family.

Cation Transport Proteins↗

PubMed2001

HYDROMIC: prediction of hydrodynamic properties of rigid macromolecular structures obtained from electron microscopy images.

We have developed a procedure for the prediction of hydrodynamic coefficients and other solution properties of macromolecules and macromolecular complexes whose volumes have been generated from electron microscopy images. Starting from the structural files generated in the three-dimensional reconstructions of such molecules, it is possible to construct a hydrodynamic model for which the solution properties can be calculated. We have written a computer program, HYDROMIC, that implements all the stages of the calculation. The use of this procedure is illustrated with a calculation of the solution properties of the volume of the cytosolic chaperonin CCT, obtained from cryoelectron microscopy images.

Biophysical Phenomena↗

PubMed2006

The structure of a filamentous bacteriophage.

Many thin helical polymers, including bacterial pili and filamentous bacteriophage, have been seen as refractory to high-resolution studies by electron microscopy. Studies of the quaternary structure of such filaments have depended upon techniques such as modeling or X-ray fiber diffraction, given that direct visualization of the subunit organization has not been possible. We report the first image reconstruction of a filamentous virus, bacteriophage fd, by cryoelectron microscopy. Although these thin ( approximately 70 A in diameter) rather featureless filaments scatter weakly, we have been able to achieve a nominal resolution of approximately 8 A using an iterative helical reconstruction procedure. We show that two different conformations of the virus exist, and that in both states the subunits are packed differently than in conflicting models previously proposed on the basis of X-ray fiber diffraction or solid-state NMR studies. A significant fraction of the population of wild-type fd is either disordered or in multiple conformational states, while in the presence of the Y21M mutation, this heterogeneity is greatly reduced, consistent with previous observations. These results show that new computational approaches to helical reconstruction can greatly extend the ability to visualize heterogeneous protein polymers at a reasonably high resolution.

Bacteriophage M13↗

PubMed2002

The structure and evolution of the major capsid protein of a large, lipid-containing DNA virus.

Paramecium bursaria Chlorella virus type 1 (PBCV-1) is a very large, icosahedral virus containing an internal membrane enclosed within a glycoprotein coat consisting of pseudohexagonal arrays of trimeric capsomers. Each capsomer is composed of three molecules of the major capsid protein, Vp54, the 2.0-A resolution structure of which is reported here. Four N-linked and two O-linked glycosylation sites were identified. The N-linked sites are associated with nonstandard amino acid motifs as a result of glycosylation by virus-encoded enzymes. Each monomer of the trimeric structure consists of two eight-stranded, antiparallel beta-barrel, "jelly-roll" domains related by a pseudo-sixfold rotation. The fold of the monomer and the pseudo-sixfold symmetry of the capsomer resembles that of the major coat proteins in the double-stranded DNA bacteriophage PRD1 and the double-stranded DNA human adenoviruses, as well as the viral proteins VP2-VP3 of picornaviruses. The structural similarities among these diverse groups of viruses, whose hosts include bacteria, unicellular eukaryotes, plants, and mammals, make it probable that their capsid proteins have evolved from a common ancestor that had already acquired a pseudo-sixfold organization. The trimeric capsid protein structure was used to produce a quasi-atomic model of the 1,900-A diameter PBCV-1 outer shell, based on fitting of the Vp54 crystal structure into a three-dimensional cryoelectron microscopy image reconstruction of the virus.

Capsid↗

PubMed2006

Nonneutralizing human rhinovirus serotype 2-specific monoclonal antibody 2G2 attaches to the region that undergoes the most dramatic changes upon release of the viral RNA.

The monoclonal antibody 2G2 has been used extensively for detection and quantification of structural changes of human rhinovirus serotype 2 during infection. It recognizes exclusively A and B subviral particles, not native virus. We have elucidated the basis of this selectivity by determining the footprint of 2G2. Since viral escape mutants obviously cannot be obtained, the structures of complexes between Fab fragments of 2G2 and 80S subviral B particles were determined by cryoelectron microscopy. The footprint of the antibody corresponds to the capsid region that we predicted would undergo the most dramatic changes upon RNA release.

Antibodies, Monoclonal↗

PubMed2001

Striking conformational change suspected within the phosphoribulokinase dimer induced by interaction with GAPDH.

A multitechnique approach was used to study the [glyceraldehyde-3-phosphate dehydrogenase](2 x 4)-[phosphoribulokinase](2 x 2) multienzymatic complex of the alga Chlamydomonas reinhardtii. On the one hand, each component of the complex was compared with known atomic structures of related enzymes or of similar enzymes originating from different organisms. On the other hand, the overall low resolution architecture of the whole complex was studied using cryoelectron microscopy and image processing techniques. The dimers of phosphoribulokinase are suspected to undergo a dramatic change in activity during a cycle of binding and detaching from tetramers of glyceraldehyde-3-phosphate dehydrogenase. This is likely supported by strong structural differences between the modeled phosphoribulokinase dimers and the counterpart in the three-dimensional reconstruction volume of the whole complex obtained from cryoelectron microscope images.

Amino Acid Sequence↗

PubMed2004

The ribosome: lifting the veil from a fascinating organelle.

It was first suggested that the ribosome is associated with protein synthesis in the 1950s. Initially, its components were revealed as surface-accessible proteins and as molecules of RNA apparently providing a scaffold for subunit shape. Attributing function to the proteins proved difficult, although bacterial protein L11 proved essential for binding one of the decoding protein release factors (RFs). With the discovery that RNA could be a catalyst, interest focussed on the rRNA that, in partnership with mRNA and tRNAs, could potentially mediate the chemical reaction underlying protein synthesis. rRNA interactions and conformational changes were invoked as key elements that facilitated function. The decoding RFs, which are proteins, are exceptions to this rule because they usurp a tRNA function in mediating stop signal recognition. Cryoelectron microscopy and associated image reconstruction technology have now given dramatic snapshots of almost every step of protein synthesis, and X-ray crystallography has revealed, at last, the subunits and monomeric ribosome in exquisite atomic detail.

Animals↗