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PubMedDate not supplied

Evidence for a distinct water-rich layer surrounding collagen fibrils in articular cartilage extracellular matrix.

Bovine articular cartilage was vitrified by high-pressure freezing. On the one hand vitrified samples were cryosectioned and investigated by cryoelectron microscopy in an unstained frozen hydrated state. On the other hand, they were freeze substituted in pure acetone, ethanol, or methanol, respectively, and subsequently embedded in Epon. Ultrathin Epon sections were poststained with uranyl acetate and lead citrate. The resulting ultrastructural representation was different for every protocol. The evaluation of the combined results provides evidence for a distinct water-rich layer surrounding collagen fibrils in articular cartilage extracellular matrix, which has not been recognized before. The possible composition and function of this layer is discussed.

Animals↗

PubMed1997

Three-dimensional reconstruction with contrast transfer function correction from energy-filtered cryoelectron micrographs: procedure and application to the 70S Escherichia coli ribosome.

Cryoelectron microscopy provides the means of studying macromolecules in their native state. However, the contrast transfer function (CTF) makes the images and the three-dimensional (3D) maps derived from them difficult to interpret. We developed methods to determine the CTF from experimental data and to obtain a CTF-corrected 3D reconstruction. The CTF correction and 3D reconstruction accomplished in one step make it easy to combine different defocus data sets and decrease the error accumulation in the computation. These methods were applied to energy-filtered images of the 70S Escherichia coli ribosome, resulting in a distortion-free 3D map of the ribosome at 1/24.5 A-1 resolution, as determined by the differential phase residual resolution criterion.

Escherichia coli↗

PubMed1997

Folding and assembly of hepatitis B virus core protein: a new model proposal.

Hepatitis B core antigen has been intensively studied. Recently, cryoelectron microscopy studies have determined the structure of human and duck hepatitis B virus nucleocapsids at low resolution. Both viruses assemble into core particles of two sizes with icosahedral dimer-clustered T = 3 and T = 4 symmetries. Both capsids present tightly clustered dimers composed of a shell and a protruding domain. The present work introduces a model for HBc folding, dimer formation, and assembly. The model is based in multiple alignments of HBc sequences from 20 mammalian and avian isolates and secondary structure predictions. The 54% alpha-helical conformation predicted is in good agreement with CD results reporting 53-71% content of alpha-helices. Despite the sequence divergence of mammalian and avian proteins, the secondary structure prediction of both shows a high degree of coincidence, according to the multiple sequence alignment. The proposed fold of HBc monomers is built from five alpha-helices. In dimers, pairs of two of those helices conform the protruding domain. The model also suggests the convergence of the region preceding the protamine domain around the sixfold symmetry axes. The model gives answers to most of the standing questions concerning the nucleocapsid assembly and antigenic behavior of HBc protein.

Amino Acid Sequence↗

PubMed1997

A method for establishing the handedness of biological macromolecules.

When biological macromolecules are imaged in the transmission electron microscope (TEM), their inherent handedness is lost because the three-dimensional (3D) structure is projected onto a two-dimensional (2D) plane, and identical 2D projections can be made from either 3D enantiomer. Nevertheless, tilt experiments in the TEM can be used to determine handedness. These experiments have been performed successfully on negatively stained specimens. More recently, the method was applied to unstained, frozen-hydrated specimens imaged by means of cryoelectron microscopy (cryoTEM) methods. Tilt experiments involve recording two micrographs of the same particles at different tilt angles, computing enantiomeric reconstructions from particle images in one micrograph, predicting orientations of corresponding particles in the second micrograph, and comparing model projections with particle images in the second micrograph. In principle, this procedure can be used to determine the handedness of any biological macromolecule imaged by cryoTEM, provided the enantiomeric reconstructions are distinguishable.

