Radial density profiles of macromolecular filaments determined from dark-field scanning transmission electron micrographs. Improvements in technique and some applications.
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Cytoskeleton↗
Biomedical subjects
J F Hainfeld
Publications and source records attributed to J F Hainfeld.
Mass mapping with the scanning transmission electron microscope.
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Macromolecular Substances↗
Quaternary structure of pyruvate dehydrogenase complex from Escherichia coli.
The pyruvate dehydrogenase complex of Escherichia coli and subcomplexes derived from it by selective removal of component enzymes have been subjected to quaternary structural analysis by scanning transmission electron microscopy. Scanning transmission electron microscopic images of the intact complex (E1E2E3), the dihydrolipoyl transacetylase-dihydrolipoyl dehydrogenase (E2E3) subcomplex, and the E2 core enzyme appear as cubic particles in various orientations. Mass distributions within this complex and its subcomplexes have been determined by radial mass analysis of similarly oriented scanning transmission electron microscopic images of each type. The data show that mass attributable to dihydrolipoyl dehydrogenase (E3) is well integrated into the structural framework of the E2 core, dihydrolipoyl transacetylase, whereas mass attributable to pyruvate dehydrogenase (E1) is located about the periphery of the core enzyme. The mass distributions are fully consistent with a structural model in which 6 E3 dimers are integrated into the six faces of the cubic E2 core, and 12 E1 dimers are associated along the 12 edges of the core enzyme.
Escherichia coli↗
Escherichia coli pyruvate dehydrogenase complex: particle masses of the complex and component enzymes measured by scanning transmission electron microscopy.
Particle masses of the Escherichia coli pyruvate dehydrogenase (PDH) complex and its component enzymes have been measured by scanning transmission electron microscopy (STEM). The particle mass of PDH complex measured by STEM is 5.28 X 10(6) with a standard deviation of 0.40 X 10(6). The masses of the component enzymes together with their standard deviations are (2.06 +/- 0.26) X 10(5) for the dimeric pyruvate dehydrogenase (E1), (1.15 +/- 0.17) X 10(5) for dimeric dihydrolipoyl dehydrogenase (E3), and (2.20 +/- 0.17) X 10(6) for dihydrolipoyl transacetylase (E2), the 24-subunit core enzyme. The latter value corresponds to a subunit molecular weight of (9.17 +/- 0.71) X 10(4) for E2. The subunit molecular weight measured by polyacrylamide gel electrophoresis in sodium dodecyl sulfate is 8.6 X 10(4). STEM measurements on PDH complex incubated with excess E3 or E1 failed to detect any additional binding of E3 but showed that the complex would bind additional E1 under forcing conditions (high concentrations with glutaraldehyde). The additional E1 subunits were bound too weakly to represent binding sites in an isolated or isolable complex. The mass measurements by STEM are consistent with the subunit composition 24:24:12 when interpreted in the light of the flavin content of the complex and assuming 24 subunits in the core enzyme (E2).
Acetyltransferases↗
Monomers through trimers of large tumor antigen bind in region I and monomers through tetramers bind in region II of simian virus 40 origin of replication DNA as stable structures in solution.
Large tumor (T) antigen and its bound multimeric states are positioned by scanning transmission electron microscopy (STEM) within a few base pairs at control sequences of the simian virus 40 DNA origin of replication region. Proximal and distal edge positions for each multimer group match the end positions of previously mapped fragments protected from DNase cleavage. Since chance correspondence is shown to be extremely unlikely, STEM mass measurements, obtained concurrently with STEM map positions, indicate that the DNase fragments arise from bound monomers, dimers, trimers, and tetramers in binding region II and monomers, dimers, and trimers in binding region I. Simultaneous binding of seven monomer-equivalent masses is observed, three in region I and four in region II, with an ordered and interpretable mass distribution in the plane of the foil. Although this observation does not prove that the six G-A-G-G-C and one T-A-G-G-C sequences, similarly distributed, function as recognition sequences for T-antigen monomer, it provides strong support for such a model. The stable existence in solution of low-and intermediate-mass structures, observed at lower T-antigen concentrations, suggests a role as assembly intermediates.
Antigens, Polyomavirus Transforming↗
Mass distribution of a probable tail-length-determining protein in bacteriophage T4.
