Insights Into Bunyavirus Architecture from Electron Cryotomography of Uukuniemi Virus

Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus

A. K. Overby¨ †‡, R. F. Pettersson†, K. Gru¨ newald§, and J. T. Huiskonen§¶ʈ

†Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institute, P.O. Box 240, S-17177 Stockholm, Sweden; §Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany; and ¶Institute of Biotechnology, University of Helsinki, P.O. Box 65, FI-00014, Helsinki, Finland

Edited by Michael G. Rossmann, Purdue University, West Lafayette, IN, and approved December 21, 2007 (received for review September 14, 2007) Bunyaviridae is a large family of viruses that have gained attention as translation and mature to form heterodimers. The glycosylated ‘‘emerging viruses’’ because many members cause serious disease in dimers are transported to the Golgi, were they are retained until humans, with an increasing number of outbreaks. These negative- budding occurs (8). Most enveloped viruses bud from the plasma strand RNA viruses possess a membrane envelope covered by glyco- membrane into the extracellular space; however, bunyavirus virions proteins. The virions are pleiomorphic and thus have not been form by budding into the Golgi (9). Bunyaviruses do not have a amenable to structural characterization using common techniques matrix protein facilitating the interaction between the membrane that involve averaging of electron microscopic images. Here, we and RNPs, as is seen in most negative-sense RNA viruses (10). determined the three-dimensional structure of a member of the Instead, the RNPs interact directly with the cytoplasmic tails of GN Bunyaviridae family by using electron cryotomography. The genome, or GC. The GN cytoplasmic tail has been shown to interact with incorporated as a complex with the nucleoprotein inside the virions, RNPs and to mediate genome packaging (11), and the cytoplasmic was seen as a thread-like structure partially interacting with the viral tails of both GN and GC contain motifs essential for virus assembly membrane. Although no ordered nucleocapsid was observed, lateral (12, 13). From the Golgi, large vesicles filled with numerous viruses interactions between the two membrane glycoproteins determine are transported to the plasma membrane, and the viruses are the structure of the viral particles. In the most regular particles, the released (14). glycoprotein protrusions, or ‘‘spikes,’’ were seen to be arranged on an Uukuniemi virus (UUKV; genus Phlebovirus) is a particularly ,triangulation. This arrangement has convenient model virus for studying bunyaviral packaging, budding 12 ؍ icosahedral lattice, with T not yet been proven for a virus. Two distinctly different spike and structure. Firstly, UUKV is not a human pathogen (15), and conformations were observed, which were shown to depend on pH. virions can be grown to high titers in cell culture and studied in This finding is reminiscent of the fusion proteins of alpha-, flavi-, and low-biosafety-level laboratories. In addition, virus-like particles can influenza viruses, in which conformational changes occur in the low be produced in different compositions, for example, with variable pH of the endosome to facilitate fusion of the viral and host mem- amounts of genome segments, for structural studies (16). This brane during viral entry. tick-borne virus was isolated in Uukuniemi, Finland, in 1964 (17).

The UUKV glycoproteins, GN (75 kDa) and GC (65 kDa) (18), are BIOPHYSICS Bunyaviridae ͉ envelope glycoproteins ͉ ribonucleoprotein particles ͉ responsible for the structural stability of the virus and have been virus assembly ͉ virus structure shown to be sufficient for formation of virus-like particles that are morphologically similar to wild-type UUKV (11). The UUKV he family Bunyaviridae consists of a diverse group of Ͼ350 glycoproteins have been depicted clustered as hollow cylindrical Tviruses and is divided into five genera: Hantavirus, Nairovirus, morphological units (19). The lateral organization of the spikes has Orthobunyavirus, Phlebovirus, and Tospovirus. Most members of the been postulated to be icosahedrally symmetric, with hexameric and ϭ Bunyaviridae family are spread by hematophagous arthopods, such pentameric clusters creating a specific so-called ‘‘T 12’’ arrange- as mosquitoes and ticks. However, members of the genus Hanta- ment (19), but the exact organization and structure of the spikes virus are spread by persistently infected rodents (1). Bunyaviruses have remained obscure. In general, bunyavirus virion structure are distributed worldwide, and many of them are considered constitutes largely uncharted territory because no crystal structure ‘‘emerging viruses,’’ with increasing numbers of outbreaks. In of the two glycoproteins—and no 3D structures of the virions— addition, some members of the family, such as La Crosse virus exist for any of the 350 members of the family. Many family (Orthobunyavirus), Rift Valley fever virus (Phlebovirus), Crimean- members have pleomorphic virion structures (20) and thus have not Congo hemorrhagic fever virus (Nairovirus), and Sin Nombre virus been amenable to structural characterization using the averaging (Hantavirus), cause severe illness in humans, including hemorrhagic techniques commonly used in the study of icosahedrally symmetric fever and encephalitis (2). virions (21). Bunyaviruses are enveloped, spherical viruses (Ϸ100 nm in In this study, we analyzed the structure of extracellular, purified, diameter), with a segmented negative-sense RNA genome. The unstained UUKV particles by using electron cryotomography. The three genome segments encode four structural proteins. The L technique allows the 3D density distribution for a biological object segment encodes the RNA-dependent RNA polymerase; the M to be resolved in a near-native, vitreous state (22, 23). The 3D segment encodes the glycoprotein precursor, which is cleaved into

