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M F Schmid

Publications and source records attributed to M F Schmid.

At least 19 recordsLinked to original sources
PubMed2001

Finding and using local symmetry in identifying lower domain movements in hexon subunits of the herpes simplex virus type 1 B capsid.

A characteristic of virus assembly is the use of symmetry to construct a complex capsid from a limited number of different proteins. Many spherical viruses display not only icosahedral symmetry, but also local symmetries, which further increase the redundancy of their structural proteins. We have developed a computational procedure for evaluating the quality of these local symmetries that allows us to probe the extent of local structural variations among subunits. This type of analysis can also provide orientation parameters for carrying out non-icosahedral averaging of quasi-equivalent subunits during three-dimensional structural determination. We have used this procedure to analyze the three types of hexon (P, E and C) in the 8.5 A resolution map of the herpes simplex virus type 1 (HSV-1) B capsid, determined by electron cryomicroscopy. The comparison of the three hexons showed that they have good overall 6-fold symmetry and are almost identical throughout most of their lengths. The largest difference among the three lies near the inner surface in a region of about 34 A in thickness. In this region, the P hexon displays slightly lower 6-fold symmetry than the C and E hexons. More detailed analysis showed that parts of two of the P hexon subunits are displaced counterclockwise with respect to their expected 6-fold positions. The most highly displaced subunit interacts with a subunit from an adjacent P hexon (P'). Using the local 6-fold symmetry axis of the P hexon as a rotation axis, we examined the geometrical relationships among the local symmetry axes of the surrounding capsomeres. Deviations from exact symmetry are also found among these local symmetry axes. The relevance of these findings to the process of capsid assembly is considered.

Capsid↗
PubMed2000

Assembly of retrovirus capsid-nucleocapsid proteins in the presence of membranes or RNA.

Retrovirus Gag precursor (PrGag) proteins direct the assembly of roughly spherical immature virus particles, while after proteolytic processing events, the Gag capsid (CA) and nucleocapsid (NC) domains condense on viral RNAs to form mature retrovirus core structures. To investigate the process of retroviral morphogenesis, we examined the properties of histidine-tagged (His-tagged) Moloney murine leukemia (M-MuLV) capsid plus nucleocapsid (CANC) (His-MoCANC) proteins in vitro. The His-MoCANC proteins bound RNA, possessed nucleic acid-annealing activities, and assembled into strand, circle (or sphere), and tube forms in the presence of RNA. Image analysis of electron micrographs revealed that tubes were formed by cage-like lattices of CANC proteins surrounding at least two different types of protein-free cage holes. By virtue of a His tag association with nickel-chelating lipids, His-MoCANC proteins also assembled into planar sheets on lipid monolayers, mimicking the membrane-associated immature PrGag protein forms. Membrane-bound His-MoCANC proteins organized into two-dimensional (2D) cage-like lattices that were closely related to the tube forms, and in the presence of both nickel-chelating lipids and RNAs, 2D lattice forms appeared similar to lattices assembled in the absence of RNA. Our observations are consistent with a M-MuLV morphogenesis model in which proteolytic processing of membrane-bound Gag proteins permits CA and NC domains to rearrange from an immature spherical structure to a condensed mature form while maintaining local protein-protein contacts.

Amino Acid Sequence↗
PubMed1999

Scaling structure factor amplitudes in electron cryomicroscopy using X-Ray solution scattering.

The structure factors derived from electron cryomicroscopic images are modified by the contrast transfer function of the microscope's objective lens and other influences. The phases of the structure factors can be corrected in a straightforward way when the positions of the contrast transfer function rings are determined. However, corrected amplitudes are also essential to yield an accurate distribution of mass in the reconstruction. The correct scale factors for the amplitudes are difficult to evaluate for data that are merged from many different micrographs. We opt to use X-ray solution scattering intensity from a concentrated suspension of the specimen to correct the amplitudes of the spherically averaged structure factors. When this approach is applied to the three-dimensional image data of ice-embedded acrosomal bundles, the core of a filament in a three-dimensional reconstruction of the acrosomal bundle becomes denser and matches more closely the outer density ascribed to scruin.

Acrosome↗
PubMed1999

The three-dimensional structure of the Limulus acrosomal process: a dynamic actin bundle.