Analysis of dark-field scanning transmission electron micrographs of unstained freeze-dried specimens established that the interior of the intact bacteriophage T4 tail tube contains extra density that is missing in tubes artificially emptied by treatment with 3 M guanidine hydrochloride. The mass of the tail tube is 3.1 X 10(6) daltons, and the central channel is 3.2 nm in diameter. Quantitative analysis of the density data is consistent with the presence of up to six strands of a protein molecule in the central channel that could serve as the template or ruler structure that determines the length of the bacteriophage tail and that could be injected into the cell with the phage DNA.
Escherichia coli↗
Quantitative structural analysis of eukaryotic ribosomal RNA by scanning transmission electron microscopy.
The conformation of 28S ribosomal RNA isolated from baby hamster kidney cells was studied by scanning transmission electron microscopy (STEM) and circular dichroic spectroscopy to establish the conditions under which STEM images of unstained freeze-dried rRNA are a meaningful representation of the conformation of rRNA in solution. We have determined the conformation of 28S rRNA under various buffer conditions, the molecular mass, the mass distribution, and the number of polynucleotide strands within the individual molecules, and the apparent radii of gyration. The 28S rRNA molecule is highly extended in water and becomes compact with increasing ionic strength. However, even in the "reconstitution buffer" (30 mM Tris/HCl/20 mM MgCl2/360 mM KCl, pH 7.6) the compactness does not reach a state in which the rRNA molecule appears structurally similar to the 60S ribosomal subunit. Our approach has a broad application in high-resolution structural studies of nucleic acids and nucleic acid-protein interactions.
Animals↗
Conformity and diversity in the structures of intermediate filaments.
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Animals↗
Mass and molecular composition of vesicular stomatitis virus: a scanning transmission electron microscopy analysis.
Dark-field scanning transmission electron microscopy was used to perform mass analyses of purified vesicular stomatitis virions, pronase-treated virions, and nucleocapsids, leading to a complete self-consistent account of the molecular composition of vesicular stomatitis virus. The masses obtained were 265.6 +/- 13.3 megadaltons (MDa) for the native virion, 197.5 +/- 8.4 MDa for the pronase-treated virion, and 69.4 +/- 4.9 MDa for the nucleocapsid. The reduction in mass effected by pronase treatment, which corresponds to excision of the external domains (spikes) of G protein, leads to an average of 1,205 molecules of G protein per virion. The nucleocapsid mass, after compensation for the RNA (3.7 MDa) and residual amounts of other proteins, yielded a complement of 1,258 copies of N protein. Calibration of the amounts of M, NS, and L proteins relative to N protein by biochemical quantitation yielded values of 1,826, 466, and 50 molecules, respectively, per virion. Assuming that the remaining virion mass is contributed by lipids in the viral envelope, we obtained a value of 56.1 MDa for its lipid content. In addition, four different electron microscopy procedures were applied to determine the nucleocapsid length, which we conclude to be 3.5 to 3.7 micron. The nucleocapsid comprises a strand of repeating units which have a center-to-center spacing of 3.3 nm as measured along the middle of the strand. We show that these repeating units represent monomers of N protein, each of which is associated with 9 +/- 1 bases of single-stranded RNA. From scanning transmission electron microscopy images of negatively stained nucleocapsids, we inferred that N protein has a wedge-shaped, bilobed structure with dimensions of approximately 9.0 nm (length), approximately 5.0 nm (depth), and approximately 3.3 nm (width, at the midpoint of its long axis). In the coiled configuration of the in situ nucleocapsid, the long axis of N protein is directed radially, and its depth corresponds to the pitch of the nucleocapsid helix.
Capsid↗
Studies on the structure of bovine factor V by scanning transmission electron microscopy.
We studied purified bovine factor V (mol wt 330,000) by scanning transmission electron microscopy (STEM) of freeze-dried unstained or negatively contrasted preparations. Freeze-dried molecules revealed discrete shapes ranging from roughly spheroidal (100 to 120 nm) to oblong (140 to 200 nm in length X 50 to 100 nm in width). Oblong shapes could often be resolved into two or three distinct domains, ranging from 60 to 100 nm in diameter. A "satellite" nodular structure (30 to 50 nm in diameter) connected to the main molecule by a thin stalk (approximately 10 nm wide) up to 80 nm in length was occasionally seen. Glutaraldehyde-treated preparations yielded the same shapes as were seen in unfixed preparations but revealed better definition of submolecular features and "satellite" nodules. STEM mass analysis confirmed that each of the different shapes represented a monomolecular form of factor V. Negatively stained images revealed objects having the same general shapes as freeze-dried molecules, although greater detail was evident. Some images suggested that molecules consist of five or more discrete parts. Taken together, these observations indicate that factor V molecules are multidomainal, flexible structures that tend to have an irregular oblong shape with an axial ratio between 3:2 and 2:1.
