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.
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