two glycoproteins (GN and GC); and the S segment encodes the Author contributions: A.K.O¨ ., R.F.P., and J.T.H. designed research; A.K.O¨ . and J.T.H. per- nucleoprotein (N protein). The N protein is associated with the formed research; K.G. and J.T.H. analyzed data; and A.K.O¨ ., K.G., and J.T.H. wrote the RNA genome and, together with the polymerase, forms the ribo- paper. nucleoproteins (RNPs) (3). The two glycoproteins are situated in The authors declare no conﬂict of interest. the envelope and form surface protrusions called ‘‘spikes.’’ This article is a PNAS Direct Submission. Bunyaviruses likely enter the cell via receptor-mediated endo- ‡Present address: Department of Virology, Institute for Medical Microbiology and Hygiene, cytosis, as has been shown for many other animal viruses. Slightly Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany. acidic conditions of the endosome are required for the fusion ʈTo whom correspondence should be addressed. E-mail: [email protected]. between the viral and cellular membrane and for subsequent This article contains supporting information online at www.pnas.org/cgi/content/full/ release of the RNPs into the cytoplasm (4–7). Nascent glycopro- 0708738105/DC1. teins are inserted into the endoplasmic reticulum membrane during © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708738105 PNAS ͉ February 19, 2008 ͉ vol. 105 ͉ no. 7 ͉ 2375–2379 Downloaded by guest on October 1, 2021 Table 1. The number of 3D tomographic reconstructions (tomograms), and particles extracted from them, for the three Uukuniemi virion samples No. of No. of Sample tomograms particles pH7 20 180 pH7* 6 196 pH6 16 40 Total 42 416

structure of the UUK virion was solved to 5.9 nm resolution, providing a structural model for a bunyavirus and insight into conformational changes in the glycoproteins during entry. Fig. 2. Size histogram for UUKV particles. The maximum diameter of particles from the pH7 sample (n ϭ 165, 92%) followed a normal distribution Results (mean ϭ 125 nm, ␴ ϭ 6 nm). UUKV Particles Display a Variable Degree of Pleiomorphy. We collected tomographic tilt series of vitrified UUKV samples, and from Glycoprotein Spikes Show Two Distinct pH-Dependent Conformations. these image series we calculated 3D density maps (tomograms). The study provided direct evidence regarding conformational Three types of samples, which differed in their pH and fixation by changes in the glycoprotein spikes and revealed that these changes glutaraldehyde (19), were used for tomography: (i) pH6, a virus are pH-dependent (Fig. 3). We analyzed the structures of UUKV suspension buffered to pH 6 and fixed with glutaraldehyde, (ii) particles extracted from the tomograms of the three samples (Table pH7, a virus suspension buffered to pH 7 and fixed with glutaral- 1). Depending on pH, the spikes appeared as either ‘‘flat’’ (Ϸ8nm dehyde, and (iii) pH7*, a virus suspension without buffering, fixed in height), with a barrel-like appearance, or ‘‘tall’’ (Ϸ13 nm in with glutaraldehyde (Table 1). Although particles in the pH7 (Fig. height), with a more pointed appearance. Virions fixed with glu- 1a) and pH7* (not depicted) samples were distributed evenly across taraldehyde at constant pH 7.0 revealed tall spikes in 34 of 40 cases the electron microscopy grids, particles in the pH6 sample (Fig. 1b) (85%) (Fig. 3a, pH7). In contrast, virions fixed at constant pH 6.0 formed large aggregates. revealed flat spikes in 7 of 9 cases (78%) (Fig. 3a, pH6). The third A slice through one of the pH7 tomograms is shown in Fig. 1c to sample (pH7*) was fixed with glutaraldehyde without buffering the illustrate general UUKV particle morphology. A variable degree of pH. This treatment caused a drop in pH from 7 to 6, presumably pleiomorphy is present in the particles. Some particles are round concomitant to fixation, and thus allowed us to capture a spectrum and regular, whereas other particles are distorted (Fig. 1, arrows), of conformations between the two extremes (pH7*; Fig. 3a). possibly as a result of the purification procedure. The outer rim of Comparison of particles with and without imposed icosahedral the particles is occupied by spikes (Fig. 1, arrowheads), which we symmetry (Fig. 3b left and right halves, respectively) verified the assign to viral membrane glycoproteins. Beneath the spike layer is presence of icosahedral symmetry in the unsymmetrized particles. a continuous layer, which we assign to the lipid bilayer. Most of the The comparison also verified the observed spike conformations in analyzed particles (pH7, n ϭ 165, 92%) were uniform in size, with the unprocessed, although much noisier, data. Both tall and flat a maximal outer diameter of 125 Ϯ 6 nm (Fig. 2). conformations of the spike revealed a cavity at the base of the spike, but in the tall conformation, the spike was closed at the top (Fig. 3c).

