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J K Stoops

Publications and source records attributed to J K Stoops.

At least 19 recordsLinked to original sources

The remarkable structural and functional organization of the eukaryotic pyruvate dehydrogenase complexes.

The three-dimensional reconstruction of the bovine kidney pyruvate dehydrogenase complex (M(r) approximately 7.8 x 10(6)) comprising about 22 molecules of pyruvate dehydrogenase (E(1)) and about 6 molecules of dihydrolipoamide dehydrogenase (E(3)) with its binding protein associated with the 60-subunit dihydrolipoamide acetyltransferase (E(2)) core provides considerable insight into the structural and functional organization of the largest multienzyme complex known. The structure shows that potentially 60 centers for acetyl-CoA synthesis are organized in sets of three at each of the 20 vertices of the pentagonal dodecahedral core. These centers consist of three E(1) molecules bound to one E(2) trimer adjacent to an E(3) molecule in each of 12 pentagonal openings. The E(1) components are anchored to the E(1)-binding domain of the E(2) subunits through an approximately 50-A-long linker. Three of these linkers emanate from the outside edges of the triangular base of the E(2) trimer and form a cage around its base that may shelter the lipoyl domains and the E(1) and E(2) active sites. The docking of the atomic structures of E(1) and the E(1) binding and lipoyl domains of E(2) in the electron microscopy map gives a good fit and indicates that the E(1) active site is approximately 95 A above the base of the trimer. We propose that the lipoyl domains and its tether (swinging arm) rotate about the E(1)-binding domain of E(2,) which is centrally located 45-50 A from the E(1), E(2), and E(3) active sites, and that the highly flexible breathing core augments the transfer of intermediates between active sites.

Animals↗

Direct evidence for the size and conformational variability of the pyruvate dehydrogenase complex revealed by three-dimensional electron microscopy. The "breathing" core and its functional relationship to protein dynamics.

Structural studies by three-dimensional electron microscopy of the Saccharomyces cerevisiae truncated dihydrolipoamide acetyltransferase (tE(2)) component of the pyruvate dehydrogenase complex reveal an extraordinary example of protein dynamics. The tE(2) forms a 60-subunit core with the morphology of a pentagonal dodecahedron and consists of 20 cone-shaped trimers interconnected by 30 bridges. Frozen-hydrated and stained molecules of tE(2) in the same field vary in size approximately 20%. Analyses of the data show that the size distribution is bell-shaped, and there is an approximately 40-A difference in the diameter of the smallest and largest structures that corresponds to approximately 14 A of variation in the length of the bridge between interconnected trimers. Companion studies of mature E(2) show that the complex of the intact subunit exhibits a similar size variation. The x-ray structure of Bacillus stearothermophilus tE(2) shows that there is an approximately 10-A gap between adjacent trimers and that the trimers are interconnected by the potentially flexible C-terminal ends of two adjacent subunits. We propose that this springlike feature is involved in a thermally driven expansion and contraction of the core and, since it appears to be a common feature in the phylogeny of pyruvate dehydrogenase complexes, protein dynamics is an integral component of the function of these multienzyme complexes.

Acetyltransferases↗

Light scattering and transmission electron microscopy studies reveal a mechanism for calcium/calmodulin-dependent protein kinase II self-association.

Calmodulin (CaM)-kinase II holoenzymes composed of either alpha or beta subunits were analyzed using light scattering to determine a mechanism for self-association. Under identical reaction conditions, only alphaCaM-kinase II holoenzymes self-associated. Self-association was detected at a remarkably low enzyme concentration (0.14 microM or 7 microg/mL). Light scattering revealed two phases of self-association: a rapid rise that peaked, followed by a slower decrease that stabilized after 2-3 min. Electron microscopy identified that the rapid rise in scattering was due to the formation of loosely packed clusters of holoenzymes that undergo further association into large complexes of several microns in diameter over time. Self-association required activation by Ca(2+)/CaM and was strongly dependent on pH. Self-association was not detected at pH 7.5, however, the extent of this process increased as reaction pH decreased below 7.0. A peptide substrate (autocamtide-2) and inhibitor (AIP) designed from the autoregulatory domain of CaM-kinase II potently prevented self-association, whereas the peptide substrate syntide-2 did not. Thus, CaM-kinase II self-association is isoform specific, regulated by the conditions of activation, and is inhibited by peptides that bind to the catalytic domain likely via their autoregulatory-like sequence. A model for CaM-kinase II self-association is presented whereby catalytic domains in one holoenzyme interact with the regulatory domains in neighboring holoenzymes. These intersubunit-interholoenzyme autoinhibitory interactions could contribute to both the translocation and inactivation of CaM-kinase II previously reported in models of ischemia.