Animals↗
Methylenetetrahydrofolate reductase. Evidence for spatially distinct subunit domains obtained by scanning transmission electron microscopy and limited proteolysis.
Scanning transmission electron microscopy of individual unfixed molecules of methylenetetrahydrofolate reductase has been used to determine the molecular mass distribution of the protein. Methylenetetrahydrofolate reductase, which has a subunit molecular mass of 77 kilodaltons, was found to exist predominantly as a dimer with an apparent molecular mass of 136 +/- 29 kilodaltons. The mass distribution of the enzyme molecules was unchanged in the presence of the allosteric inhibitor S-adenosylmethionine. Examination of negatively stained protein molecules suggested that each subunit of the dimer consists of two globular domains of approximately equal size. Limited proteolysis of the enzyme by trypsin gave results which were entirely consistent with the presence of two domains per subunit. In the presence of 1% trypsin, the enzyme was cleaved into two fragments. The masses of these fragments were 39 and 36 kilodaltons as assessed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Tryptic cleavage did not lead to loss of NADPH-menadione or NADPH-methylenetetrahydrofolate oxidoreductase activity, and the flavin prosthetic group remained bound to the protein. However, the cleaved protein was completely desensitized with respect to inhibition by S-adenosylmethionine. These results suggest that each subunit of methylenetetrahydrofolate reductase contains two domains and that allosteric inhibition requires specific interactions between these domains. The region between these two domains appears to be very sensitive to proteolysis, while the domains themselves are relatively resistant to further degradation.
5,10-Methylenetetrahydrofolate Reductase (FADH2)↗
Structure of haptoglobin and the haptoglobin-hemoglobin complex by electron microscopy.
The human serum protein, haptoglobin, forms a stable, irreversible complex with hemoglobin. Haptoglobin is composed of two H chains, which are connected via two smaller L chains to give a protein of 85,000 Mr. In the complex, each H chain binds an alpha beta dimer of hemoglobin for a total molecular weight of 150,000. The scanning transmission electron microscope has been used to derive new information about the shape and structure of haptoglobin and hemoglobin, and about their relative orientation in the complex. The micrographs of negatively stained images show that haptoglobin has the shape of a barbell with two spherical head groups, which are the H chains. These are connected by a thin filament with a central knob, which corresponds to the L chains. The overall length of the molecule is about 124(+/- 8) A and the interhead distance is 87 (+/- 7) A. In the haptoglobin-hemoglobin complex, the head groups are ellipsoidal and under optimal staining conditions bilobal . Thus, the alpha beta dimers are binding to the H chains, but off the long axis of the barbell by 127 degrees in a trans configuration. This angle considerably restricts the region on the surface of the H chain structure that can contain the hemoglobin binding site. The interhead group distance for complex is 116.5(+/- 6.3) A or 30 A greater than for haptoglobin. The N terminus of the beta chain was located on the trans off-axis configured barbell structure of complex by using a hemoglobin that was crosslinked between the alpha beta dimers in the region of the beta N terminus. The distances and angles that are measured on the micrographs for the native and crosslinked complex molecules permit the directions of two of the alpha beta dimer ellipsoid axes to be assigned. Taken together, these data provide an approximate relative orientation for the binding of the alpha beta dimer to the H chain of haptoglobin.
Binding Sites↗
Structure and assembly of haptoglobin polymers by electron microscopy.
