Journal of Structural Biology 127, 169–176 (1999) Article ID jsbi.1999.4120, available online at http://www.idealibrary.com on Tetrairidium, a Four-Atom Cluster, Is Readily Visible as a Density Label in Three-Dimensional Cryo-EM Maps of Proteins at 10–25 Å Resolution N. Cheng,* J. F. Conway,* N. R. Watts,* J. F. Hainfeld,† V. Joshi,‡ R. D. Powell,‡ S. J. Stahl,§ P. E. Wingfield,§ and A. C. Steven*,1 *Laboratory of Structural Biology and §Protein Expression Laboratory, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892; †Department of Biology, Brookhaven National Laboratory, Upton, New York 11973; and ‡Nanoprobes Incorporated, Stony Brook, New York 11790 Received December 17, 1998; and in revised form April 2, 1999 interest. In addition to considerations of specificity and efficiency of labelling, the resolution achieved depends primarily on the size of the metal particle and the distance between its center-of-mass (the feature detected) and its point of attachment (the targeted site). For instance, in colloidal gold-based immunolabelling (reviews, Griffiths, 1994; Bendayan, 1995), which is widely used to map the distributions of molecules in cells, the gold particles are typically 50–200 Å in diameter, and there may be up to 200–400 Å between their projected centers and the epitope marked, depending on whether intermediaries such as protein A or secondary antibodies are used. For structural analysis of isolated macromolecules, smaller labels and more precise localizations are needed. In this context, metal clusters derivatized to bind directly to groups such as sulfhydryls or amines are finding a growing number of applications (reviews, Hainfeld, 1996; Hainfeld and Powell, 1997; see other papers in this issue). Nanogold (Hainfeld and Furuya, 1992) has a core of 55–75 or so gold atoms, ⬃14 Å in diameter; in maleimide linkage to cysteine, there is a spacing of ⬃20 Å between its center and the sulfur of the labelled amino acid. Undecagold (Bartlett et al., 1978; Hainfeld, 1989) has 11 atoms in a cluster ⬃8 Å across, and ⬃17 Å separate its center and the targeted residue. Nanogold has sufficient scattering power to be directly visible in micrographs even against a background of negative stain (Wenzel and Baumeister, 1995; Grigori et al., 1997). Undecagold is smaller and only marginally visible in unprocessed micrographs of iceembedded specimens. However, it has been detected in averaged two-dimensional (Crum et al., 1994) and three-dimensional (Milligan et al., 1990; Steinmetz et al., 1998) density maps at 15–30 Å resolution. Typically, the metal cluster of a labelling compound is surrounded by an organic shell that contains the Heavy metal clusters derivatized to bind to designated chemical groups on proteins have great potential as density labels for cryo-electron microscopy. Smaller clusters offer higher resolution and penetrate more easily into sterically restricted sites, but are more difficult to detect. In this context, we have explored the potential of tetrairidium (Ir4 ) as a density label by attaching it via maleimide linkage to the C-terminus of the hepatitis B virus (HBV) capsid protein. Although the clusters are not visible in unprocessed cryo-electron micrographs, they are distinctly visible in three-dimensional density maps calculated from them, even at only partial occupancy. The Ir4 label was clearly visualized in our maps at 11–14 Å resolution of both size variants of the HBV capsid, thus confirming our previous localization of this site with undecagold (Zlotnick, A., Cheng, N., Stahl, S. J., Conway, J. F., Steven, A. C., and Wingfield, P. T., Proc. Natl. Acad. Sci. USA 94, 9556–9561, 1997). Ir4 penetrated to the interior of intact capsids to label this site on their inner surface, unlike undecagold for which labelling was achieved only with dissociated dimers that were then reassembled into capsids. The Ir4 cluster remained visible as the resolution of the maps was lowered progressively to ⬃25 Å. r 1999 Academic Press Key Words: metal cluster; cryo-electron microscopy; molecular marker; three-dimensional image