MRC2020: improvements to Ximdisp and the MRC image-processing programs.

The great success of single-particle electron cryo-microscopy (cryoEM) during the last decade has involved the development of powerful new computer programs and packages that guide the user along a recommended processing workflow, in which the wisdom and choices made by the developers help everyone, especially new users, to obtain excellent results. The ability to carry out novel, non-standard or unusual combinations of image-processing steps is sometimes compromised by the convenience of a standard procedure. Some of the older programs were written with great flexibility and are still very valuable. Among these, the original MRC image-processing programs for structure determination by 2D crystal and helical processing alongside general-purpose utility programs such as Ximdisp, label, imedit and twofile are still available. This work describes an updated version of the MRC software package (MRC2020) that is freely available from CCP-EM. It includes new features and improvements such as extensions to the MRC format that retain the versatility of the package and make it particularly useful for testing novel computational procedures in cryoEM.

Tubular crystals of acetylcholine receptor.

Well-ordered tubular crystals of acetylcholine receptor were obtained from suspensions of Torpedo marmorata receptor-rich vesicles. They are composed of pairs of oppositely oriented molecules arranged on the surface lattice with the symmetry of the plane group p2 (average unit cell dimensions: a = 90 A, b = 162 A, gamma = 117 degrees). The receptor in this lattice has an asymmetric distribution of mass around its perimeter, yet a regular pentagonal shape; thus its five transmembrane subunits appear to have different lengths, but approximately equal cross sections. The tubes grow by lateral aggregation on the vesicle surface of ribbons of the paired molecules. Both ribbons and tubes were sensitive to dispersal by the disulphide reductant, dithiothreitol. This observation and other evidence suggest that the basic pairing interaction in the tubes may be that of the physiological dimer, involving contact between delta-subunits.

Two-dimensional structure of the light-harvesting chlorophyll a/b complex by cryoelectron microscopy.

The light-harvesting chlorophyll a/b complex (LHC-II) found in green plants has at least three functions: it absorbs light energy for transfer to the reaction centers, it is involved in keeping the photosynthetic membranes stacked, and it regulates energy distribution between the two photosystems. We have developed a procedure to produce large vesicles consisting almost exclusively of two-dimensional crystalline domains of LHC-II in which LHC-II is biochemically and structurally intact, as shown by SDS-PAGE, response to cations, and 77K fluorescence excitation spectra. The vesicles were examined by cryoelectron microscopy and analyzed, in projection, to a resolution of 17 A. Their surface lattice consists of trimers arranged in interlocking circles; the two-sided plane group is p321 (unit cell dimension, a = 124 A) with two, oppositely facing trimers/unit cell. Individual trimers consist of matter arranged in a ring, around a central cavity, an appearance similar to that obtained in some conditions using negative stain (Li, J., 1985. Proc. Natl. Acad. Sci. USA. 82:386-390). The monomer (approximately 45 x 20 A) is seen as two domains of slightly different size at this resolution. The thickness of single layers is approximately 48 A, measured from edge-on views of the frozen vesicles. Based on these dimensions, the molecular mass of the monomer is approximately 30 kD. Therefore, each monomer appears to be composed of a single polypeptide and its associated pigments.

Calcium-mediated changes in gap junction structure: evidence from the low angle X-ray pattern.

Rat liver gap junctions were isolated in Ca2+-free media and analyzed in controlled environments by x-ray diffraction of partially oriented pellets. Different treatments of the same preparations were compared. The ordered hexagonal lattices gave rise to detail that was sensitive to low Ca2+ concentrations (0.05 mM), but not to Mg2+ (up to 0.16 mM) or pH (between 6.0 and 8.0). The major Ca2+-mediated responses were reductions in the intensity of the (1, 0) peak and in the off-equatorial contributions to the (2, 1) peak, and changes of scale equivalent to a decrease (approximately 2%) in lattice dimension, but an increase (approximately 4%) in the dimension perpendicular to the lattice. A simple structural interpretation of these findings is that Ca2+ induces the subunits of the channel-forming assembly, the connexon, to align more nearly parallel to the channel, thereby causing the connexon to become slightly longer and more radially compact. The rearrangement is of the same nature as one found under less physiological circumstances by electron microscopy (Unwin, P. N. T., and G. Zampighi, 1980, Nature (Lond.)., 283:545-549), and may be part of a coordinated mechanism by which the channel closes.