Limulus sperm contains a dynamic macromolecular structure that rapidly extends a 50 microm process called the true discharge. The core of this structure is a bundle of ordered filaments composed of a complex of actin, scruin and calmodulin. We determined its structure by electron crystallographic reconstruction. The three-dimensional map reveals an actin-scruin helix that is azimuthally modulated by the influence of the interactions of a filament with its neighbors. There are a variety of density connections with neighboring filaments involving scruin. Scruin commonly contacts one neighbor, but we observe up to three interfilament connections involving both domains of the 28 scruin molecules in the unit cell. Our structure indicates that promiscuous scruin-scruin contacts are the major determinants of bundle stability in the true discharge. It also suggests that rearrangements would be permitted, which can facilitate the transition from the coiled to the true discharge form.

Acrosome↗
PubMed1999

High-resolution electron cryomicroscopy of macromolecular assemblies.

Electron cryomicroscopy is a high-resolution imaging technique that is particularly appropriate for the structural determination of large macromolecular assemblies, which are difficult to study by X-ray crystallography or NMR spectroscopy. For some biological molecules that form two-dimensional crystals, the application of electron cryomicroscopy and image reconstruction can help elucidate structures at atomic resolution. In instances where crystals cannot be formed, atomic-resolution information can be obtained by combining high-resolution structures of individual components determined by X-ray crystallography or NMR with image-derived reconstructions at moderate resolution. This can provide unique and crucial information on the mechanisms of these complexes. Finally, image reconstructions can be used to augment X-ray studies by providing initial models that facilitate phasing of crystals of large macromolecular machines such as ribosomes and viruses.

Actin Cytoskeleton↗
PubMed1998

beta-Filagenin, a newly identified protein coassembling with myosin and paramyosin in Caenorhabditis elegans.

Muscle thick filaments are stable assemblies of myosin and associated proteins whose dimensions are precisely regulated. The mechanisms underlying the stability and regulation of the assembly are not understood. As an approach to these problems, we have studied the core proteins that, together with paramyosin, form the core structure of the thick filament backbone in the nematode Caenorhabditis elegans. We obtained partial peptide sequences from one of the core proteins, beta-filagenin, and then identified a gene that encodes a novel protein of 201-amino acid residues from databases using these sequences. beta-Filagenin has a calculated isoelectric point at 10.61 and a high percentage of aromatic amino acids. Secondary structure algorithms predict that it consists of four beta-strands but no alpha-helices. Western blotting using an affinity-purified antibody showed that beta-filagenin was associated with the cores. beta-Filagenin was localized by immunofluorescence microscopy to the A bands of body-wall muscles, but not the pharynx. beta-filagenin assembled with the myosin homologue paramyosin into the tubular cores of wild-type nematodes at a periodicity matching the 72-nm repeats of paramyosin, as revealed by immunoelectron microscopy. In CB1214 mutants where paramyosin is absent, beta-filagenin assembled with myosin to form abnormal tubular filaments with a periodicity identical to wild type. These results verify that beta-filagenin is a core protein that coassembles with either myosin or paramyosin in C. elegans to form tubular filaments.

Amino Acid Sequence↗
PubMed1998

Muscle thick filaments are rigid coupled tubules, not flexible ropes.

Understanding the structures of thick filaments and their relation to muscle contraction has been an important problem in muscle biology. The flexural rigidity of natural thick filaments isolated from Caenorhabditis elegans as determined by statistical analysis of their electron microscopic images shows that they are considerably more rigid (persistence length=263 microm) than similarly analyzed synthetic actin filaments (6 microm) or duplex DNA (0.05 microm), which are known to be helical ropes. Indeed, cores of C. elegans thick filaments, having only 11% of the mass per unit length of intact thick filaments, are quite rigid (85 microm) compared with the thick filaments. Cores comprise the backbones of the thick filaments and are composed of tubules containing seven subfilaments cross-linked by non-myosin proteins. Microtubules reconstituted from tubulin and microtubule-associated proteins are nearly as rigid (55 microm) as the cores. We propose a model of coupled tubules as the structural basis for the observed rigidity of natural thick filaments and other linear structures such as microtubules. A similar model was recently presented for microtubules [Felgner et al., 1997]. This coupled tubule model may also explain the differences in flexural rigidity between natural rabbit skeletal muscle thick filaments (27 microm) or synthetic thick filaments reconstituted from myosin and myosin binding protein C (36 microm) and those reconstituted from purified myosin (9 microm). The more flexible myosin structures may be helical ropes like F-actin or DNA, whereas the more rigid muscle or synthetic thick filaments which contain myosin and myosin binding protein C may be constructed of subfilaments coupled into tubules as in C. elegans cores. The observed thick filament rigidity is necessary for the incompressibility and lack of flexure observed with thick filaments in contracting skeletal muscle.