Freezing↗

PubMed1998

Molecular imaging of halocynthia papillosa cellulose

The molecular organization of cellulose Ibeta microfibrils in the tunic of Halocynthia papillosa was analyzed by high-resolution cryoelectron microscopy on ultrathin cross sections of artificially highly oriented microfibrils. The arrangement of cellulose chains intersected by the 0.6-, 0.53-, and 0.39-nm equatorial lattice planes was clearly imaged over the whole area of a parallelogram-shaped cross section of a microfibril. One, edge of the parallelogram was parallel to the 0.6-nm lattice plane, while the other did not correspond to a crystallographic plane. Such organization is distinct from previous findings on algal cellulose Ialpha-rich microfibrils, which have an almost square cross section bounded by both 0.6- and 0.53-nm crystallographic planes. A tentative model for microfibril formation is proposed by introducing a two-step biocrystallization mechanism: the formation of molecular sheets spaced by 0.53 nm between adjacent molecules, followed by self-deposition of these sheets by hydrogen bonding between them. Copyright 1998 Academic Press.

Journal Article↗

PubMed2001

Composition and mass of the bacteriophage phi29 prohead and virion.

The protein composition of the Bacillus subtilis bacteriophage phi29 prohead and virion was determined by combustion of gel bands of (3)H-labeled proteins. Copy numbers of individual proteins were calculated relative to the 12 copies of the head-tail connector protein. The mean numbers of copies of the major capsid protein in the prohead and virion were 241 and 218, respectively, approaching the 235 copies determined previously by cryoelectron microscopy. The mean numbers of copies of the dimeric head fiber on the prohead and virion were 24 and 31, respectively, demonstrating partial occupancy of the 55 fiber binding sites. Measured copies of neck and tail proteins in the virion included 11 of the lower collar, 58 of the appendage, and 9 of the tail; if the true copies of these proteins are 12, 60, and 9, respectively, the entire neck and tail of phi29 has quasi-sixfold symmetry. The mass of the fiberless prohead with pRNA was about 14.2 MDa, and the mass of the prohead determined by scanning transmission electron microscopy was consistent with the biochemical data. The mass of the fiberless virion containing the 12.8-MDa DNA genome was about 30.4 MDa. A full complement of dimeric fibers on the prohead or virion would increase the mass of the particle by about 3.2 MDa. The data complement studies relating the structure of phi29 components to dynamic functions in morphogenesis and infection.

Bacillus Phages↗

PubMed2001

Mutational screen identifies critical amino acid residues of beta-actin mediating interaction between its folding intermediates and eukaryotic cytosolic chaperonin CCT.

The three-dimensional reconstruction of apo-CCT-alpha-actin by cryoelectron microscopy shows that actin binds either the CCTbeta-CCTdelta or the CCTepsilon-CCTdelta subunit pairs of the chaperonin in an open and apparently quasi-native conformation. The CCT-binding sites are seen located at the tips of the two arms of actin and these same regions of actin have been implicated in CCT binding through beta-actin peptide-array screening. Three main CCT binding regions exist: actin Sites I, II, and III, which are composed of loops that are surface-exposed in native actin. Sixty-eight amino acid residues on beta-actin have been screened by mutagenesis for effects on CCT interaction in quantitative in vitro translation assays in rabbit reticulocyte lysate. Actin seems to be folding cooperatively on chaperonin, since certain mutants discriminate CCT binding from processing. Actin Site II, located at the tip of actin subdomain 4, is the major determinant for CCT binding. Site II is composed of two anti-parallel extended beta-strands, with F200-T203 and D244 contributing substantially to the binding site. The substrate recognition chemistry of CCT thus seems different from that of Group I chaperonins and probably reflects the fact that it needs to be highly specific to enable capture and folding of the actins and tubulins.

Actins↗

PubMed1995

In vitro assembly of cowpea chlorotic mottle virus from coat protein expressed in Escherichia coli and in vitro-transcribed viral cDNA.