reconstruction; hepatitis B virus INTRODUCTION Heavy metal particles, whose high electron density renders them visible in electron micrographs, have been extensively used to label molecules of 1 To whom correspondence should be addressed at Building 6, Room B2-34, MSC 2717, National Institutes of Health, Bethesda, MD 20892-2717. Fax: (301) 480-7629; E-mail: Alasdair_Steven@nih.gov. 169 1047-8477/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved. 170 CHENG ET AL. functional group required for specific attachment to proteins and also governs its solubility. Nanogold and undecagold have triphenylphosphine shells that give them full diameters of ⬃26 and ⬃20 Å, respectively. However, because this shell poses a steric hindrance on a cluster’s ability to reach potentially labellable sites, it is of interest to have cluster labels that are smaller than undecagold. Two such candidates are the four-atom clusters, tetramercury (Hg4 ) and tetrairidium (Ir4 ). The mercury compound has been used to label the filamentous bacteriophage fd (Lipka et al., 1979), but it was concluded that the Hg was too volatile under the electron beam to be generally suitable as an EM label, and little subsequent use has been made of this reagent. Ir4 was originally developed for X-ray crystallography of large proteins whose native reflections are insufficiently perturbed by single heavy atom derivatives to provide adequate phases (Jahn, 1989; Weinstein et al., 1989). In EM, Ir4 has been visualized in dark-field STEM micrographs (Furuya et al., 1988), but its scope as a protein label was limited by the state of preservation of freeze-dried specimens. Here, we report that Ir4 is a highly effective label for ice-embedded specimens, which retain their native conformations (Adrian et al., 1984). Recently we localized the C-terminus of the hepatitis B virus (HBV) capsid protein by appending a cysteine residue at this site and labelling it with undecagold (Zlotnick et al., 1997). HBV capsids are icosahedral shells of two sizes—280 Å in diameter (90 dimers of 17-kDa subunits) and 320 Å (120 dimers), respectively. Unusually for viral capsids, these shells are perforated with sizable holes whose diameters, at ⬃20 Å, are similar to that of undecagold. Our attempts to label intact capsids were unsuccessful, but dissociated dimers were readily labelled and could be reassembled into capsids. In the density map, undecagold was conspicuously present at a specific site on the inner surface of the capsid; on this basis, we attributed the nonlabelling of intact capsids to failure of the cluster to diffuse through the holes in the capsid wall. In the experiments reported here, we were able to label intact capsids with Ir4, demonstrating the greater penetrating power of this smaller cluster. MATERIALS AND METHODS Preparation of the tetrairidium cluster. To prepare Ir4 (CO) 85P( pC6H4C(O) NHMe) 363(P( p-C6H4C(O)NH Me) 2 ( p-C6H4C(O)NH(CH2 ) 3NH2 )6, and its maleimido derivative. One-hundred milligrams of Ir4 (CO) 12, 69 mg of Tris( p-N-methylcarboxamidophenyl)phosphine, P( p-C6H4C(O)NHMe) 3, and 62 mg of the amino-substituted phosphine, P(p-C6H4C(O)NHMe)2 (p-C6H4C(O)NH(CH2 ) 3NH2 ), were refluxed for 4 h in 50 mL of anhydrous toluene under N2, cooled to room temperature, and then separated by gel filtration on a 500-mL column (LH-20, Pharmacia-Biotech, Piscataway, NJ) eluted with methanol. Fractions with a yellow–orange color were pooled and evaporated to dryness. Fractions with similar UV-Vis and IR spectra were combined. Pure product was characterized using [1H], [13C], and [31P]NMR spectral data. A total of 1.4 mg of this cluster and 11.4 mg (150-fold excess) of N-methoxycarbonylmaleimide were each dissolved in 0.5 mL of methanol, and the two solutions were combined and left at 5°C for 30 min. The maleimido cluster was isolated by gel filtration (LH-20 column, 80 mL, eluted with methanol at 2-mL/min flow rate). Fractions 8–11 (5-mL fractions) were pooled, and the solvent was quickly removed under reduced pressure. Preparation and labelling of capsids. Capsid protein constructs were expressed