Animals↗
PubMed1997

Structural analysis of membrane-bound retrovirus capsid proteins.

We have developed a system for analysis of histidine-tagged (His-tagged) retrovirus core (Gag) proteins, assembled in vitro on lipid monolayers consisting of egg phosphatidylcholine (PC) plus the novel lipid DHGN. DHGN was shown to chelate nickel by atomic absorption spectrometry, and DHGN-containing monolayers specifically bound gold conjugates of His-tagged proteins. Using PC + DHGN monolayers, we examined membrane-bound arrays of an N-terminal His-tagged Moloney murine leukemia virus (M-MuLV) capsid (CA) protein, His-MoCA, and in vivo studies suggest that in vitro-derived His-MoCA arrays reflect some of the Gag protein interactions which occur in assembling virus particles. The His-MoCA proteins formed extensive two-dimensional (2D) protein crystals, with reflections out to 9.5 A resolution. The image-analyzed 2D projection of His-MoCA arrays revealed a distinct cage-like network. The asymmetry of the individual building blocks of the network led to the formation of two types of hexamer rings, surrounding protein-free cage holes. These results predict that Gag hexamers constitute a retrovirus core substructure, and that cage hole sizes define an exclusion limit for entry of retrovirus envelope proteins, or other plasma membrane proteins, into virus particles. We believe that the 2D crystallization method will permit the detailed analysis of retroviral Gag proteins and other His-tagged proteins.

Amino Acid Sequence↗
PubMed1997

A strategy for electron tomographic data collection and crystallographic reconstruction of biological bundles.

Structures of highly ordered biological bundles have unique features which call for special experimental and computational methods in electron cryomicroscopy. They can be considered as three-dimensional quasi-crystals and reconstructed using a crystallographic approach. However, they are neither "infinitely" large with respect to the borders of the bundle, nor are they a single unit cell in thickness along the viewing direction. Also, because of their shape, bundles do not generally have a preferred azimuthal orientation, which poses challenges for orientation estimation and refinement. We developed a strategy for recording and processing electron cryomicroscopic images that differs from classical two-dimensional crystalline reconstruction techniques. These developments allowed us to merge data from tomographic tilt series of ice-embedded acrosomal bundles. The goal is to determine accurately amplitudes and phases at the diffraction maxima in terms of hkl indices, and compute a three-dimensional map from the diffraction data.

Acrosome↗
PubMed1997

Electron crystallography of macromolecular periodic arrays on phospholipid monolayers.

Electron crystallography has the potential of yielding structural information equivalent to x-ray diffraction. The major difficulty has been preparing specimens with the required structural order and size for diffraction and imaging in the electron microscope. 2D crystallization on phospholipid monolayers is capable of fulfilling both of these requirements. Crystals can form as a result of specific interactions with a protein's ligand or an analog, suitably linked to a lipid tail; or on a surface of complementary head-group charge. With such choices, the availability of a suitable lipid is limited only by synthetic chemistry. Ultimately, it is the quality and regularity of the protein-protein interactions that determine the crystalline order, as it is with any protein crystal. In the case of streptavidin, the monolayer crystal diffracts beyond 2.5 A. A 3 A projection map reconstructed from electron diffraction amplitudes and phases from images shows density which can be interpreted as beta-sheets and clusters of side chains. It remains to be shown that the monolayer crystals are flat and diffract as well at high tilt angle as untilted. Technological issues such as charging must be resolved. With parallel advances in data collection and processing, electron crystallography of monolayer macromolecular crystals will eventually take its place beside x-ray crystallography and NMR as a routine and efficient structural technique.