The small spherical plant virus, cowpea chlorotic mottle virus (CCMV), provides an ideal system to examine spherical virus assembly. We have modified the CCMV in vitro assembly system to produce virions from coat protein expressed in Escherichia coli and viral RNA transcribed in vitro from full-length cDNAs. Examination of the in vitro-assembled particles with cryoelectron microscopy and image reconstruction techniques demonstrates that the particles are indistinguishable from plant purified particles at 2.5 nm resolution. Mutational analysis of the coat protein N- and C-terminal extensions demonstrate their respective roles in virus assembly. The N-terminus is required for assembly of RNA containing particles but not for the assembly of empty virions. The C-terminus is essential for coat protein dimer formation and particle assembly.

Base Sequence↗

PubMed1996

The capsid architecture of channel catfish virus, an evolutionarily distant herpesvirus, is largely conserved in the absence of discernible sequence homology with herpes simplex virus.

Although herpesviruses have a wide host range and their genomes vary substantially in size, the nucleocapsid appears to be a conservative element of viral design. The capsid shell is icosahedrally symmetric (T = 16), and 125 nm in diameter and 15nm thick in the case of herpes simplex virus 1 (HSV-1). Channel catfish virus (CCV) has the gross morphology of a herpesvirus, although no relationship to other herpesviruses is evident from the sequences of its proteins. To examine CCV capsid architecture more closely, we have determined its structure by cryoelectron microscopy and three-dimensional image reconstruction. The CCV capsid is smaller than that of HSV-1, but its 12% smaller genome is packed to essentially the same average density; its icosahedral facets are flatter, and its shell is about 20% thinner, consistent with the smaller size of its major capsid protein. Otherwise, their major features are remarkably similar: CCV has the same triangulation number; its hexons and pentons also have chimney-like protrusions with an axial channel through each capsomer; and there are "triplexes" on the outer surface at the sites of local threefold symmetry. The basic herpesvirus capsid architecture is, therefore, remarkably well conserved in CCV and implies a utilitarian basis to this design. The protein composition of CCV mirrors that of HSV-1, except for the absence of the 12-kDa protein, VP26, which is dispensable for assembly in the HSV-1 system and, apparently, wholly dispensable for CCV.

Animals↗

PubMed1996

Orbivirus structure and assembly.

Orbiviruses (Reoviridae family) are complex nonenveloped RNA viruses with seven structural proteins and a RNA genome consisting of 10 variously sized double-stranded RNA segments. Significant advances in orbivirus research have been made in recent years through the use of gene manipulation techniques coupled with the baculovirus expression system. Several orbivirus proteins have yielded to crystallization and X-ray crystallographic structure determination and, when combined with the three-dimensional image reconstruction of virion particles and cores obtained by cryoelectron microscopy, considerable insight has been gained into the intricate organization and topography of the individual viral components. Formal identification of the sites of interaction has been obtained through protein-protein interaction studies on the components of the virion particle, including those that are involved in capsid assembly. Finally, a beginning of the understanding of the sequence of assembly events has also been obtained.

Animals↗

PubMed1997

Structure of the herpes simplex virus capsid: peptide A862-H880 of the major capsid protein is displayed on the rim of the capsomer protrusions.

The herpes simplex virus-1 (HSV-1) capsid shell has 162 capsomers arranged on a T = 16 icosahedral lattice. The major capsid protein, VP5 MW = 149,075) is the structural component of the capsomers. VP5 is an unusually large viral capsid protein and has been shown to consist of multiple domains. To study the conformation of VP5 as it is folded into capsid promoters, we identified the sequence recognized by a VP5-specific monoclonal antibody and localized the epitope on the capsid surface by cryoelectron microscopy and image reconstruction. The epitope of mAb 6F10 was mapped to residues 862-880 by immunoblotting experiments performed with (1) proteolytic fragments of VP5, (2) GST-fusion proteins containing VP5 domains, and (3) synthetic VP5 peptides. As visualized in a three-dimensional density map of 6F10-precipitated capsids, the antibody was found to bind at sites on the outer surface of the capsid just inside the openings of the trans-capsomeric channels. We conclude that these sites are occupied by peptide 862-880 in the mature HSV-1 capsid.