in Escherichia coli and isolated in a procedure that involves dissociation to dimers, further purification, and reassembly (Wingfield et al., 1995; Zlotnick et al., 1997). All labelling experiments were performed on preformed particles prepared as follows: 2 mL of dimeric protein at 1 mg/mL in 100 mM NaHCO3, 20 mM DTT, pH 9.6, was mixed with 0.4 mL of 500 mM Hepes, 2 M NaCl, pH 5.0, and incubated for 1 h at room temperature. For labelling, the buffer was changed to 20 mM Hepes, 2 mM EDTA, pH 6.9, with a PD10 gel filtration column (Pharmacia-Biotech). The protein concentration was determined by absorbance, using ⑀280 ⫽ 2.95 ⫻ 104 M⫺1 · cm⫺1 (Zlotnick et al., 1997). Fifty nanomoles of tetrairidium monomaleimide dissolved in 200 µL of DMSO was immediately added to 50 nmol of protein in a final volume of 1200 µL. After an overnight incubation at room temperature, MgCl2 was added to a final concentration of 100 mM to alleviate capsid aggregation. Unbound label and unassembled protein were then removed by gel filtration on a 12-mL, 15-cm-long column of CL-6B (Pharmacia-Biotech) equilibrated with 20 mM Hepes, 50 mM MgCl2, pH 8.0. Particles were concentrated to ⬃2 mg/mL by ultrafiltration (Ultrafree-15, Millipore, Bedford, MA). Labelling was quantitated as follows: the concentration of Ir4 was calculated from the absorbance at 320 nm (corrected for the contribution by light scattering alone, estimated from the UV spectrum of the reduced unlabelled particles) using ⑀320 ⫽ 2.55 ⫻ 104 M⫺1 · cm⫺1. Protein concentrations were calculated from the absorbance at 280 nm, corrected for the contribution by Ir4 at this wavelength, using A280/A320 ⫽ 2.08. Cryo-electron microscopy. Capsid samples were frozen in a thin film of vitrified ice suspended over holey carbon films, as described by Zlotnick et al. (1996). Micrographs were recorded at a magnification of ⫻38 000 on a CM200-FEG microscope (Philips, Eindhoven, Netherlands), fitted with a model 626 cryoholder (Gatan, Pleasanton, CA) and operating at 120 keV. Pairs of micrographs at different defocus values were recorded using low-dose techniques. In the focal pairs analyzed in calculating the reconstructions, the first exposure had a defocus value in the range 0.8–1.1 µm, so that the first zeros of the contrast transfer function (CTF) were at spatial frequencies of (17 Å) ⫺1 ⫺ (20 Å) ⫺1, which was increased by 0.4 µm for the second exposure. Calculation of density maps. Micrographs were digitized on an SCAI scanner (Zeiss, Englewood, CO) at 7 µm per pixel, corresponding to 1.8 Å per pixel at the specimen. Particle images were extracted and processed using a semiautomated procedure (Conway et al., 1993). Their orientations were determined by means of the PFT algorithm (Baker and Cheng, 1996), using as starting models preexisting maps of Cp147 capsids: T ⫽ 4 (Conway et al., 1997); T ⫽ 3 (Conway et al., 1998). The CTF was corrected and the focal pairs were combined as described by Conway et al. (1997). The maps were calculated using methodology reviewed by Fuller et al. (1996), including all particles with correlation coefficients of 0.5 or higher for each of the three statistics calculated by the PFT program (Baker & Cheng, 1996). The number of particles included in each reconstruction and the resulting resolution are listed in Table I. TETRAIRIDIUM AS A CRYO-EM DENSITY LABEL 171 RESULTS Labelling HBV Capsids with Ir4. The capsids studied represent variants of the HBV assembly domain expressed in E. coli. The complete assembly domain is 149 residues long. In the native capsid protein, it is coupled to a 34-residue, C-terminal domain that is highly basic and binds RNA in nucleocapsid assembly. The wildtype assembly domain has cysteines at positions 48, 61, and 107 that are replaced by alanines in the 149-residue construct, Cp*149. Cp*150 has an additional residue, a cysteine, appended at the C-terminus. Labelling was performed by adding Ir4 (Fig. 1) dissolved in DMSO to preassembled, freshly reduced capsids at a 1:1 molar ratio of Ir4 clusters:protein monomers and incubating for 12 h at room temperature. At