Crystallography↗
PubMed1997

Assemblases and coupling proteins in thick filament assembly.

Thick filaments are stable assemblies of myosin that are characteristic of specific muscle types from both vertebrates and invertebrates. In general, their structure and assembly require remarkably precise determination of lengths and diameters, structural differentiation and nonequivalence of myosins, a high degree of inelasticity and rigidity, and dynamic regulation of assembly and disassembly in response to both extracellular and intracellular signals. Directed assembly of myosin in which additional proteins function in key roles, therefore, is more likely to be significant than the simple self assembly of myosin into thick filaments. The nematode Caenorhabditis elegans permits a wide spectrum of biochemical, genetic, molecular and structural approaches to be applied to the experimental testing of this hypothesis. Biochemical analysis of C. elegans thick filaments reveals that paramyosin, a homologue of the myosin rod that is the unique product of a single genetic locus, exists as two populations which differ by post-translational modification. The major paramyosin species interacts with the two genetically specified myosin heavy chain isoforms. The minor paramyosin species is organized within the cores of the thick filaments, where it is associated stoichiometrically with three recently identified proteins P20, P28 and P30. These proteins have now been characterized molecularly and contain unique, novel amino acid sequences. Structural analysis of the core shows that seven paramyosin subfilaments are crosslinked by additional internal proteins into a highly rigid tubule. P20, P28 and P30 are proposed to couple the paramyosin subfilaments together into the core tubule during filament assembly. Mutants that affect paramyosin assembly are being characterized for alterations in the core proteins. A fourth protein has been identified recently as the product of the unc-45 gene. Computational analysis of this gene's DNA suggests that the predicted protein may exhibit protein phosphatase and chaperone activities. Genetic analysis shows that three classes of specific unc-45 mutant proteins differentially interact with the two myosins during thick filament assembly. The unc-45 protein is proposed to be a myosin assemblase, a protein catalyst of thick filament assembly.

Actin Cytoskeleton↗
PubMed1995

Three-dimensional structure of the acrosomal filament of Limulus sperm by 400 kV electron cryomicroscopy.

The acrosomal bundle of Limulus sperm was imaged by electron cryomicroscopy, and the three-dimensional structure of a filament computationally isolated from the bundle was determined by helical image reconstruction. The actin model of Holmes was fit into the map, and its interactions with scruin, the actin-binding and cross-linking protein, were studied. Scruin binds to two consecutive actins along the helix via subdomains 1 and 3. These interactions involve helix-loop-beta motifs that are present in both actin subdomains (in different monomers) in positions available for binding by the same scruin molecule as it wraps around the actin. Taking first the structural motif homology and then looking for sequence pattern similarities, a stretch of 12 out of 20 matches in hydrophobicity is found. Scruin itself has been found to have an internal tandem homology.

Acrosome↗
PubMed1994

Electron cryomicroscopy of Bacillus stearothermophilus 50 S ribosomal subunits crystallized on phospholipid monolayers.

50 S ribosomal subunits from Bacillus stearothermophilus have been crystallized as 2-dimensional periodic arrays on phospholipid monolayer films at the water-air interface. These crystals were preserved in vitreous ice and imaged with 100 keV electrons under low dose and low temperature conditions. The unit cell parameters of the crystals are a = 371.3(+/- 3.8) A, b = 152.3(+/- 1.6) A, gamma = 96.3(+/- 1.0) degrees. Some of the image arrays of these crystals have twofold rotational symmetry with a phase residual of less than 25 degrees. The mean figure of merit of the merged structure factors from these image arrays out to 20 A resolution is higher than 0.87. The 2-dimensional projection map shows a level of detail not seen in previous structural studies of the 50 S ribosome subunit. Some of these features may be related to the current 3-dimensional model of the subunit. This analysis illustrates the potential of using the electron crystallographic approach for determining the 3-dimensional structure of the 50 S ribosomal subunit crystallized on a monolayer surface. In addition, the structural information retrieved by electron crystallography might be useful for phasing X-ray data towards an atomic resolution model of the ribosome.

Crystallography↗
PubMed1994

Structural studies of viruses by electron cryomicroscopy.

