Structural states of myelin observed by x-ray diffraction and freeze- fracture electron microscopy

Coordinated freeze-fracture electron microscopy and x-ray diffraction were used to visualize the morphological relation between compacted and native period membrane arrays in myelinated nerves treated with dimethylsulfoxide (DMSO). Comparison of x-ray diffraction at room temperature and at low temperature was used as a critical measure of the extent of structural preservation. Our x-ray diffraction patterns show that in the presence of cryoprotective agents, it is possible to preserve with only small changes the myelin structure which exists at room temperature. These changes include a slight increase in packing disorder of the membrane, a small, negative thermal expansion of the membrane unit, and some reorganization in the cytoplasmic half of the bilayer. The freeze-fracture electron microscopy clearly demonstrates continuity of compact and native period phases in DMSO-treated myelin. Finally, the use of freezing to trap the transient, intermediate structure during a structural transition in glycerol is demonstrated.     

Structural studies of tropomyosin by cryoelectron microscopy and x-ray diffraction.

A comparison has been made between cryoelectron microscope images and the x-ray structure of one projection of the Bailey tropomyosin crystal. The computed transforms of the electron micrographs extend to a resolution of approximately 18 A compared with the reflections from x-ray crystallography which extend to 15 A. After correction of the images for lattice distortions and the contrast transfer function, the structure factors were constrained to the plane group (pmg) symmetry of this projection. Amplitude and phase data for five images were compared with the corresponding view from the three-dimensional x-ray diffraction data (Phillips, G.N., Jr., J.P. Fillers, and C. Cohen. 1986. J. Mol. Biol. 192: 111-131). The average R factor between the electron microscopy and x-ray amplitudes was 15%, with an amplitude-weighted mean phase difference of 4.8 degrees. The density maps derived from cryoelectron microscopy contain structural features similar to those from x-ray diffraction: these include the width and run of the filaments and their woven appearance at the crossover regions. Preliminary images obtained from frozen-hydrated tropomyosin/troponin cocrystals suggest that this approach may provide structural details not readily obtainable from x-ray diffraction studies.     

Gap junction structures. I. Correlated electron microscopy and x-ray diffraction

X-ray crystallographic methods and electron microscope image analysis have been used to correlate the structure and the chemical composition of gap junction plaques isolated intact from mouse liver. The requirement that the interpretations of X-ray, electron microscope, and chemical measurements be consistent reduces the uncertainties inherent in the separate observations and leads to a unified picture of the gap junction structures. Gap junctions are built up of units called connexons that are hexagonally arrayed in the pair of connected cell membranes. X-ray diffraction and electron microscope measurements show that the lattice constant of this array varies from about 80 to 90 A. Analysis of electron micrographs of negatively stained gap junctions shows that there is significant short range disorder in the junction lattice. even though the long range order of the array is remarkably regular. Analysis of the disorder provides information about the nature of the intermolecular forces that hold the array together.     

Electron microscopy in structural studies of Photosystem II

Various techniques of electron microscopy (EM) such as ultrathin sectioning, freeze-fracturing, freeze-etching, negative staining and (cryo-)electron crystallography of two-dimensional crystals have been employed, since now, to obtain much of the structural information of the Photosystem II (PS II) pigment–protein complex at both low and high resolution. This review summarizes information about the structure of this membrane complex as well as its arrangement and interactions with the antenna proteins in thylakoid membranes of higher plants and cyanobacteria obtained by means of EM. Results on subunit organization, with the emphasis on the proteins of the oxygen-evolving complex (OEC), are compared with the data obtained by X-ray crystallography of cyanobacterial PS II. This revised version was published online in August 2006 with corrections to the Cover Date.     

Electron microscopy and X-ray diffraction of a hexagonal net of light meromyosin

Light meromyosin, prepared by brief digestion of rabbit myosin, forms at low ionic strength tactoids with a 43 nm periodicity and open nets. These nets, when negatively stained, show strands intersecting at intervals of ~ 60 nm and at an angle of 120 ° to form hexagonal arrays (Huxley, 1963).By slow dialysis of light meromyosin from 0.35 to 0.1 m-KCl we have obtained large, highly ordered hexagonal nets, which we have subjected to structural analysis by electron microscopy of both negatively stained and sectioned material, and by X-ray diffraction. The net is a three-dimensional crystalline array whose overall shape is that of an oblate ellipsoid. Viewed down the short axis, a hexagonal appearance is seen. Analysis of other views of the net suggests that it has a simple layered structure, each layer consisting of a set of parallel strands of diameter about 10 nm. Each strand crosses over those in neighbouring layers at intervals of 64.4 nm and at an angle of 120 °, so that in the whole structure there is a 3-fold screw axis through each node of the net. A model for a strand is described in which light meromyosin molecules, ~ 100 nm in length, are arranged in an anti-parallel manner, each molecule having one end at a node of the lattice. If this end corresponds to the free end of the myosin tail, one of the interactions is similar to that found in type 1 segments of myosin rod (Harrison et al., 1971). The molecular packing within strands may be related to the packing of myosin tails in the bare zone of muscle thick filaments.     

Electron microscopy and structural model of human fibronectin receptor.

Highly-purified human fibronectin receptor (a heterodimer of two distinct subunits, alpha and beta) was studied using electron microscopy and a variety of preparative procedures. It was found that the receptor consists of a globular head approximately 80 by 120 A and two tails about 20 A thick and 180-200 A long. The whole complex is approximately 280 A long. At low concentrations of detergent the receptor forms doublets, triplets or rosettes associated with the tails which possess the transmembrane portion of the molecule. Computer-assisted structure prediction using the published amino acid sequence of both subunits showed differences in the secondary structure of the tails, the alpha-tail being rich in beta-strands, the beta-tail having five cysteine-rich repeats analogous to the EGF-like repeats of laminin. Estimates of the length of the tails from the predicted structure conformed well with the dimensions obtained from electron micrographs.     