lower temperatures or at Ir4 concentrations much above 50 nmol/mL, the reagent began to precipitate. In early experiments, spectroscopy gave erratic estimates of labelling levels, which were explained by the presence of microprecipitates of Ir4 seen by cryo-EM (e.g., Fig. 2d). Subsequently, precipitation of the reagent was largely alleviated by including up to 20% DMSO or 2-propanol, additives that are well tolerated by most (but not all) proteins. No adverse effects of the DMSO on HBV capsid structure were observed, at least to ⬃11 Å resolution (see below). After the incubation, unbound label was removed TABLE I Summary of Three-Dimensional Reconstructions Resolution (Å) b Sample Total number of particles a FSC FRC T ⫽ 3: Cp ⴱ 149 T ⫽ 3: Cp ⴱ 150 ⫹ Ir4: Expt 1 T ⫽ 4: Cp149 T ⫽ 4: Cp ⴱ 150 ⫹ Ir4: Expt 1 T ⫽ 4: Cp ⴱ 150 ⫹ Ir4: Expt 2 1041 (2) 1096 (4) 1296 (2) 1460 (4) 600 (4) 13.7 14.8 11.3 12.6 14.7 13.2 14.5 10.9 11.5 10.5 a The total number of particles used in each reconstruction is given. The number of focal pairs combined to yield these data is given in parentheses. All particles present were analyzed; of these, all that had all three PFT correlation coefficients (PFT, PRJ, and CMP—see Baker and Cheng, 1996) of 0.5 or higher were included in the reconstruction. This fraction varied from ⬃40 to ⬃80% in different focal pairs. b Resolution was calculated by two criteria—the Fourier shell correlation (FSC, van Heel, 1987) and the Fourier ring correlation coefficient (FRC, Saxton and Baumeister, 1982; Conway et al., 1993). In both cases, the criterion used to define the resolution limit was the frequency at which the correlation dropped below 0.5 for the last time. The relatively large discrepancy between the two measures for the last reconstruction (T ⫽ 4, Expt 2) simply reflects that the correlation hovers about the 0.5 value over a considerable range, i.e., between approx (14 Å)⫺1 and (10 Å)⫺1, in this analysis. FIG. 1. Schematic diagram of the tetrairidium cluster compound used. The diameter of the cluster core is 5 Å and its full diameter, including the organic shell, is ⬃17 Å (Weinstein et al., 1989). The electron density in the metal core is 4.7 electrons/Å3. The average Ir–Ir bond distance is expected to be 2.736 Å. Although the Ir atom at the apex of the cluster is directly bonded to six atoms or ligands, it satisfies the 18-electron rule (as applied to coordinate-covalent bonds in organometallic clusters), as do the other three iridium atoms (Ros et al., 1986). by gel filtration and capsid labelling was assessed by measuring absorbance by Ir4 at 320 nm and by protein at 280 nm. These data reproducibly indicated a labelling efficiency of ⬃11% for Cp*150 under the conditions used. Nonspecific binding, estimated by treating Cp*149 capsids—which have no cysteines—in the same way, was found to be less than 0.5%. Cryo-EM of Ir4-labelled capsids. Micrographs of labelled and unlabelled capsids are compared in Fig. 2a and Figs. 2b and 2c, respectively. There is no clear-cut difference between them, and we conclude that individual Ir4 clusters are not directly visible in cryo-micrographs. The images shown were recorded quite close to focus (⬃0.8 µm), conditions that we 172 CHENG ET AL. considered to be conducive to visualizing the small (5 Å) metal clusters while still conveying the capsids, albeit with relatively low contrast. The clusters were no more visible at other defocus values used, up to ⬃2 µm (data not shown). Density maps of Ir4-labelled capsids. In an experiment in which 11% labelling of Cp*150 was obtained, density maps were calculated for both the T ⫽ 4 and the T ⫽ 3 capsids to 12 and 14 Å resolution, respectively (Table I, Fig. 3). In both cases, conspicuous focal densities are present beneath the fivefold and quasi-sixfold lattice sites. These densities are absent from Cp*149 control capsids that were subjected to the same labelling reaction (cf., Fig. 3). This localization of the Ir4 clusters and, consequently, of the C-termini of the capsid protein confirms the result previously obtained by visualizing undecagold