Icosahedral Lattice. In the 12 ؍ Glycoprotein Spikes Organize on a T a c most regular particles of the three samples (pH7, pH7*, and pH6), we observed 122 glycoprotein spikes on the positions of a T ϭ 12 icosahedral lattice. In this lattice type, three hexavalent positions not related to each other by icosahedral symmetry (Fig. 4, positions numbered 1–3) are located between the pentavalent positions (Fig. 4, pentagons). Position 2 is on an icosahedral 2-fold axis of symmetry, and position 3 is on an icosahedral 3-fold axis of b symmetry. No other lattice types were observed in any of the 416 * analyzed UUKV particles. The spikes at the pentavalent positions * are slightly smaller (Ϸ12 nm in diameter) than spikes at other positions (Ϸ15 nm in diameter). Center-to-center spacing between the spikes is Ϸ17 nm, and an Ϸ3-nm-wide gap is present between the external parts of the spikes. Thin densities bridge this gap at all of the spike-to-spike interfaces, creating the major interspike Fig. 1. UUKV particle morphology. (a and b) Low-magnification electron contacts throughout the T ϭ 12 lattice (Fig. 4, arrows). The spikes cryomicroscopy images of UUKV samples at pH 7 (a) and pH 6 (b). Both images at hexavalent positions 1 and 2 were very similar in their structures. show a representative area of the carbon support film with four 2-␮m holes Because these positions are not related by icosahedral symmetry, and vitrified virus sample. One virion is indicated with an arrowhead in a.At the observed structural similarity between them proves that the pH 7, the virions were distributed evenly, but at pH 6 they aggregated heavily. applied symmetry is valid. (Scale bars, 1 ␮m.) (c) A 5-nm-thick slice through a 3D tomogram of the pH7 sample is shown. The interior of UUK virions is visible, revealing the membrane RNP Interacts with the Membrane. and RNP density (black asterisk). Similar-appearing density, possibly leaked The tomograms revealed not from the particles, was also identified (white asterisk). A few representative only the surface structures but also the UUKV lipid bilayer and spikes around the UUKV particles are indicated (arrowheads). Some particles inner RNP densities (Figs. 3b and 5). The lipid bilayer appeared show deformations from a spherical shape, and often the membrane has a as a continuous Ϸ5-nm thick layer at the average radii of 45 nm negative curvature (arrows). (Scale bar, 100 nm.) in the pH7 sample and 43 nm in the pH6 sample (Fig. 5). This