Amino Acid Sequence↗

PubMed1998

Internal/structures containing transcriptase-related proteins in top component particles of mammalian orthoreovirus.

The structure of mammalian orthoreovirus top component particles, which are profoundly deficient in the content of double-stranded RNA genome, was determined at 30 A resolution by transmission cryoelectron microscopy and three-dimensional image reconstruction. Previously undetected, ordered densities, appearing primarily as pentameric flowers in the reconstruction, were seen to extend 65 A inwardly from the inner capsid at the icosahedral fivefold axes. Identically positioned but lower density elements were observed in two types of partially uncoated top component particles obtained by limited proteolysis. The levels of three inner-capsid proteins-lamda 1, lamda 3, and mu 2-were reduced in concert with the internal densities during proteolytic uncoating. Since lamda 3 contains the catalytic regions of the viral RNA polymerase and since both lamda 1 and mu 2 appear to play roles in transcription or mRNA capping, the internal structures are concluded to be complexes of the viral transcriptase-related enzymes. The findings have implications for the mechanisms of transcription and mRNA capping by orthoreovirus particles.

Animals↗

PubMed1994

Crystallographic and cryo EM analysis of virion-receptor interactions.

Cryoelectron microscopy has been used to determine the first structure of a virus when complexed with its glycoprotein cellular receptor. Human rhinovirus 16 (HRV16) complexed with the two amino-terminal, immunoglobulin-like domains of the intercellular adhesion molecule-1 (ICAM-1) shows that ICAM-1 binds into the 12 A deep "canyon" on the surface of the virus. This is consistent with the prediction that the viral receptor attachment site lies in a cavity inaccessible to the host's antibodies. The atomic structures of HRV14 and CD4, homologous to HRV16 and ICAM-1, showed excellent correspondence with observed density, thus establishing the virus-receptor interactions.

Cell Adhesion Molecules↗

PubMed1996

Vitrified articular cartilage reveals novel ultra-structural features respecting extracellular matrix architecture.

The quality of cryosections prepared from high pressure frozen bovine articular cartilage has been recently evaluated by systematic electron diffraction analysis, and vitrification found to be zone-dependent. The lower radial layer was optimally frozen throughout the entire section thickness (150 microns), whereas in the upper radial, transitional and superficial layers this was achieved down to a depth of only approximately 5-50 microns. These differences were found to correlate proportionally with proteoglycan concentration and inversely with water content. In the current investigation, extracellular matrix ultrastructure was examined in high pressure frozen material (derived from the lower radial zone of young adult bovine articular cartilage), by both cryoelectron microscopy of cryosections and by conventional transmission electron microscopy of freeze-substituted and embedded samples. Several novel features were revealed, in particular, the existence of a fine filamentous network; this consisted of elements 10-15 nm in diameter and with a regular cross-banded structure similar to that characterising collagen fibrils. These filaments were encountered throughout the entire extracellular space, even within the pericellular region, which is generally believed to be free of filamentous or fibrillar components. The proteoglycan-rich interfibrillar/filamentous space manifested a fine granular appearance, there being no evidence of the reticular network previously seen in suboptimally frozen material.

Animals↗

PubMed2006

The Membrane Protein Data Bank.