bound to Cp*150 at ⬃20 Å resolution (Zlotnick et al., 1997). Consistent results were obtained for a T ⫽ 4 capsid reconstruction in an earlier labelling experiment in which the DMSO was omitted (Table I, cf., Figs. 4a and 4b). Although the labelling efficiency of the latter experiment could not be determined reliably by spectroscopy (see above), it must have been very similar to that of the experiment illustrated in Fig. 3, to judge by the intensity of the clusterassociated densities in the respective maps. Dependence of cluster visibility on resolution. To estimate the minimum resolution required to visualize Ir4 labels by cryo-EM, we progressively lowered the resolution of these density maps. As shown in Fig. 4, the cluster-associated density becomes progressively more diffuse as the resolution falls, but it remains significantly above the level of residual background noise to at least 25 Å resolution. DISCUSSION FIG. 2. Cryo-micrographs, recorded relatively close to focus (⌬f ⬃0.9 µm), of (a) Ir4-labelled Cp*150 capsids and (b, c) unlabelled capsids. The two controls are (b) Cp*149 capsids that were subjected, with negative results, to the labelling protocol and (c) Cp147 capsids that were not so treated. Also shown (d), attached to a carbon film, are microprecipitates of Ir4 clusters formed when the labelling reaction was attempted in the absence of DMSO, which are large enough to be directly visible. Bar ⫽ 500 Å. Visibility of tetrairidium as a density label. Hitherto, undecagold is the smallest metal cluster to have been used as a density label in cryo-EM. Ir4 is significantly smaller (2 vs 6.5 kDa, 4 vs 11 heavy atoms). Although it was essentially invisible in micrographs covering a considerable range of defocus values (Fig. 2), Ir4 was clearly visualized in threedimensional density maps, even at incomplete occupancy and moderate resolution (Figs. 3 and 4), reflecting the large improvement in signal-to-noise ratio in such a density map compared to the original projection images. We reproducibly obtained a labelling stoichiometry of ⬃11% for Ir4 relative to the capsid protein under our conditions of incubation, corresponding to ⬃26 clusters per T ⫽ 4 capsid and ⬃20 clusters per T ⫽ 3 capsid. However—as with undecagold (Zlot- TETRAIRIDIUM AS A CRYO-EM DENSITY LABEL 173 FIG. 4. Effect of progressively lowering the resolution on the visibility of the Ir4 cluster label on the T ⫽ 4 Cp*150 capsid. (a, b) Reconstructions from independent labelling experiments at a resolution of ⬃11 Å (see Table I). (d, e, f ) The resolution of reconstruction a was lowered to (d) 15 Å, (e) 20 Å, and (f ) 25 Å, respectively. (c) A 25-Å resolution reconstruction based on 100 particles (cf., the 1460 particles used for the reconstructions in a, d, e, and f ), whose statistical properties (residual noise level) is more typical of a density map at this resolution. Bar ⫽ 50 Å. nick et al., 1997)—the bound clusters are located on the fivefold and quasi-sixfold axes, where occupancy by one cluster blocks the binding of clusters to the other four (or five) subunits surrounding the lattice point. Thus, the maximum binding to be expected is 42 (T ⫽ 4) and 32 (T ⫽ 3) clusters per capsid, so that the binding obtained represents about 60% of maximum. As visualized (Figs. 3, 4a, and 4b), the 5 Ådiameter clusters appear considerably larger than expected; in fact, their full width at half-height is ⬃20 Å. Only part of this broadening may be attributed to the limited resolution (11 Å), because a step function 5 Å across has a full width at a half-height of ⬃9 Å when band-limited to this resolution. The remainder of the broadening must arise from other effects, such as local disorder of the C-termini, or slight displacement of each cluster from the adjacent symmetry axis in a direction that depends on which of the surrounding subunits it is bound to. The latter effect would give rise to averaging-related smearing of the cluster density as depicted in the reconstruction. Initially, we were surprised that a label as small as Ir4 should be so conspicuous under these conditions of