2376 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708738105 O¨ verby et al. Downloaded by guest on October 1, 2021 Fig. 3. pH-dependent conformational differences in the glycoprotein spikes. (a) Individual particles with icosahedral symmetry applied are shown along the icosahedral 2-fold axis of symmetry. Representa- tive particles from three different types of samples are shown, as indicated above the columns: pH7 was buffered at pH 7 and fixed with glutaraldehyde; pH7* was fixed with glutaraldehyde without buffering; pH6 was buffered at pH 6 before fixation. Each column represents data from a single particle. Isosur- faces were rendered at 1.5␴ above the mean density. (Scale bar, 100 nm.) (b) Consecutive 8-nm-thick slices, 24 nm apart, are shown for the particles displayed in a. Within each panel, the icosahedrally symmetrized version of the particle is shown on the left, with the unsymmetrized, denoised particle on the right for comparison. The top row shows the central section. Arrows indicate RNP interactions with the membrane. (Scale bar, 100 nm.) (c) Close-ups from b (central section, top row) show the conformation of the spike in more detail. A cavity at the base of the spike is indicated with an arrow in pH7 and pH6 particles. (Scale bar, 50 nm.)

difference in the position of the membrane indicates a change membrane center. This spacing likely reflects the extensive contacts not only in the conformation of the external part of the glyco- seen between the membrane and the RNP (Fig. 3b, arrows). protein spikes but also in the shape of the membrane when the Discussion

pH is lowered from 7 to 6. BIOPHYSICS The volume enclosed by the bilayer is Ϸ3 ϫ 105 nm3. RNPs UUKV presents a unique enveloped virus architecture with glyco- occupy this volume as densely packed, thread-like structures, filling protein spikes at T ϭ 12 positions (Fig. 6). Because the nucleopro- the interior volume and often closely following the curvature of the tein did not appear particularly organized under the membrane, membrane. At times, several globular densities were evident, but no and because UUKV lacks a matrix protein, the interspike contacts separation into three classes of RNPs—corresponding to the three likely have a major role in determining virion shape. However, different lengths of genome segments—was detected. Although the because the virion structure seems fairly flexible, the structure UUKV RNP density is not organized on regular shells, in the most could only be captured by using electron cryotomography. In contrast, in all previously studied enveloped viruses, such as the ordered particles it located preferentially at radii 34, 19, and 9 nm flaviviruses, icosahedral organization of the continuous outer cap- in the pH7 particles and at radii 32, 18, and 10 nm in the pH6 sid results in a well defined structure and thus nearly identical particles (Fig. 5). Thus, in both samples, the preferred radius of the virions (24). Therefore, UUKV appears to represent a borderline RNP density proximal to the membrane was 11 nm from the case between strictly icosahedrally symmetric and truly pleiomorphic viruses. Before the development of electron cryotomographic techniques, pleomorphic viruses were not amenable to structural a b

1 1 32 32

Fig. 4. Glycoprotein spike organization. Shown are virions with spikes in tall conformation (a) and in ﬂat conformation (b). Insets show close-ups of the area indicated with dashed lines. Flat spikes have a barrel-like appearance, whereas taller spikes have a more pointed appearance. The relationship between the ﬁve-coordinated positions (indicated with a pentagon) and six-coordinated positions (numbered 1–3) is consistent with T ϭ 12 triangulation (43). Bridging densities (indicated by arrows) are present between the Fig. 5. Radial density distribution. The radii for the RNPs, membrane, and spikes at every position and in both conformations. Isosurfaces were rendered spike protein layer are indicated for pH6 and pH7 samples. The RNP is pref- at 1.5␴ above the mean density. (Scale bar, 100 nm.) erentially located at three radii, as indicated by the three density maxima.