Amino Acid Sequence↗

Three-dimensional reconstructions of calcium/calmodulin-dependent (CaM) kinase IIalpha and truncated CaM kinase IIalpha reveal a unique organization for its structural core and functional domains.

Studies of the structural organization of calcium/ calmodulin-dependent protein kinase IIalpha (CaM KIIalpha) and truncated CaM KIIalpha by three-dimensional electron microscopy and protein engineering show that the structures consist of 12 subunits that are organized in two stacked hexameric rings with 622 symmetry. The body of CaM KIIalpha is gear-shaped, consisting of six slanted flanges, and has six foot-like processes attached by narrow appendages to both ends of the flanges. Truncated CaM KIIalpha that lacks functional domains has a structure that is very similar to the body of CaM KIIalpha. Thus, the functional domains reside in the foot-like processes, and the association domain comprises the gear-shaped core. The ribbon diagram of the bilobate structure of CaM KI fits nicely in the envelope of the foot-like component and indicates that the crevice between the two lobes comprising the functional domains is near the middle portion of the foot. The clustering of the functional domains provides a favorable arrangement for the autophosphorylation reaction, and the unusual arrangement of the catalytic domain on extended tethers appears to be significant for the remarkable functional diversity of CaM KIIalpha in cellular regulation.

Animals↗

The structure of the C949S mutant human alpha(2)-macroglobulin demonstrates the critical role of the internal thiol esters in its proteinase-entrapping structural transformation.

A three-dimensional reconstruction of a protein-engineered mutant alpha(2)-macroglobulin (alpha(2)M) in which a serine residue was substituted for the cysteine 949 (C949S), making it unable to form internal thiol ester moieties, was compared with native and methylamine-transformed alpha(2)Ms. The native alpha(2)M structure consists of two oppositely oriented Z-shaped strands. Thiol ester cleavage following an encounter with a proteinase or a nucleophilic attack by methylamine causes a structural transformation in which the strands assume an opposite handedness and a significant portion of the protein density migrates from the distal ends of the molecule toward the center. The C949S mutant showed a protein density distribution very similar to that of transformed alpha(2)M, with a compact central region of protein density connected to two receptor-binding arms on each end of the molecule. Since no particle shapes characteristic of native or half-transformed alpha(2)Ms were seen in electron micrographs and the C949S mutant and alpha(2)M-methylamine structures are highly similar, we conclude that the intact thiol esters maintain native alpha(2)M in a quasi-stable state. In their absence, alpha(2)M folds into the more stable transformed structure, which displays the functionally important receptor-binding domains and contains the proteinase-entrapping internal cavity.

Amino Acid Substitution↗

Three-dimensional structure of the human herpesvirus 8 capsid.

Human herpesvirus 8 (HHV-8), or Kaposi's sarcoma-associated herpesvirus, is a gammaherpesvirus implicated in all forms of Kaposi's sarcoma and certain lymphomas. HHV-8 has been extensively characterized, both biochemically and immunologically, since its first description in 1994. However, its three-dimensional (3D) structure remained heretofore undetermined largely due to difficulties in viral purification. We have used log-phase cultures of body cavity-based lymphoma 1 cells induced with 12-O-tetradecanoylphorbol-13-acetate to obtain HHV-8 capsids for electron cryomicroscopy and computer reconstruction. The 3D structure of the HHV-8 capsids revealed a capsid shell composed of 12 pentons, 150 hexons, and 320 triplexes arranged on a T=16 icosahedral lattice. This structure is similar to those of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV), which are prototypical members of alpha- and betaherpesviruses, respectively. The inner radius of the HHV-8 capsid is identical to that of the HSV-1 capsid but is smaller than that of the HCMV capsid, which is consistent with the relative sizes of the genomes they enclose. While the HHV-8 capsid exhibits many structural similarities to the HSV-1 capsid, our reconstruction shows two major differences: its hexons lack the "horn-shaped" VP26 densities bound to the HSV-1 hexon subunits, and the HHV-8 triplexes appear smaller and less elongated than those of HSV-1. These differences are in excellent agreement with our sequence comparisons of HHV-8 and HSV-1 capsid proteins. This gammaherpesvirus capsid structure complements previous structural studies on alpha- and betaherpesviruses in providing an account of structural similarities and differences among capsids representing all human herpesvirus subfamilies.

