Biomedical subjects
Publications and source records attributed to M F Schmid.
Actin and scruin are present in a highly ordered array in the acrosomal bundle from Limulus sperm. Frozen, hydrated acrosomal bundle can be imaged in a 400 kV electron cryomicroscope using the spot-scan technique. Quantitative analysis of the h0l projection images shows diffraction data beyond 7 A.
SPECTRA is a program system that combines well tested and familiar algorithms and advanced graphics tools for (i) the display and indexing of the Fourier transform of images of crystalline specimens, (ii) refinement of the reciprocal lattice, (iii) output of amplitudes and phases of reflections and (iv) lattice distortion correction through cross-correlation map analysis. Because of the ease of identifying and indexing reflections, the user can include many reflections for reciprocal lattice refinement, leading to more accurate lattice determination. Automatic indexing is much faster than entering indices by hand, with less operator error. The user can run the procedure that corrects for lattice distortions with either standard parameters or storable parameters tailored to a particular specimen. SPECTRA's primary function is as a control program which acts as an interface between existing programs and the user. It was written in C with routines for the display, user interface and peak list search. Communication between the separate processes that make up SPECTRA is via the UNIX pipe mechanism and command files, the latter so that users may run, test or replace them or even run them as stand-alone programs outside this system.
Numerous technical advances in electron crystallography have facilitated determination of the three-dimensional structures of macromolecules, especially those that form two-dimensional or helical periodic arrays. Several recent studies have demonstrated the utility of this technique for visualizing secondary structure such as alpha-helices and beta-sheets of membrane proteins and, in one case, the entire polypeptide backbone. Electron crystallography, therefore, has great potential as a tool for studying structural problems that are relevant to both molecular biology and biotechnology.
Electron microscopic analysis can be used to determine the three-dimensional structures of macromolecules at resolutions ranging between 3 and 30 A. It differs from nuclear magnetic resonance spectroscopy or x-ray crystallography in that it allows an object's Coulomb potential functions to be determined directly from images and can be used to study relatively complex macromolecular assemblies in a crystalline or noncrystalline state. Electron imaging already has provided valuable structural information about various biological systems, including membrane proteins, protein-nucleic acid complexes, contractile and motile protein assemblies, viruses, and transport complexes for ions or macromolecules. This article, organized as a series of lectures, presents the biophysical principles of three-dimensional analysis of objects possessing different symmetries.
The acrosomal process of Limulus sperm contains a bundle of filaments composed of actin and a 102 kDa protein in a 1:1 molar ratio. The structure of the bundle in true discharge was investigated by electron cryomicroscopy, X-ray scattering and crystallographic image analysis. A bundle can be characterized as a quasi-crystal with continuously varying views along the bundle axis. Each segment of the bundle is found to obey the symmetry of space group P1, with a = b = 147 A, c = 762 A, alpha = 90 degrees, beta = 90.6 degrees, gamma = 120 degrees. A unit cell contains a helical repeat of the filament with a selection rule following that of an actin filament. A 24 A projection map based on the h0l view was reconstructed after averaging 5300 unit cells from six electron images. Filaments in this projection are well separated and clearly display a 21 screw symmetry. This screw symmetry results from the helical parameters of the bundle filament and is found to be a non-crystallographic symmetry element present in the unit cell. Our structural analysis has led to the proposal that the assembly of a stable bundle with a defined maximum diameter can be controlled by the crystallographic packing of the twisted filaments.
Thin, multilayered crystals of gp32*I were analyzed by negative stain electron microscopy and image processing. Images of untilted crystals exhibited different projection symmetries and structural motifs. Systematic analysis of these images categorized the projections into four types. Areas producing the type 1 projection were reconstructed in three-dimensions from four tilt series containing 111 images. The three-dimensional data has excellent p121 plane group symmetry and reveals that the gp32*I molecule contains two large domains linked together by a small domain. Computer simulations utilizing projection data suggested that the type 2 and 3 projections arise from superposition of type 1 projections related by a 21 screw axis along the projection axis. The three-dimensional reconstruction was utilized in a final simulation that explained the occurrence of the fourth type of projection. This work provides a firm foundation for future high-resolution analysis of the crystal by electron cryomicroscopy.
The 11 S form of acetylcholinesterase from Electrophorus electricus was purified by affinity chromatography. The protein was crystallized from polyethylene glycol solutions. One crystal form proved suitable for x-ray diffraction studies. Preliminary x-ray analysis demonstrates that the space group of this crystal is F222. The unit cell dimensions are a = 141.0 +/- 0.2, b = 202.4 +/- 0.2, and c = 237.4 +/- 0.1 A. The diffraction is anisotropic, extending to at least 3.5 A along the a* and b* axes, but becoming weak beyond about 6 A along the c* axis. Crystal density measurements suggest that one complete 11 S tetramer occupies the asymmetric unit of the crystal.
Two-dimensional crystalline arrays of native tetanus toxin have been formed at the interface between a solution of the toxin and a phospholipid monolayer containing a ganglioside. Electron crystallographic analysis has been used to study these periodic arrays. The arrays obey the symmetry of plane group p12(1), with a = 126 A and b = 84 A, and a thickness of 90 A (1 A = 0.1 nm). The three-dimensional structure of tetanus toxin in negative stain is reconstructed to a nominal resolution of 14 A from multiple tilt images. The molecule presents an asymmetric three-lobed structure and could interact with the monolayer in two possible orientations.