O¨ verby et al. PNAS ͉ February 19, 2008 ͉ vol. 105 ͉ no. 7 ͉ 2377 Downloaded by guest on October 1, 2021 proteins purified from the virions (26). GN exists in a purely dimeric form, independent of pH, whereas GC exists as both dimers and monomers at pH Ϸ6.2 or above but will monomerize if pH is decreased below 6.0. The monomerization is thought to be caused by a pH-dependent conformational change. In addition, we observed aggregation of the virions at pH 6 (Fig. 1b), probably due to exposure of hydrophobic areas of the glycoproteins. This observation agrees with a previous study (33) in which another bunyavirus, La Crosse virus, aggregated at pH 6.2 and below. It is also known that Uukuniemi virus entry is pH-dependent and occurs under conditions similar to those for Semliki Forest virus (at pH 6 or below) (26, 34). We propose that the conformational change induced by low pH exposes hydrophobic parts of the spike glycoproteins, possibly in the GC protein. Furthermore, this conformational change would facilitate fusion between the viral and the host endosomal membrane, similar to many other membrane-containing viruses Fig. 6. Composite model of the UUK virion in a native state. During entry to entering via an endocytic pathway (35). the host cell via the endocytotic pathway, the glycoprotein spikes (blue, tall Our UUKV structural model (Fig. 6) is directly applicable for the conformation) are thought to change their conformation to facilitate fusion phleboviruses in the family Bunyaviridae. Phleboviruses include between the viral membrane (yellow) and a host endosomal membrane and, further, to release the RNPs (brown) into host cell cytoplasm. A part of the several human pathogens, such as Rift Valley fever virus, sandfly glycoprotein shell, and a slightly smaller part of the membrane, are removed fever virus, and Punta Toro virus (2, 3). The surface morphology to reveal the virion interior. The glycoprotein spikes and the membrane are of these viruses is very similar to UUKV, as shown by electron rendered at 1.5␴ and the RNP at 1.0␴ above the mean density. The resolution microscopy of negative-stained samples (20). However, at the level of the reconstruction is 5.9 nm. For visualization, the RNP density was of the whole Bunyaviridae family, a great degree of plasticity seems denoised. to exist in the glycoprotein lattices. Although phleboviruses appear to be the most regular members of the family, having a constant size and being only mildly pleiomorphic, other members vary in size and analysis at this level of detail. However, only after more virus shape and have nonicosahedral glycoprotein lattice organizations. families are studied by using tomographic techniques will it be For example, the glycoprotein lattice in Hantaan virus (Hantavirus) known whether this combination of pleomorphism and order is a has revealed a grid-like pattern, and the virions are usually elon- surprising anomaly. gated (20). A electron cryomicroscopy study on La Crosse virus has Although glycoproteins form heterodimers during transport shown that the majority of the particles are round but heteroge- from the endoplasmic reticulum to the Golgi (25), and form neous in size (36). Further electron cryomicroscopy studies, in homodimers in solution when purified from virions (26), the combination with tomographic reconstruction, will be required in biochemical composition of the glycoprotein spikes in the order to determine the level of structural variation in the members virion remains unknown. In the icosahedral lattice, higher of family Bunyaviridae. Together with x-ray crystallographic struc- multimers would be expected to be present, creating the ture determination of the viral proteins, these studies will form the observed pentavalent and hexavalent structures; however, no basis for understanding the virus–host interactions on cell entry and oligomers of glycoproteins higher than a dimer have been for a rational drug design against bunyaviral diseases. characterized. On the other hand, hexameric glycoproteins at hexavalent positions are not strictly required, contrary to what Materials and Methods has been previously suggested (19). In fact, pseudohexameric Cell Culture and Virus Purification. BHK-21 cells (American Type Culture Collec- dimers or trimers could occupy these positions—a phenome- tion) were grown in MEM, supplemented with 5% FCS, 5% tryptose phosphate non seen in many other viruses (27). For instance, dimers of broth, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 ␮g/ml streptomycin (Invitro- glycoproteins at hexavalent positions 1 and 2 (Fig. 4), or spikes gen). The cells were infected with UUKV at an infectious virus-per-cell ratio with different biochemical compositions at different positions, (‘‘multiplicity of infection’’) of 3. The stock virus, diluted in 5 ml PBS with 0.02% would give rise to a so-called ‘‘pseudo-T ϭ 12’’ triangulation. BSA, was incubated for1hinaT75flask (37°C, 5% CO2). Unadsorbed virus was The tomograms also revealed RNP densities in the interior of the removed, and 10 ml of cell culture medium (with 2% FCS and antibiotics) was virions. Although globular densities were sometimes evident, the added. The supernatant containing UUKV was collected 48 h after infection and ϫ RNP seemed mainly organized as a thread-like structure and was clarified by low-speed centrifugation (1,000 g for 10 min). The pH of the ␮ located preferentially at three radii. Very dense packing of the supernatant was set by adding phosphate buffer to give pH 6.0 or pH 7.0 (50 M final concentration). No phosphate buffer was added to the pH7* sample. To RNP, and limited resolution in the tomograms, hampered tracing preserve the particle surface morphology at different pH, acidic glutaraldehyde of the continuity in the RNP densities. However, the contacts was added to the medium to a final concentration of 0.5% (19). The virus observed between the RNP and the membrane (Fig. 3b), and a suspension was concentrated through a sucrose cushion [20% sucrose in TN constant distance between them (Fig. 5), likely correspond to the buffer (0.05 M Tris⅐HCl, 0.1 M NaCl, pH 6 or 7)] at 100,000 ϫ g for 1 h. The pellet interaction between the cytoplasmic tails of the GN glycoprotein was washed once in TN buffer before resuspending in the same buffer. and the N protein in the RNP (11). Because the RNP segments did not reveal segregation into separate densities, their organization Electron Cryotomography and Image Processing. For electron cryomicroscopy, a may be principally different from that of influenza virus, in which 3-␮l aliquot of virus suspension was pipetted on a glow-discharged, holey carbon- seven rod-like segments seem to surround the eighth segment in the coated EM grid (37). Excess suspension was blotted with filter paper, and the grid middle (28, 29). The number of each genome segment, and the was vitrified by plunging into liquid ethane, before transfer into the microscope packaging mechanism in UUKV, remain to be determined. under liquid nitrogen temperature. Electron cryotomography was performed using a 300-keV electron microscope (Polara; FEI) with an energy filter (GIF 2002; The different pH-dependent conformations of the glycopro- Gatan) operated at zero-energy-loss mode with a slit width of 10–20 eV. Forty- tein spikes—tall vs. flat (Fig. 3)—likely reflect a pH-dependent two single-axis tomographic tilt series (Table 1) were collected, covering the conformational change, primarily in the GC glycoprotein, a angular range of approximately Ϫ66° to ϩ66° at 2° increments, using TOM putative class II fusion protein (7, 30–32). Both GN and GC form acquisition software (38) or serialEM (39) under low-dose conditions, giving a homodimers, but they differ in their pH sensitivity, as shown for total dose of Ϸ65–140 electrons per square angstrom per series. The images were