The Membrane Protein Data Bank (MPDB) is an online, searchable, relational database of structural and functional information on integral, anchored and peripheral membrane proteins and peptides. Data originates from the Protein Data Bank and other databases, and from the literature. Structures are based on X-ray and electron diffraction, nuclear magnetic resonance and cryoelectron microscopy. The MPDB is searchable online by protein characteristic, structure determination method, crystallization technique, detergent, temperature, pH, author, etc. Record entries are hyperlinked to the PDB and Pfam for viewing sequence, three-dimensional structure and domain architecture, and for downloading coordinates. Links to PubMed are also provided. The MPDB is updated weekly in parallel with the Protein Data Bank. Statistical analysis of MPDB records can be performed and viewed online. A summary of the statistics as applied to entries in the MPDB is presented. The data suggest conditions appropriate for crystallization trials with novel membrane proteins.

Databases, Protein↗

PubMed2003

Robert Feulgen Lecture. Microscopic assessment of membrane protein structure and function.

Membrane proteins represent an important class of proteins that are encoded by about 40% of all genes, but compared to soluble proteins structural information is sparse. Most of the atomic coordinates currently available are from bacterial membrane proteins and have been obtained by X-ray crystallography. Recent results demonstrate the imaging power of the atomic force microscope and the accuracy of electron crystallography. These methods allow membrane proteins to be studied while embedded in the bilayer, and thus in a functional state. The low signal-to-noise ratio of cryoelectron microscopy is overcome by crystallizing membrane proteins in a two-dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at subnanometer resolution, and their conformational states to be sampled. This review discusses examples of microscopic membrane protein structure determination and illuminates recent progress.

Animals↗

PubMed2004

CoMFA, HQSAR and molecular docking studies of butitaxel analogues with beta-tubulin.

Results from biochemical analyses for a series of 20 butitaxel analogues, paclitaxel and docetaxel were used to build two- and three-dimensional quantitative structure-activity relationship (QSAR) models in order to investigate the properties associated with microtubule assembly and stabilization. A comparative molecular field analysis (CoMFA) model was built using steric and electrostatic fields. The CoMFA model yielded an r2 of 0.943 and a cross-validated r2 (i.e. q2) of 0.376. Hologram quantitative structure-activity relationship (HQSAR) modeling of these same data generated an r2 of 0.919 and a q2 of 0.471. Contour maps used to visualize the steric and electrostatic contributions associated with activity or lack thereof were, as expected, localized to the varied position of the taxane system. Each analogue was docked successfully into a model of beta-tubulin derived from previously determined cryoelectron microscopy analyses of the tubulin alpha/beta heterodimer. All analogues superimposed well with paclitaxel bound to the protein, as well as with each other. Defining the variable region of each structure as the ligand and docking it separately into the paclitaxel site revealed a modest correlation (r2 = 0.53) between activity and docking energy of all the compounds in the dataset. When only the butitaxel derivatives were considered, the correlation increased to 0.61. The mathematical models derived here provide information for the future development of taxanes.

Animals↗

PubMed1990

Characterization of microtubule protofilament numbers. How does the surface lattice accommodate?

Frozen-hydrated specimens of microtubules assembled in vitro were observed by cryoelectron microscopy. Specimens were of both pure tubulin, and of microtubule protein isolated by three cycles of assembly and disassembly. It is shown that the characteristic image contrast of individual microtubules allows the microtubule protofilament number to be determined unambiguously. Microtubules with 13, 14 and 15 protofilaments are observed to coexist in specimens prepared under various assembly conditions. Confirmation of these results is obtained by observations of thin sections of pelleted samples fixed and stained using the glutaraldehyde/tannic acid technique. Images of individual microtubules show both characteristic contrast profiles across their width and typical variations of these profiles along their length. The profiles across the images indicate the protofilament number of the microtubule. The lengthwise variations indicate how the protofilaments are aligned with respect to the microtubule axis giving what has previously been called a supertwist. In 13 protofilament microtubules the protofilaments are paraxial. In 14 and 15 protofilament microtubules, the protofilaments are skewed with respect to the microtubule axis. The skew is greater for the 15 protofilament case than for 14 protofilaments. The skew allows the extra protofilaments to be accommodated by the surface lattice. These results should also be relevant to situations in vivo.

Histocytochemistry↗