Haptoglobin (Hp) consists of light (L) and heavy (H) chains, the latter of which combine with hemoglobin alpha beta dimers to form a highly stable complex. Human haptoglobin assembles as HL units that occur in two allelic forms; HL1 , which is monovalent, and HL2 , which is divalent. As a result, three phenotypic forms exist in the human population: Hp1-1, the homozygous form in which the monovalent HL1 unit occurs as a dimer; Hp2-2, the homozygous form of the divalent HL2 unit, which gives a series of polymers; and the heterozygous Hp2-1 form, which gives a different series of polymers. We have investigated the structures and assembly properties of these two haptoglobin polymeric series in their complexes with hemoglobin using high-resolution scanning transmission electron microscopy. Polymers of complex are composed of ellipsoidal or bilobal head groups, which are the H alpha beta subunits connected by thin filament-like structures, which are the L chains. Polymers of size up to pentamers can be identified easily by counting the number of head groups in the molecule. Complex 2-1 and complex 2-2 trimers were studied extensively. The differences in detailed morphology show that while the 2-1 trimer is a linear polymer, the 2-2 trimer is a closed circular molecule. The micrograph images suggest that complex 2-2 tetramers and pentamers, and perhaps higher forms may also be cyclic. The structure of the L2 subunit of haptoglobin is shown to be composed of two domains, which may be similar in structure to the single domain of the monovalent L1 chain. The two L2 domains are connected by a hinge that has quite limited flexibility. Using these structural models, assembly characteristics and structural properties of the trimers and tetramers of complex 2-1 and complex 2-2 are described.
Biopolymers↗
Radial distributions of density within macromolecular complexes determined from dark-field electron micrographs.
A procedure has been developed for direct determination of radial distributions of density in filamentous and spheroidal particles by analyzing dark-field scanning transmission electron micrographs of unstained freeze-dried specimens. Unlike electron microscopic methods based on staining or shadowing with heavy atoms, this approach can be used to probe the internal structure of macromolecular complexes. As an experimental proving ground, we have applied the procedure to tobacco mosaic virus (TMV) and to RNA-free helical polymers of TMV coat protein. Both structures are found to project outermost diameters of 17.6 +/- 0.4 nm, to have empty axial holes approximately equal to 3.5 nm in diameter, and to have density peaks at radii of 2.5 +/- 0.5 and 6.7 +/- 0.3 nm. Thus visualized, the only significant difference between them is the presence in the virion of an additional density peak at 4.1 +/- 0.5 nm contributed by its internalized RNA molecule. We have also used the procedure to monitor the structural expression of radiation damage in the low electron dose regime prior to the onset of significant mass loss. Changes in the radial density profiles are detected at average doses as low as approximately equal to 400 electrons per nm2: the trend is for the internal structure of these particles to fuse toward a state of uniform density, although the values of their outermost diameters remain unaffected.
Densitometry↗
The distribution of mass in heteropolymer intermediate filaments assembled in vitro. Stem analysis of vimentin/desmin and bovine epidermal keratin.
We have studied the distribution of mass in several types of intermediate filaments (IF) assembled in vitro, by analyzing scanning transmission electron micrographs (STEM) of unstained specimens imaged in dark-field mode. Bovine epidermal keratin IF, which are obligate heteropolymers, were thus characterized and compared with facultative heteropolymers of vimentin and desmin and with earlier observations of homopolymer vimentin IF. The major components of each type of IF have linear densities of 37 kilodaltons/nm. Minor polymorphic variants are also present in each case, with linear densities of approximately 25 and 48 kilodaltons/nm, respectively. In view of the known subunit masses, these results are consistent with the proposition that at least three IF types (bovine epidermal keratin, vimentin, and desmin) are structurally homologous. For each type of IF studied, measurements taken near the ends of long IF or from short IF tend to have lower densities, characteristic of the least dense polymorphic variant. This form may represent a precursor "minimal core" structure in the sense of a minimal aggregate of protofilaments sufficiently stable to permit elongation. In the course of in vitro assembly, this form would be maturable to the normal IF structure at 37 kilodaltons/nm by the accretion of further protein. We also find the projected widths of these IF as measured from the STEM images to be significantly greater than the corresponding diameters determined in previous studies by conventional electron microscopy. Whereas the conventional techniques have routinely yielded diameters of 7-11 nm for IF contrasted by heavy metal staining, the STEM images of unstained IF provide estimates of 15 +/- 1 nm for the outer diameter of the major density class for each IF type studied and average values of 13.8 and 16.1 nm for the less dense and more dense variants, respectively.
Animals↗
Undecagold labeling of a glycoprotein: STEM visualization of an undecagoldphosphine cluster labeling the carbohydrate sites of human haptoglobin-hemoglobin complex.