Electron cryomicroscopy is a unique biophysical technique for studying molecular structures of viruses which are difficult to analyze by x-ray diffraction. The structural information derived from the low resolution reconstructions of viruses has so far been useful to understand various functional properties of the viruses such as antibody neutralization, receptor binding and assembly. Electron cryomicroscopy has enabled the visualization of the four core alpha helices of the coat protein in tobacco mosaic virus. This represents the highest resolution detail of a virus studied by electron cryomicroscopy. The prospects of attaining similar resolution beyond 10 A for spherical viruses as well are encouraging, with newly available instrumentation, data collection and processing procedures.

Cryopreservation↗
PubMed1994

Three-dimensional structure of a single filament in the Limulus acrosomal bundle: scruin binds to homologous helix-loop-beta motifs in actin.

Frozen, hydrated acrosomal bundles from Limulus sperm were imaged with a 400 kV electron cryomicroscope. Segments of this long bundle can be studied as a P1 crystal with a unit cell containing an acrosomal filament with 28 actin and 28 scruin molecules in 13 helical turns. A novel computational procedure was developed to extract single columns of superimposed acrosomal filaments from the distinctive crystallographic view. Helical reconstruction was used to generate a three-dimensional structure of this computationally isolated acrosomal filament. The scruin molecule is organized into two domains which contact two actin subunits in different strands of the same actin filament. A correlation of Holmes' actin filament model to the density in our acrosomal filament map shows that actin subdomains 1, 2, and 3 match the model density closely. However, actin subdomain 4 matches rather poorly, suggesting that interactions with scruin may have altered actin conformation. Scruin makes extensive interactions with helix-loop-beta motifs in subdomain 3 of one actin subunit and in subdomain 1 of a consecutive actin subunit along the genetic filament helix. These two actin subdomains are structurally homologous and are closely spaced along the actin filament. Our model suggests that scruin, which is derived from a tandemly duplicated gene, has evolved to bind structurally homologous but non-identical positions across two consecutive actin subunits.

Acrosome↗
PubMed1994

Metastatic adenocarcinoma to the brain: MR with pathologic correlation.

PURPOSE: To describe the appearance on T2-weighted scans of metastatic adenocarcinoma to the brain and to show that the hypointensity frequently associated with these lesions is not related to the presence of mucin, blood products, iron, or calcium. METHODS: The MR scans of 14 patients with metastatic adenocarcinoma to the brain were reviewed retrospectively. The signal intensity on T2-weighted scans of the solid enhancing portion of the tumors was compared with white matter. Histologic examination of the surgical specimens included special stains to search for calcium, mucin, and iron. RESULTS: Eight of nine surgical and all six nonsurgical lesions were either iso- or hypointense to white matter on T2-weighted scans. There was no correlation with tumor histology or the presence of mucin, blood products, iron, or calcium. CONCLUSIONS: The presence of a hypointense intraaxial mass on T2-weighted scans strongly suggests the possibility of metastatic adenocarcinoma. The MR appearance is not explained by the presence of mucin, blood products, iron, or calcium. This phenomenon most likely reflects the relaxation parameters of the tissue from which the metastasis arose.

Adenocarcinoma↗
PubMed1993

Direct visualization of botulinum neurotoxin-induced channels in phospholipid vesicles.

The seven botulinum neurotoxin (NT) serotypes produced by strains of Clostridium botulinum inhibit neurotransmitter release from synaptic vesicles. Neurotoxin is synthesized as a roughly 150K single-chain protein. Proteolysis produces two fragments, the 50K L-chain and 100K H-chain, that remain linked by a disulphide bond. Intoxication involves membrane attachment by the C-terminal half of the H-chain, endocytotic/lysosomal internalization, vesicle channel formation mediated by the 50K N-terminal half of the H-chain at low pH, and finally blockade of synaptic vesicle fusion after the L-chain reaches the cytosol. We report here the visualization of the neurotoxin-membrane complex by electron cryomicroscopy and image processing. Three-dimensional reconstructions show the neurotoxin bound to the exterior of ganglioside/PC lipid vesicles and show channels entirely perforating the vesicle wall. Each channel appears to arise from the interaction of four neurotoxin molecules.

Botulinum Toxins↗