2378 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708738105 O¨ verby et al. Downloaded by guest on October 1, 2021 acquired by a CCD camera (2,048 ϫ 2,048 pixels; Gatan) at Ϫ7 ␮m defocus and a rials and Methods. Briefly, an iterative approach was used in which a computa- final magnification of ϫ37,000, giving a pixel size of 0.81 nm. The data were tionally created model for the T ϭ 12 lattice served as an initial template. In the computationally down-sampled to give a final pixel size of 1.6 nanometers per subsequent iterations, care was taken to avoid model bias. The particle orienta- pixel. A low-pass filter to 1/3.6-nm spatial frequency was imposed during recon- tions were refined to 3° accuracy. Radial density profiles (Fig. 5) were calculated struction, to account for the contrast reversals due to the contrast transfer for the averages of the most ordered particles (34 particles from the pH7 sample function of the microscope at higher frequencies. Gold particles (10-nm diame- and 7 particles from the pH6 sample). ter), added to the sample as fiducial markers, were used to align the tilt images The visualized particles (Fig. 3) were subjected to resolution assessment. Two with respect to each other, and reconstructions were calculated in IMOD (40). half-maps were calculated for each particle, using one half, or the other half, of Further image processing was carried out in Bsoft (41), unless stated otherwise. the 60 icosahedral symmetry operators. The spatial frequency at which the If indicated, noise was computationally reduced in the tomograms by using calculated Fourier shell correlation coefficient between the two half-maps nonlinear anisotropic diffusion in a combined edge-enhancing diffusion /coher- dropped below 0.5 determined the resolution of the particle. The analyzed ence-enhancing diffusion mode for 10 iterations (42). A total of 416 smaller particles were 5.9–7.8 nm in resolution. This measure reflected not only the volumes, each containing one virus particle, were extracted from 42 tomograms. resolution of the original tomogram but also the degree of icosahedral symmetry Particles were centered iteratively against their own 3D radial density profile, in each particle and is thus a lower estimate for the overall resolution of the independent of each other and the presence or absence of any symmetry. The tomograms. position at which the radially averaged density dropped to the level of back- ground density determined the maximal particle radius. A histogram of diame- ACKNOWLEDGMENTS. This work was supported by Academy of Finland Post- ters was calculated, and a Gaussian function was fitted to give an average doctoral Researcher’s Grant 114649 (to J.T.H.); European Molecular Biology Or- diameter and standard deviation (Fig. 2). T ϭ 12 lattice was identified by using a ganization Long-Term Fellowship ALTF 820-2006 (to J.T.H.); and Deutsche For- template-matching approach, as described in supporting information (SI) Mate- schungsgemeinschaft Grants GR1990/1-2 and 2-1 (to K.G.).

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