A new type of label for electron microscopy has been introduced recently which consists of 11 gold atoms in a compact stable cluster with an organic shell composed of primary amine-substituted phosphine ligands. The radius of the cluster is about 10 A. The (phosphine ligand) amines can be derivatized or allowed to react directly forming covalent bonds to specific sites of other molecules. This report describes the specific labeling of carbohydrate moietis on the glycoprotein human haptoglobin (Hp) in the haptoglobin-hemoglobin complex (Hp X Hb). The Hp X Hb complex is easily recognized in the EM as a barbell-shaped molecule. Only the Hp portion contains carbohydrate (eight carbohydrate chains per Hp X Hb). The carbohydrate moieties of the Hp X Hb complex were oxidized by sodium periodate to produce aldehydes. The primary amines on the undecagold cluster were allowed to react with the aldehyde residues to produce Schiff's base linkages which were subsequently reduced with sodium borohydride. Micrographs obtained on the Brookhaven National Laboratory high-resolution scanning transmission electron microscope (STEM) showed the undecagold label to be localized in a region known to be occupied by the heavy chains of haptoglobin. The amount of labeling was found to be two to four gold clusters per molecule when excess label was reacted. The variation in position of the label is discussed and may be due to flexibility of the carbohydrate chains. Control experiments ruled out nonspecific binding of the gold cluster to the Hp X Hb. The high chemical specificity of the reaction and the high resolution of the gold cluster should make this new label of widespread value in studies of other glycoproteins or carbohydrate-bearing molecules.
Affinity Labels↗
Solution and surface effects on plasma fibronectin structure.
As assessed by electron microscopy, the reported shape of the plasma fibronectin molecule ranges from that of a compact particle to an elongated, rod-like structure. In this study, we evaluated the effects of solution and surface conditions on fibronectin shape. Freeze-dried, unstained human plasma fibronectin molecules deposited at pH 7.0-7.4 onto carbon films and examined by scanning transmission electron microscopy appeared relatively compact and pleiomorphic, with approximate average dimensions of 24 nm X 16 nm. Negatively stained molecules also had a similar shape but revealed greater detail in that we observed irregular, yarn-like structures. Glutaraldehyde-induced intramolecular cross-linking did not alter the appearance of plasma fibronectin. Molecules deposited at pH 2.8, pH 9.3, or after succinylation were less compact than those deposited at neutral pH. In contrast, fibronectin molecules sprayed onto mica surfaces at pH 7, rotary shadowed, and examined by transmission electron microscopy were elongated and nodular with a contour length of 120-130 nm. Sedimentation velocity experiments and electron microscopic observations indicate that fibronectin unfolds when it is succinylated, when the ionic strength is raised at pH 7, or when the pH is adjusted to 9.3 or 2.8. Greater unfolding is observed at pH 2.8 at low ionic strength (less than 0.01) compared with material at that pH in 0.15 M NaCl solution. We conclude that (a) the shape assumed by the fibronectin molecule can be strongly affected by solution conditions and by deposition onto certain surfaces; and that (b) the images of fibronectin seen by scanning transmission electron microscopy at neutral pH on carbon film are representative of molecules in physiologic solution.
Electrophoresis, Polyacrylamide Gel↗
Mass distributions of coated vesicles isolated from liver and brain: analysis by scanning transmission electron microscopy.
Populations of coated vesicles purified from bovine brain (BCV) and from rat liver (LCV) have been characterized with respect to the parameters of mass and diameter by analysis of scanning transmission electron micrographs of unstained specimens. Coated vesicles from both sources are heterogeneous, particularly in their masses. The respective distributions, compiled from mass measurements of many individual particles, are complex and markedly different. BCV range from 20 Mdaltons to approximately 100 Mdaltons with a weighted average of 35 Mdaltons: most BCV (80%) lie between 20 and 40 Mdaltons, including peaks at approximately 26 Mdaltons and at approximately 34 Mdaltons. In contrast, LCV masses tend to be substantially higher, ranging from 20 to 220 Mdaltons with a weighted average of 66 Mdaltons. There is a prominent subpopulation at approximately 35 Mdaltons, and 59% of all LCV belong to a broad peak between 50 and 120 Mdaltons. The Kolmogorov-Smirnov distribution-free test was used to affirm the statistical reproducibility of these isolates. BCV diameters vary from 50 to 90 nm, and those of LCV from 50 to 150 nm. Both protein compositions, determined by SDS PAGE, are dominated by clathrin and they are generally similar, except that corresponding secondary bands, notably the clathrin-associated light chains, appear to have lower molecular weights in the case of LCV. From consideration of the joint mass-diameter distribution, it is apparent that coated vesicles of a given diameter vary considerably in mass and that this variation is due primarily to widely differing amounts of material enclosed within the clathrin coat.
Animals↗