Electron microscopy,electron diffraction,and element analysis of wet biological specimens

Temperature controlled differentially pumped environmental chambers now allow more routine examination of wet specimens in the electron microscope. A sensitive test of their efficiency is the ability to provide high resolution electron diffraction patterns from wet, unfixed protein microcrystals. Fortunately, wet specimens can be prepared with only a few tens of nanometers thickness of remaining water, so extraneous electron scattering by liquid water can be kept to a minimum. It still remains to be determined whether microprobe analysis (X-ray or electron energy-loss spectroscopy) using wet specimens gives better element localization in cells than the current freezing methods. More extensive comparisons are also required of the ultrastructural preservation and visibility of macromolecules immersed in a thin layer of water vs immersion in a thin layer of amorphous ice. However, the recent introduction of commercial forms of the necessary equipment now make these comparisons more feasible.     

Electron microscopy, electron diffraction, and element analysis of wet biological specimens

Temperature controlled differentially pumped environmental chambers now allow more routine examination of wet specimens in the electron microscope. A sensitive test of their efficiency is the ability to provide high resolution electron diffraction patterns from wet, unfixed protein microcrystals. Fortunately, wet specimens can be prepared with only a few tens of nanometers thickness of remaining water, so extraneous electron scattering by liquid water can be kept to a minimum. It still remains to be determined whether microprobe analysis (X-ray or electron energy-loss spectroscopy) using wet specimens gives better element localization in cells than the current freezing methods. More extensive comparisons are also required of the ultrastructural preservation and visibility of macromolecules immersed in a thin layer of water vs immersion in a thin layer of amorphous ice. However, the recent introduction of commercial forms of the necessary equipment now make these comparisons more feasible.     

Low angle x-ray diffraction and biology

The use of the low-angle x-ray diffraction method in biology is described. Some x-ray diffraction patterns have been known since the early 1930's but, in the last decade, many new patterns, which show more reflections than previously, have been recorded. An interpretation of these improved patterns of biological systems provides detailed information on the molecular structure of living cells. The theory and the current status of the low-angle x-ray diffraction method is presented. A brief account of the x-ray studies on collagen, muscle, nerve and membranes is given. Emphasis is given to the history of ideas underlying progress in this area and the current unsolved problems in this field of biophysical research are indicated and discussed.     

Electron diffraction analysis of structural changes in the photocycle of bacteriorhodopsin.

Structural changes are central to the mechanism of light-driven proton transport by bacteriorhodopsin, a seven-helix membrane protein. The main intermediate formed upon light absorption is M, which occurs between the proton release and uptake steps of the photocycle. To investigate the structure of the M intermediate, we have carried out electron diffraction studies with two-dimensional crystals of wild-type bacteriorhodopsin and the Asp96-->Gly mutant. The M intermediate was trapped by rapidly freezing the crystals in liquid ethane following illumination with a xenon flash lamp at 5 and 25 degrees C. Here, we present 3.5 A resolution Fourier projection maps of the differences between the M intermediate and the ground state of bacteriorhodopsin. The most prominent structural changes are observed in the vicinity of helices F and G and are localized to the cytoplasmic half of the membrane.     

Method of x-ray anomalous diffraction for lipid structures

The structures of the unit cells of lipid phases that exhibit long-range crystalline order but short-range liquid-like disorder are of biological interests. In particular, the recently discovered rhombohedral phase has a unit cell containing either the structure of a membrane fusion intermediate state or that of a peptide-induced transmembrane pore, depending on the lipid composition and participating peptides. Diffraction from such systems generally presents a difficult phase problem. The existing methods of phase determination all have their limitations. Therefore it is of general interest to develop a new phasing method. The method of multi-wavelength anomalous dispersion is routinely used in protein crystallography, but the same method is difficult for lipid systems for the practical reason that the commonly used lipid samples for diffraction do not have a well-defined thickness. Here we describe a practical approach to use the multi-wavelength anomalous dispersion method for lipid structures. The procedure is demonstrated with the lamellar phase of a brominated lipid. The method is general to all phases as long as anomalous diffraction is applicable.     

Crystallization and preliminary x-ray diffraction studies of two new antigen-antibody (lysozyme-Fab) complexes

The complexes between the Fab fragments of two monoclonal anti-lysozyme antibodies, Fab10.6.6 (high affinity) and D44.2 (lower affinity), and their specific antigen, hen egg-white lysozyme, have been crystallized. The antibodies recognize an antigenic determinant including Arg68, but differ significantly in their association constants for the antigen. Two crystalline forms were obtained for the complex with FabF10.6.6, the higher affinity antibody. One of them is monoclinic, space group P21, with unit cell dimensions a = 145.6 A, b = 78.1 A, c = 63.1 A, beta = 89.05 degrees, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 3 A making this form suitable for high-resolution X-ray diffraction studies. The second form crystallizes in the triclinic space group P1, with unit cell dimensions a = 134.0 A, b = 144.7 A, c = 98.6 A, alpha = 90.30 degrees, beta = 97.1 degrees, gamma = 90.20 degrees, consistent with the presence of 10 to 12 molecules of the complex in the unit cell. These crystals do not diffract X-rays beyond 5 A resolution. The antigen-antibody complex between FabD44.2, the lower affinity antibody, and hen egg-white lysozyme crystallizes in space group P2(1)2(1)2(1), with unit cell dimensions a = 99.7 A, b = 167.3 A, c = 84.7 A, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 2.5 A resolution.