Molecular structure of human TFIIH.

TFIIH is a multiprotein complex required for both transcription and DNA repair. Single particles of human TFIIH were revealed by electron microscopy and image processing at a resolution of 3.8 nm. TFIIH is 16 x 12.5 x 7.5 nm in size and is organized into a ring-like structure from which a large protein domain protrudes out. A subcomplex assembled from five recombinant core subunits also forms a circular architecture that can be superimposed on the ring found in human TFIIH. Immunolabeling experiments localize several subunits: p44, within the ring structure, forms the base of the protruding protein density which includes the cdk7 kinase, cyclin H, and MAT1. Within the ring structure, p44 was flanked on either side by the XPB and XPD helicases. These observations provide us with a quartenary organizational model of TFIIH.

Three-dimensional structures of the TAFII-containing complexes TFIID and TFTC.

TBP (TATA-binding protein)-associated factors (TAF(II)s) are components of large multiprotein complexes such as TFIID, TFTC, STAGA, PCAF/GCN5, and SAGA, which play a key role in the regulation of gene expression by RNA polymerase II. The structures of TFIID and TFTC have been determined at 3.5-nanometer resolution by electron microscopy and digital image analysis of single particles. Human TFIID resembles a macromolecular clamp that contains four globular domains organized around a solvent-accessible groove of a size suitable to bind DNA. TFTC is larger and contains five domains, four of which are similar to TFIID.

Structural analysis of a MIP family protein from the digestive tract of Cicadella viridis.

Homopteran insects, and especially Cicadella viridis, display in their digestive tract a specialized epithelial differentiation, the filter chamber (FC) acting as a water-shunting complex. The main intrinsic membrane protein of the FC is a 25,000-Da polypeptide (P25). In this paper we demonstrate that this P25 polypeptide is a member of the MIP family of membrane channel proteins, and that P25 forms homotetramers in the native membranes. Using polymerase chain reaction, a 360-base pair cDNA, named cic, was isolated from RNA of the FC. cic encodes a 119-amino acid polypeptide (CIC) whose homologies with MIP26, AQP1 (CHIP), AQP2, and gamma-TIP are 38, 38, 34, and 20%, respectively. Using a specific antibody raised against a 15-amino acid peptide from the CIC sequence, we concluded that CIC and P25 are identical entities, and hence that P25 belongs to the MIP family. We investigated the quaternary structure of P25 in the membranes of the FC using biophysical analysis of P25 nondenaturing detergent micelles, scanning transmission electron microscopy, and image processing of conventional transmission electron microscopic images. All those different approaches converged to the conclusion that P25 exists as an homotetramer forming a regular two-dimensional array in the membranes.

Two-dimensional structure of membrane-bound annexin V at 8 A resolution.

Two-dimensional (2-D) crystals of annexin V, grown by specific binding to phosphatidylserine-containing planar lipid films, were studied by electron image analysis. Images of negatively stained two-dimensional crystals showed diffraction peaks extending to 11 A. After correcting lattice distorsions and averaging over several crystalline areas, the resolution of the analysis was extended up to 8 A. Observed along a direction perpendicular to the membrane plane, the four homologous domains characteristic of annexin V exhibit a noticeable difference in their distribution of protein density. An unambiguous assignment of the domains was possible due to the similar packing of annexin V molecules in the 2-D crystals and in a 3-D crystal form with pseudo-R3 symmetry. The domains I and IV (numbering according to Huber et al., Embo J., 1990, 9, 3867-3874) appear well resolved. On the other hand, the two other domains, II and III, present an almost continuous density, with a protrusion extending outwards the annexin V molecule. In addition, no hydrophilic opening is resolved at the center of the molecule, yet a stain-filled 13-A structure is present, surrounded by domains I, II, and IV and distant by 5 A from the center of the molecule. We interpret these structural features as reflecting a conformational change in the annexin V structure resulting from its membrane binding.

Three-dimensional model of Escherichia coli gyrase B subunit crystallized in two-dimensions on novobiocin-linked phospholipid films.

Two-dimensional crystals of the Escherichia coli DNA gyrase B subunit were obtained upon specific interactions with novobiocin linked phospholipid films. A three-dimensional surface model of the protein was generated by analysing images of tilted negatively stained crystals. The structure showed, at 2.5 to 3.0 nm resolution, two elongated arms organised as a V-shaped protein: the bottom of the V contains the novobiocin binding site, and the extremities of the arms mediate protein-protein interactions between the two monomers in the unit cell. Image analysis of frozen hydrated two-dimensional crystals resulted in a 1.0 nm resolution projection map that shows structural elements not revealed with negative staining. Electron microscopic structural data were compared with the crystallographic structure of the 43 kDa N-terminal fragment of the B subunit complexed with a non hydrolysable ATP analogue.

A 9 A two-dimensional projected structure of cholera toxin B-subunit-GM1 complexes determined by electron crystallography.

Highly ordered two-dimensional crystals of cholera toxin B-subunit pentamers have been grown by specific interaction with planar lipid films containing monosialoganglioside GM1. Electron diffractograms of frozen-hydrated crystals show diffraction peaks extending to beyond 4 A, while electron images diffract to 8 A. A two-dimensional projected structure of cholera toxin B-subunit-GM1 complex has been calculated at 9 A resolution by combining electron diffraction and image data. Crystals present an approximate pgg projection symmetry, with unit cell dimensions a = 119(+/- 1) A, b = 123(+/- 1) A, gamma = 90 degrees. Each pentameric assembly presents two concentric rings of electron scattering density, separated by an area of lower density. The outer and inner rings are centered at 25 A and and 11 A from the pentamer centre, respectively. The apparent projected density of the outer ring is larger than that of the inner ring. We propose that the outer and inner density rings correspond respectively to the peripheral beta-sheet arrangement and the central alpha-helix barrel, recently identified in the crystal structure of the heat-labile enterotoxin from Escherichia coli.