Capsid↗

Structural details of proteinase entrapment by human alpha2-macroglobulin emerge from three-dimensional reconstructions of Fab labeled native, half-transformed, and transformed molecules.

Three-dimensional electron microscopy reconstructions of native, half-transformed, and transformed alpha2-macroglobulins (alpha2Ms) labeled with a monoclonal Fab Fab offer new insight into the mechanism of its proteinase entrapment. Each alpha2M binds four Fabs, two at either end of its dimeric protomers approximately 145 A apart. In the native structure, the epitopes are near the base of its two chisel-like features, laterally separated by 120 A, whereas in the methylamine-transformed alpha2M, the epitopes are at the base of its four arms, laterally separated by 160 A. Upon thiol ester cleavage, the chisels on the native alpha2M appear to split with a separation and rotation to give the four arm-like extensions on transformed alpha2M. Thus, the receptor binding domains previously enclosed within the chisels are exposed. The labeled structures further indicate that the two protomeric strands that constitute the native and transformed molecules are related and reside one on each side of the major axes of these structures. The half-transformed structure shows that the two Fabs at one end of the molecule have an arrangement similar to those on the native alpha2M, whereas on its transformed end, they have rotated. The rotation is associated with a partial untwisting of the strands and an enlargement of the openings to the cavity. We propose that the enlarged openings permit the entrance of the proteinase. Then cleavage of the remaining bait domains by a second proteinase occurs with its entrance into the cavity. This is followed by a retwisting of the strands to encapsulate the proteinases and expose the receptor binding domains associated with the transformed alpha2M.

Endopeptidases↗

On the structural changes of native human alpha2-macroglobulin upon proteinase entrapment. Three-dimensional structure of the half-transformed molecule.

The reconstructions of an intermediate form of human alpha2-macroglobulin (half-transformed alpha2M) in which two of its four bait regions and thiol ester sites were cleaved by chymotrypsin bound to Sepharose were obtained by three-dimensional electron microscopy from stain and frozen-hydrated specimens. The structures show excellent agreement and reveal a structure with approximate dimensions of 195 (length) x 135 (width) and 130 A (depth) with an internal funnel-shaped cavity. The structure shows that a chisel-shaped body is connected to a broad base at the opposing end by four stands. Four approximately 45 A diameter large openings in the body of the structure result in a central cavity that is more accessible to the proteinase than those associated with the native or fully transformed structures. The dissimilarity in the shapes between the two ends of alpha2M half-transformed and the similarity between its chisel-shaped body and that of native alpha2M indicate that the chymotrypsin has cleaved both bait regions in the bottom-half of the structure. Consequently, its functional division lies on the minor axis. The structural organization is in accord with biochemical studies, which show that the half-transformed alpha2M migrates on native polyacrylamide gels at a rate intermediate to the native and fully transformed alpha2M and is capable of trapping 1 mol of proteinase. Even though its upper portion is similar to the native molecule, significant differences in their shapes are apparent and these differences may be related to its slower reaction with a proteinase than the native structure. These structural comparisons further support the view that the transformation of alpha2M involves an untwisting of its strands with an opening of the cavity for entrance of the proteinase and a retwisting of the strands around the proteinase resulting in its encapsulation.

Binding Sites↗

Three-dimensional structure of the human plasmin alpha2-macroglobulin complex.

The three-dimensional reconstructions of the human plasmin alpha2-macroglobulin binary complex were computed from electron microscopy images of stain and frozen-hydrated specimens. The structures show excellent agreement and reveal a molecule with approximate dimensions of 170 (length) x 140 (width) x 140 A (depth). The asymmetric plasmin structure imparts significant asymmetry to the plasmin alpha2-macroglobulin complex not seen in the structures resulting from the reaction of alpha2-macroglobulin with methylamine or chymotrypsin. The structure shows, when combined with other studies, that the C-terminal catalytic domain of the rod-shaped plasmin molecule is entrapped inside of the alpha2-macroglobulin cavity, whereas its N-terminal kringle domains protrude outside one end between the two arm-like features of the transformed alpha2-macroglobulin structure. This arrangement ensures that the catalytic site of plasmin is prevented from degrading plasma proteins. The internalized C-terminal portion of the plasmin structure resides primarily on the major axis of alpha2-macroglobulin, suggesting that after the initial cleavage of the two bait domains and the thiol esters, the rod-shaped plasmin molecule enters the alpha2-macroglobulin cavity through the large openings afforded by the half-transformed structure. This mode of entrapment requires the untwisting and the separation of the two strands that constitute the alpha2-macroglobulin structure.