observation (Fig. 3). However, the difference in specific density between the cluster and the adjacent protein (⌬ ⬃21.5 ⫽ 22.8 vs 1.3) is so much higher than that between protein and vitreous ice (⌬ ⬃0.35 ⫽ 1.3 vs 0.95) that it remained distinctly visible in the map, despite attenuation by partial occupancy and delocalization arising from averaging (see above). We anticipate that Hg4, which is similar to Ir4 in electron scattering power, should also be detectable under similar conditions, i.e., in 3D density maps of ice-embedded specimens imaged at low electron doses. Resolution of metal cluster labels. The resolution attainable with these markers depends more on the spacing between the center of the cluster-associated density and the labelled residue than it does on the 174 CHENG ET AL. FIG. 3. Density maps of Ir4-labelled and unlabelled HBV capsids. (Top) T ⫽ 4 capsids of the labelled Cp*150 construct and the control Cp*149 construct. The small black arrowhead (middle panel, top row) points to the organic linker connecting the Ir4 cluster to the capsid protein. (Bottom) A similar comparison for the corresponding T ⫽ 3 capsids. For each density map, isodensity renderings of the outer and inner surfaces are shown, together with a central section depicting local variations in density. The capsids are viewed along a twofold symmetry axis and are contoured to enclose 100% of expected mass. The specifics of the reconstructions are given in Table I. For both capsids, labels are visible at corresponding sites on their inner surfaces. Bar ⫽ 50 Å. TETRAIRIDIUM AS A CRYO-EM DENSITY LABEL FIG. 5. Distribution of protein subunits over the surface lattice of the HBV T ⫽ 4 capsid, adapted from Zlotnick et al. (1997). The symmetry axes are marked and the four quasiequivalent subunits (A, B, C, D) are distinguished. The building blocks are A–B and C–D dimers. Because six quasiequivalent subunits are clustered around the twofold axis, it is referred to (Discussion) as the quasi-sixfold axis. size of the cluster. With undecagold, this spacing is ⬃17 Å, and with Ir4, it is slightly less, at ⬃15 Å. However, it is possible to achieve more precise localizations than these figures imply because, under favorable circumstances, the organic linker connecting the cluster to the labelled protein may be visualized (arrowhead in Fig. 3, middle panel, top row; see also Fig. 4 of Zlotnick et al., 1997). Extrapolating an appropriate distance along this vector from the cluster center should give a precise localization of the residue of interest, i.e., to within ⫾5 Å or so. While clusters as small as Ir4 are readily visible in density maps at 25–30 Å resolution (cf., Fig. 4), one advantage of extending their resolution to 10–15 Å is enhanced prospects of visualizing this linker and, consequently, of effecting a more precise localization of the targeted residue. Although shifting to progressively smaller clusters does not bring a major gain in resolution, these reagents have the advantage of being less susceptible to steric constraints that may limit their reactivity. The relatively bulky organic shell surrounding the cluster prevents it from accessing confined spaces in much the same way that an atomic force microscope is limited in the vertical dimension by the size of its tip. Thus, smaller clusters have superior penetrating power, as illustrated by the observed positive Ir4 labelling of a residue inside HBV capsids 175 that is inaccessible to undecagold. An alternative approach to this problem of steric impedance may be to engineer variants of undecagold or Ir4 with longer linkers between the relatively bulky label and the functional group that may be better able to reach into restricted sites, albeit at possible cost in terms of resolution (see above). Preferential labelling of B-subunits in capsids assembled from labelled dimers; implications for regulation of capsid assembly. In the T ⫽ 4 capsid, pairs of three nonequivalent subunits surround the quasi-sixfold axis. They are called the B-, C-, and D-subunits, respectively (Fig. 5). For capsids assembled from dimers