Complexes of RecA protein with phi X174 circular single-stranded DNA (cssDNA) with and without ATP gamma S were rapidly frozen and embedded in a thin layer of vitreous ice. The electron micrographs of these frozen-hydrated complexes clearly show visible helicity. Quantitative image analyses of these micrographs reveal the helical pitch and the axial rise between DNA bases of these complexes. Both of these structural parameters of RecA-cssDNA complexes increase significantly when ATP gamma S is present. These observations agree qualitatively but not quantitatively with those from negative stained specimens and confirm the general model that the interactions among RecA molecules and between RecA and DNA could change according to the functional states of the RecA-cssDNA complex.
The structure of the water-soluble bacteriochlorophyll a protein (Bchl protein) from the green photosynthetic bacterium Prosthecochloris aestuarii has been refined at 1.9 A resolution to a crystallographic residual of 18.9%. The refinement was carried out without knowledge of the amino acid sequence and has led to an "X-ray sequence". The structure consists of seven Bchl molecules enclosed within a protein "bag" and the refinement supports the general conformation of the molecule described previously. The refinement also supports the previous suggestion that the ligands to the seven Bchl magnesiums are, respectively, five histidines, a carbonyl oxygen from the polypeptide backbone of the protein, and a bound water molecule. The conformations of the seven Bchl head-groups are described in detail. In two cases the magnesium atoms are approximately 0.48 A "below" the plane of the conjugated macrocycle while in the other five cases the atoms are, on average, 0.48 A "above" the plane. The acetyl ring substituents are more-or-less coplanar with the dihydrophorbin macrocycle, consistent with a previous resonance Raman study. The conjugated atoms in each of the seven macrocycles have significant departures from strict planarity. These deviations are similar for Bchls 1, 2 and 3 (class I) and are also similar for Bchls 4, 5, 6 and 7 (class II). Ethylchlorophillide also belongs to class II. The out-of-plane deformations for the class I and class II bacteriochlorophylls appear to correspond to two distinct modes of bending or curvature of the dihydrophorbin macrocycle.
Low dose cryoelectron microscopy has been used to record images and electron diffraction patterns of frozen hydrated crystals of the single-stranded DNA binding protein gp32*I. Fourier transforms from 13 image areas, corresponding to approximately 40,000 unit cells, were aligned by a minimal phase residual search and merged by vector addition in reciprocal space. Phases from the resulting composite transform were combined with amplitudes from electron diffraction patterns to reconstruct the projected mass density of the gp32*I crystal at 8.4 A resolution.
As spatial frequency increases, the electron microscope "image" deviates increasingly from a true projection of the specimen's structure. This is due to the finite radius of the Ewald sphere. Quantitative estimates of these deviations of the reconstruction from the true projection are presented for a range of accelerating voltages, spatial frequencies, specimen thicknesses, and specimen tilts.
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The amino acid sequence of human plasma alpha1-acid glycoprotein, upon comparison with the sequences of other blood proteins, was shown to possess significant similarity with the immunoglobulins. Employing direct and corrected sequence identity, the average mutation value and two different computer comparisons for the evaluation of sequence similarity, the following two regions of this alpha-globulin, which account for approximately half of the total amino acid sequence of the protein, were found to possess sequence similarity with the immunoglobulins. a) The region from residues 77 through 125 proved to be related to the variable region of several human H and L chains, and b) the region from residues 136 through 166 was found to be related not only to the constant region of a human and a mouse L chain but also to the third and fourth constant region of a rabbit and a human H chain, respectively. These results suggest that alpha1-acid glycoprotein is probably related to the immunoglobulins and further suggest that it possibly diverged from the immunoglobulin evolutionary tree prior to the formation of the primitive L chain.
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Electron images and diffraction patterns of ice-embedded tropomyosin crystalline sheets have been recorded at 100 and 400 kV. Optical diffractograms from the images indicated an elongated, centered unit cell with a = 799.2 +/- 10.6 A, b = 55.1 +/- 3.5 A. Evaluation of the phases in the computed Fourier transforms up to 7 A resolution revealed the presence of symmetry axes consistent with two-dimensional space group cmm. Electron diffraction patterns show diffuse arcs and discrete sampling at a resolution of 5.1 A, arising from the alpha-helical coiled-coil features of the molecule. These results demonstrate that tropomyosin thin sheets are highly ordered and suggest that retrieval of its high-resolution three-dimensional structure may be feasible by electron crystallography.
Ice-embedded crystals of bacteriophage T4 DNA helix-destabilizing protein gp32*I were imaged by computer-controlled spot-scanning on a 400-kV electron cryomicroscope. gp32*I crystals generally have different steps of thickness within a crystal; each step can have different symmetry. Multivariate statistical analysis enabled us to unambiguously select spot-scan images that consist entirely of one motif which were processed subsequently by crystallographic Fourier-averaging techniques. The computed phases of the resulting reflections were evaluated for symmetry in projection, and some of those images were correlated with independent thickness measurements of freeze-dried samples of the same crystals. The structure factors with pgg symmetry from nine spot-scan images were merged, and the mean figure of merit of merged phases was better than 0.9 for data at resolution up to 4 A. A projection map was generated and showed multiple density peaks that corresponded to the high-resolution features of gp32*I.