A large particle associated with the perimeter of the nuclear pore complex.

The three-dimensional structure of the nuclear pore complex has been determined to a resolution of approximately 90 A by electron microscopy using nuclear envelopes from Xenopus oocytes. It is shown to be an assembly of several discrete constituents arranged with octagonal symmetry about a central axis. There are apparent twofold axes perpendicular to the octad axis which suggest that the framework of the pore complex is constructed from two equal but oppositely facing halves. The half facing the cytoplasm is in some instances decorated by large particles, similar in appearance and size to ribosomes.

In vitro crystallization of ribosomes from chick embryos.

A new two-dimensional ribosome crystal, having the tetragonal space group P42(1)2 (a = 593 A), has been grown from ribosome tetramers extracted from hypothermic chick embryos. It is of particular interest because of its larger size (up to 3 x 3 micron2) and greater stability compared to other related polymorphic forms, and because it can easily be grown in large amounts. X-ray diffraction shows the order in the crystal to extend to a resolution of at least 60 A. The crystalline ribosomes appear to contain a full complement of small and large ribosomal subunit proteins and an additional four proteins not characteristic of chick embryo polysomes.

Molecular structure determination of crystalline specimens in frozen aqueous solutions.

Images were obtained of eukaryotic ribosome crystals, bladder membranes and gap junctions preserved in frozen aqueous solutions under conditions where either amorphous or crystalline ice is formed. Evaluation of these images by optical diffraction showed that specimens containing the largest open spaces were sensitive to ice crystal damage during freezing, whereas those containing the smallest open spaces were not. Projection maps were calculated from the images and compared to maps obtained from the same specimens at the same resolution in negative stain. Significant differences were apparent between each pair of maps. These were attributed to details being revealed of the RNA and protein (ribosomes) or the complete protein (membranes) when using frozen solutions, compared to just the hydrophilic surfaces when using stain. Thus the freezing method appears to provide the most complete and accurate descriptions of these structures.

A cold stage for the Philips EM300 electron microscope.

A cold stage has been constructed for the Philips EM300 (and EM301) electron microscopes for investigating the structure of frozen-hydrated biological specimens. The stage entails minimal alterations to the instrument and is capable of a resolution better than 10 A at the normal operating temperature of -120 degrees C. Frozen specimens can be readily exchanged without condensation or warming up, and maintained in the stage over periods of several hours without detectable deterioration.

Three-dimensional model of membrane-bound ribosomes obtained by electron microscopy.

A low-resolution three-dimensional man has been obtained from crystalline arrays of membrane-bound eukaryotic ribosomes. It shows both ribosomal subunits to be adjacent to the membrane surface, attached to it by a part protruding from the large subunit.

Gap junction structure and the control of cell-to-cell communication.

Gap junctions are collections of oligomeric membrane proteins (connexons), which interact across the space between neighbouring cells to form continuous cell-to-cell pathways for ions and small molecules. The connexon is constructed from six identical subunits, arranged symmetrically in the plane of the membrane and delineating the channel along their common sixfold axis. The subunits are rod-shaped and 7-8 nm long; they protrude about 1.5 nm into the extracellular space, but somewhat less into the cell interior. Their cross-section within the membrane corresponds most closely to that of four closely packed alpha-helical rods. The channel is narrowest near the cytoplasmic surface and widest in the extracellular region. Changes between alternative quaternary configurations are most pronounced in the cytoplasmic region, and involve a coordinated tilting of the subunits, predominantly tangential to the central symmetry axis. The observed molecular details suggest that switching between open and closed states of the channel may entail a cooperative mechanism in which a localized effect induced by ligand binding triggers a long-range concerted rearrangement of the subunits. Other membrane channels have similar molecular designs and may act in an analogous way.