Chymotrypsin↗

On the unique structural organization of the Saccharomyces cerevisiae pyruvate dehydrogenase complex.

Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components attach. We have determined the structures of the truncated 60-mer core dihydrolipoamide acetyltransferase (tE2) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex and complexes of the tE2 core associated with a truncated binding protein (tBP), intact binding protein (BP), and the BP associated with its dihydrolipoamide dehydrogenase (BP.E3). The tE2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges. Previous studies have given rise to the generally accepted belief that the other components are bound on the outside of the E2 scaffold. However, this investigation shows that the 12 large openings in the tE2 core permit the entrance of tBP, BP, and BP.E3 into a large central cavity where the BP component apparently binds near the tip of the tE2 trimer. The bone-shaped E3 molecule is anchored inside the central cavity through its interaction with BP. One end of E3 has its catalytic site within the surface of the scaffold for interaction with other external catalytic domains. Though tE2 has 60 potential binding sites, it binds only about 30 copies of tBP, 15 of BP, and 12 of BP.E3. Thus, E2 is unusual in that the stoichiometry and arrangement of the tBP, BP, and E3.BP components are determined by the geometric constraints of the underlying scaffold.

Freeze Fracturing↗

Utility of Butvar support film and methylamine tungstate stain in three-dimensional electron microscopy: agreement between stain and frozen-hydrated reconstructions.

A random conical tilt reconstruction of negatively stained Saccharomyces cerevisiae fatty acid synthase was used as a model to compute a three-dimensional reconstruction from untilted stain specimens of the molecules in multiple orientations using a three-dimensional projection alignment method. The resulting structure (24 A resolution) has a more uniform resolution than the initial structure and the handedness revealed in the random conical tilt method is preserved. In a similar approach, this model was used to compute a 21-A-resolution frozen-hydrated structure from untilted specimens of the molecules in multiple orientations. Even though the reconstructions are in close agreement, the stain structure appears to enhance the protein density associated with less robust features. These procedures significantly reduce the time and effort required to obtain a three-dimensional reconstruction from frozen-hydrated data with a resolution that is comparable to the best obtained by more laborious methods. The agreement between the stain and frozen-hydrated reconstructions affords convincing evidence concerning the validity of the structure and the information afforded by the two reconstructions significantly enhances the structural analysis of the molecule.

Coloring Agents↗

Assembly of the gigantic hemoglobin of the earthworm Lumbricus terrestris. Roles of subunit equilibria, non-globin linker chains, and valence of the heme iron.

The extracellular hemoglobin of the earthworm Lumbricus terrestris has four major kinds of O2-binding chains: a, b, and c (forming a disulfide-linked trimer), and chain d. Non-heme, non-globin structural chains, "linkers," are also present. Light-scattering techniques have been used to show that the ferrous CO-saturated abc trimer and chain d form an (abcd)4 complex of 285 kDa at neutral pH. Formation of the full-sized 4-MDa molecule requires the addition of linker chains in the proportion of two linkers per (abcd)4 and occurs much more rapidly in the presence of 10 mM calcium. This stoichiometry is supported not only by direct quantitative analysis of the intact hemoglobin but also by the fact that the addition of 50% of the proposed stoichiometric quantity of linkers results in the conversion of 50% of the (abcd)4 to full-sized molecules. Isolated CO-saturated abc trimers self-associate to (abc)2 and higher aggregates up to an apparent limit of (abc)10 approximately 550 kDa. The CO-saturated chain d forms dimers, (d)2, and tetramers, (d)4. Oxidation of the (abcd)4 complex with ferricyanide causes complete dissociation of chain d from the abc trimer, but addition of CN- maintains the (abcd)4 complex. Valence hybrids have also been studied. The ferrous CO-saturated abc trimer and met (ferric) chain d also associate to form (abcd)4, but the met abc trimer and ferrous CO-saturated chain d do not. Oxidation of the abc trimer and chain d to the ferric form causes the formation of a characteristic hemichrome spectrum with a maximum at 565 nm and a shoulder near 530 nm. These results show that interactions between the abc trimer and chain d are strongly dependent on the ligand and valence state of the heme iron. Light-scattering measurements reveal that oxidation of the intact Hb produces a significant drop in molecular mass from 4.1 to 3.6 MDa. Inclusion of CN- prevents this drop. These experiments indicate that oxidation causes the Hb to shed subunits. The observations provide an explanation for the wide variations in the molecular mass of L. terrestris Hb that have been observed previously.