labelled in solution with undecagold, it was observed that the linker connecting the cluster to the B-subunit was markedly more visible than that for the C- and D-subunits, implying that the single cluster per quasi-sixfold site was preferentially associated with a B-subunit (Zlotnick et al., 1997). Although this linker is also discernible in our map of preformed capsids labelled with Ir4 (Figs. 3 and 4), the preference for B-subunits is much less pronounced. This distinction may reflect a feature of the assembly pathway; for instance, binding of the cluster to the C-termini of unassembled dimers might switch them into a state of enhanced receptivity for binding other dimers, leading to a cooperative aggregation around a local symmetry axis, and in so doing, may mimic an effect of RNA binding to the C-terminal domain in nucleocapsid assembly. If the symmetry axis in question were fivefold, this would explain the preferential labelling of B-subunits, with additional dimers subsequently adding on to connect the resulting pentamers, thereby completing the formation of either a T ⫽ 4 or a T ⫽ 3 capsid. Prospects for mapping the 3D structures of protein complexes by cysteine scanning and cluster labelling. Although ample scope remains for further development of clusters with other functionalities, the reaction of maleimide with the sulfhydryl of cysteine has been found to work well with undecagold (Hainfeld, 1988), Nanogold (Hainfeld and Furuya, 1992), and Ir4 (Jahn, 1989). In addition to naturally occurring cysteines, current molecular biology techniques readily allow the insertion or substitution of this amino acid at virtually any site of interest. In principle, therefore, the possibility already exists to use cluster labelling to delineate the overall path of a polypeptide chain in any protein complex amenable to analysis by cryo-EM. Given enough constraints of this kind, it should then be possible to fold in information on conformational preferences of specific peptides, secondary structure predictions, and other data such as intramolecular cross-links to build detailed molecular models, starting from cryo-EM density maps in the 10 Å-resolution range 176 CHENG ET AL. (Böttcher et al., 1997; Conway et al., 1997; Trus et al., 1997). We thank Drs. A. Zlotnick, D. Belnap, and B. Trus for helpful discussions and Dr. T. Baker (Purdue) for software. REFERENCES Adrian, M., Dubochet, J., Lepault, J., and McDowall, A. W. (1984) Cryo-electron microscopy of viruses, Nature 308, 32–36. Baker, T. S., and Cheng, R. H. (1996) A model-based approach for determining orientations of biological macromolecules imaged by cryoelectron microscopy, J. Struct. Biol. 116, 120–130. Bartlett, P. A., Bauer, B., and Singer, S. J. (1978) Synthesis of water-soluble undecagold cluster compounds of potential importance in electron microscopic and other studies of biological systems, J. Am. Chem. Soc. 100, 5085–5092. Bendayan, M. (1995) Colloidal gold post-embedding immunocytochemistry, Prog. Histochem. Cytochem. 9, 1–25. Böttcher, B., Wynne, S. A., and Crowther, R. A. (1997) Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy, Nature 386, 88–91. Conway, J. F., Trus, B. L., Booy, F. P., Newcomb, W. W., Brown, J. C., and Steven, A. C. (1993) The effects of radiation damage on the structure of frozen hydrated HSV-1 capsids, J. Struct. Biol. 111, 222–233. Conway, J. F., Cheng, N., Zlotnick, A., Wingfield, P. T., Stahl, S. J., and Steven, A. C. (1997) Visualization of a 4-helix bundle in the hepatitis B virus capsid by cryo-electron microscopy, Nature 386, 91–94. Conway, J. F., Cheng, N., Zlotnick, A., Stahl, S. J., Wingfield, P. T., and Steven, A. C. (1998) Localization of the N-terminus of hepatitis B virus capsid protein by peptide-based difference mapping from cryoelectron microscopy, Proc. Natl. Acad. Sci. USA 95, 14622–14627. Crum, J., Gruys, K. J., and Frey, T. G. (1994) Electron microscopy of cytochrome c oxidase crystals: Labeling of subunit III with a monomaleimide undecagold cluster compound, Biochemistry 33, 13719–13726. Fuller, S. D., Butcher, S. J., Cheng, R. H., and Baker, T. S. (1996) Three-dimensional reconstruction of icosahedral particles: ‘‘The uncommon line,’’ J. Struct. Biol. 116, 48–55. Furuya, F. R., Miller, L. L., Hainfeld, J. F., Christopfel, W. C., and Kenny, P. W. (1988) Use of Ir4 (CO) 11 to measure the lengths of organic molecules with a scanning transmission electron microscope, J. Am. Chem. Soc. 110, 641–643. Griffiths, G. (1994) Fine Structure Immunocytochemistry, Springer-Verlag, Berlin. Gregori, L., Hainfeld, J. F., Simon, M. N., and Goldgaber, D. (1997) Amyloid beta-protein inhibits ubiquitin-dependent protein degradation in vitro, J. Biol. Chem. 272, 58–62. Hainfeld, J. F. (1989) Undecagold-antibody method, in Hayat, M. A. (Ed.), Colloidal Gold: Principles, Methods, and Applications., Vol. 2, pp. 413–429, Academic Press, San Diego. Hainfeld, J. F. (1992) Site specific cluster labels, Ultramicroscopy 46, 135–144. Hainfeld, J. F., and Furuya, F. R. (1992) A 1.4-nm gold cluster covalently attached to antibodies improves immunolabeling, J. Histochem. Cytochem. 40, 177–184. Hainfeld, J. F. (1996) Labeling with Nanogold and undecagold: Techniques and results, Scanning Microsc. Suppl. (Proc. 14th Pfefferkorn Conf.) 10, 309–322. [Malecki, M., and Roomans, G. M. (Eds.), Scanning Microscopy Int’l, Chicago]. Hainfeld, J. F., and Powell, R. D. (1997) Nanogold technology: New frontiers in gold labeling, Cell Vision 4, 408–432. Jahn, W. (1989) Synthesis of water soluble tetrairidium clusters suitable for heavy atom labelling of proteins, Z. Naturforsch. B 44, 79–82. Lipka, J. J., Lippard, S. J., and Wall, J. S. (1979) Visualization of polymercuri-methane-labeled fd bacteriophage in the scanning transmission electron microscope, Science 206, 1419–1421. Milligan, R. A., Whittaker, M., and Safer, D. (1990) Molecular structure of F-actin and location of surface binding sites, Nature 348, 217–221. Ros, R., Scrivanti, A., Albano, V. G., Braga, D., and Garlaschelli, L. (1986) Chemistry of tetrairidium carbonyl clusters. Part 1. Synthesis, chemical characterization, and nuclear magnetic resonance study of mono- and di-substituted phosphine derivatives. X-Ray crystal structure determination of the diaxial isomer of [Ir4 (CO) 7 (µ-CO) 3 (Me2PCH 2CH2PMe2 )], J. Chem. Soc. Dalton Trans. 2411–2421. Saxton, W. O., and Baumeister, W. (1982) The correlation averaging of a regularly arranged bacterial cell envelope protein, J. Microsc. (Oxford) 127, 127–138. Steinmetz, M. O., Stoffler, D., Muller, S. A., Jahn, W., Wolpensinger, B., Goldie, K. N., Engel, A., Faulstich, H., and Aebi, U. (1998) Evaluating atomic models of F-actin with an undecagoldtagged phalloidin derivative, J. Mol. Biol. 276, 1–6. Trus, B. L., Roden, R. B., Greenstone, H. L., Vrhel, M., Schiller, J. T., and Booy, F. P. (1997) Novel structural features of bovine papillomavirus capsid revealed by a three-dimensional reconstruction to 9 Å resolution, Nat. Struct. Biol. 4, 413–420. Van Heel, M. (1987) Similarity between images, Ultramicroscopy 21, 95–100. Weinstein, S., Jahn, W., Hansen, H., Wittmann, H. G., and Yonath, A. (1989) Novel procedures for derivatization of ribosomes for crystallographic studies, J. Biol. Chem. 264, 19138– 19142. Wenzel, T., and Baumeister, W. (1995) Conformational constraints in protein degradation by the 20S proteasome, Nat. Struct. Biol. 2, 199–204. Wingfield, P. T., Stahl, S. J., Williams, R. W., and Steven, A. C. (1995) Hepatitis core antigen produced in Escherichia coli: Subunit composition, conformational analysis, and in vitro capsid assembly, Biochemistry 34, 4919–4932. Zlotnick, A., Cheng, N., Conway, J. F., Booy, F. P., Steven, A. C., Stahl, S. J., and Wingfield, P. T. (1996) Dimorphism of hepatitis B virus capsids is strongly influenced by the C-terminus of the capsid protein, Biochemistry 35, 7412–7421. Zlotnick, A., Cheng, N., Stahl, S. J., Conway, J. F., Steven, A. C., and Wingfield, P. T. (1997) Localization of the C terminus of the assembly domain of hepatitis B virus capsid protein: Implications for morphogenesis and organization of encapsidated RNA, Proc. Natl. Acad. Sci. USA 94, 9556–9561.