Alcohol Dehydrogenase↗

Structure-function relationships of the Saccharomyces cerevisiae fatty acid synthase. Three-dimensional structure.

The three-dimensional structure of the Saccharomyces cerevisie fatty acid synthase was computed from electron microscopy of stain images. The barrel-shaped structure (point group symmetry 32) has major and minor axes of approximately 245 x 220 A, respectively, and consists of two different subunits organized in an alpha6beta6 complex (Mr = 2.5 x 10(6)). Two sets of three beta subunits form triangle-shaped caps that enclose the ends of the barrel. The wall of the barrel appears to consist of three N-shaped alpha subunit pairs each with an over and underlying arch-shaped beta subunit. Inside the molecule there are three major interconnected cavities that are tilted approximately 20 degrees with respect to its major axis. An axle-shaped structure extends the length of the cavity on the 3-fold axis and is connected to the two ends of the barrel. The cavities are partially divided on the equator of the molecule by three spokes that extend from the axle on the 2-fold axis to the exterior wall. We propose that these six cavities constitute the six equivalent sites of fatty acid synthesis resulting in an extraordinary structure-function relationship with the 42 catalytic sites involved in fatty acid synthesis inside the molecule. The six cavities each have two funnel-shaped openings ( approximately 20 A in diameter) which may serve to permit the diffusion of substrates and products in and out of these functional units. The subunits appear to be arranged in a manner that affords extensive intermolecular interactions contributing to the stability of this macromolecular complex.

Fatty Acid Synthases↗

Characterization of the cytoplasmic filament protein gene (cfpA) of Treponema pallidum subsp. pallidum.

Treponema pallidum and other members of the genera Treponema, Spirochaeta, and Leptonema contain multiple cytoplasmic filaments that run the length of the organism just underneath the cytoplasmic membrane. These cytoplasmic filaments have a ribbon-like profile and consist of a major cytoplasmic filament protein subunit (CfpA, formerly called TpN83) with a relative molecular weight of approximately 80,000. Degenerate DNA primers based on N-terminal and CNBr cleavage fragment amino acid sequences of T. pallidum subsp. pallidum (Nichols) CfpA were utilized to amplify a fragment of the encoding gene (cfpA). A 6.8-kb EcoRI fragment containing all but the 5' end of cfpA was identified by hybridization with the resulting PCR product and cloned into Lambda ZAP II. The 5' region was obtained by inverse PCR, and the complete gene sequence was determined. The cfpA sequence contained a 2,034-nucleotide coding region, a putative promoter with consensus sequences (5'-TTTACA-3' for -35 and 5'-TACAAT-3' for -10) similar to the sigma70 recognition sequence of Escherichia coli and other organisms, and a putative ribosome-binding site (5'-AGGAG-3'). The deduced amino acid sequence of CfpA indicated a protein of 678 residues with a calculated molecular mass of 78.5 kDa and an estimated pI of 6.15. No significant homology to known proteins or structural motifs was found among known prokaryotic or eukaryotic sequences. Expression of a LacZ-CfpA fusion protein in E. coli was detrimental to survival and growth of the host strain and resulted in the formation of short, irregular filaments suggestive of partial self-assembly of CfpA. The cytoplasmic filaments of T. pallidum and other spirochetes appear to represent a unique form of prokaryotic intracytoplasmic inclusions.

Amino Acid Sequence↗

Three-dimensional structure of the truncated core of the Saccharomyces cerevisiae pyruvate dehydrogenase complex determined from negative stain and cryoelectron microscopy images.

Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components are attached. We have imaged by negative stain and cryoelectron microscopy the truncated dihydrolipoamide acetyltransferase core (60 subunits; M(r) = 2.7 x 10(6)) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. Using icosahedral particle reconstruction techniques, we determined its structure to 25 A resolution. Although the model derived from the negative stain reconstruction was approximately 20% smaller than the model derived from the frozen-hydrated data, when corrected for the effects of the electron microscope contrast transfer functions, the reconstructions showed excellent correspondence. The pentagonal dodecahedron-shaped macromolecule has a maximum diameter, as measured along the 3-fold axis, of approximately 226 A (frozen-hydrated value), and 12 large openings (approximately 63 A in diameter) on the 5-fold axes that lead into a large solvent-accessible cavity (approximately 76-140 A diameter). The 20 vertices consist of cone-shaped trimers, each with a flattened base on the outside of the structure and an apex directed toward the center. The trimers are interconnected by 20 A thick "bridges" on the 2-fold axes. These studies also show that the highest resolution features apparent in the frozen-hydrated reconstruction are revealed in a filtered reconstruction of the stained molecule.

Acetyltransferases↗

Structure-function relationships of the yeast fatty acid synthase: negative-stain, cryo-electron microscopy, and image analysis studies of the end views of the structure.

The yeast fatty acid synthase (M(r) = 2.5 x 10(6)) is organized in an alpha 6 beta 6 complex. In these studies, the synthase structure has been examined by negative-stain and cryo-electron microscopy. Side and end views of the structure indicate that the molecule, shaped similar to a prolate ellipsoid, has a high-density band of protein bisecting its major axis. Stained and frozen-hydrated average images of the end views show an excellent concordance and a hexagonal ring having three each alternating egg- and kidney-shaped features with low-protein-density protrusions extending outward from the egg-shaped features. Images also show that the barrel-like structure is not hollow but has a Y-shaped central core, which appears to make contact with the three egg-shaped features. Numerous side views of the structure give good evidence that the beta subunits have an archlike shape. We propose a model for the synthase that has point-group symmetry 32 and six equivalent sites of fatty acid synthesis. The protomeric unit is alpha 2 beta 2. The ends of each of the two archlike beta subunits interact with opposite sides of the two dichotomously arranged disclike alpha subunits. Three such protomeric units form the ring. We propose that the six fatty acid synthesizing centers are composed of two complementary half-alpha subunits and a beta subunit, an arrangement having all the partial activities of the multifunctional enzyme required for fatty acid synthesis.

Fatty Acid Synthases↗

Comparisons of the low-resolution structures of ornithine decarboxylase by electron microscopy and X-ray crystallography: the utility of methylamine tungstate stain and Butvar support film in the study of macromolecules by transmission electron microscopy.

The structure of ornithine decarboxylase (Mr approximately 1.04 x 10(6] from Lactobacillus 30a was investigated by electron microscopy and x-ray crystallography. Electron micrographs showed the structure to be well preserved in methylamine tungstate stain. The molecules interacted little with the Butvar support film, yielding three unique projections: a hexagonal ring (front view) and two rod-shaped projections (edge views). Stereo pairs revealed a novel feature of the Butvar film in that some molecules were suspended in the stain in random orientations. Consequently, the relatedness of the hexagonal ring and the rod-shaped particles could be demonstrated since some particle shapes interconverted when the stage was tilted +/- 45 degrees. The two edge views were related by a 30 degrees rotation about the sixfold axis. Image averaging of the three primary views suggested a dodecamer (point group symmetry 622) composed of two hexameric rings, apparently in an eclipsed configuration. To investigate the structural organization of the complex, the dissociation of the enzyme was studied by electron microscopy. The dissociation process involved the initial breakage of the ring followed by separation of dimers from the ring (one subunit from each of the two hexamers). Thus, the dodecamer forms as a hexamer of dimers rather than a dimer of hexamers. These structural studies were confirmed and extended by x-ray crystallographic analysis. A 4.0-A resolution electron density map revealed two hexameric rings, consisting of six closely associated dimers, tilted approximately 10 degrees with respect to the molecular twofold axis. Electron density projections of the three primary views of the molecule derived from the x-ray data corresponded closely to those obtained from image averaging of the electron microscopy data, thereby establishing in a novel way the reliability of the electron microscopy studies. Methylamine tungstate stain and Butvar support film therefore offer unique advantages for investigating protein structures by electron microscopy.

Image Processing, Computer-Assisted↗