Insights into bunyavirus architecture from electroncryotomography of Uukuniemi virusA. K. Overby†‡, R. F. Pettersson†, K. Grunewald§, and J. T. Huiskonen§¶�

†Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institute, P.O. Box 240, S-17177 Stockholm, Sweden; §Department of MolecularStructural 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‘‘emerging viruses’’ because many members cause serious disease inhumans, with an increasing number of outbreaks. These negative-strand RNA viruses possess a membrane envelope covered by glyco-proteins. The virions are pleiomorphic and thus have not beenamenable to structural characterization using common techniquesthat involve averaging of electron microscopic images. Here, wedetermined the three-dimensional structure of a member of theBunyaviridae family by using electron cryotomography. The genome,incorporated as a complex with the nucleoprotein inside the virions,was seen as a thread-like structure partially interacting with the viralmembrane. Although no ordered nucleocapsid was observed, lateralinteractions between the two membrane glycoproteins determinethe structure of the viral particles. In the most regular particles, theglycoprotein protrusions, or ‘‘spikes,’’ were seen to be arranged on anicosahedral lattice, with T � 12 triangulation. This arrangement hasnot yet been proven for a virus. Two distinctly different spikeconformations were observed, which were shown to depend on pH.This finding is reminiscent of the fusion proteins of alpha-, flavi-, andinfluenza viruses, in which conformational changes occur in the lowpH of the endosome to facilitate fusion of the viral and host mem-brane during viral entry.

Bunyaviridae � envelope glycoproteins � ribonucleoprotein particles �virus assembly � virus structure

The family Bunyaviridae consists of a diverse group of �350viruses and is divided into five genera: Hantavirus, Nairovirus,

Orthobunyavirus, Phlebovirus, and Tospovirus. Most members of theBunyaviridae family are spread by hematophagous arthopods, suchas mosquitoes and ticks. However, members of the genus Hanta-virus are spread by persistently infected rodents (1). Bunyavirusesare distributed worldwide, and many of them are considered‘‘emerging viruses,’’ with increasing numbers of outbreaks. Inaddition, some members of the family, such as La Crosse virus(Orthobunyavirus), Rift Valley fever virus (Phlebovirus), Crimean-Congo hemorrhagic fever virus (Nairovirus), and Sin Nombre virus(Hantavirus), cause severe illness in humans, including hemorrhagicfever and encephalitis (2).

Bunyaviruses are enveloped, spherical viruses (�100 nm indiameter), with a segmented negative-sense RNA genome. Thethree genome segments encode four structural proteins. The Lsegment encodes the RNA-dependent RNA polymerase; the Msegment encodes the glycoprotein precursor, which is cleaved intotwo glycoproteins (GN and GC); and the S segment encodes thenucleoprotein (N protein). The N protein is associated with theRNA genome and, together with the polymerase, forms the ribo-nucleoproteins (RNPs) (3). The two glycoproteins are situated inthe envelope and form surface protrusions called ‘‘spikes.’’

Bunyaviruses likely enter the cell via receptor-mediated endo-cytosis, as has been shown for many other animal viruses. Slightlyacidic conditions of the endosome are required for the fusionbetween the viral and cellular membrane and for subsequentrelease of the RNPs into the cytoplasm (4–7). Nascent glycopro-teins are inserted into the endoplasmic reticulum membrane during

translation and mature to form heterodimers. The glycosylateddimers are transported to the Golgi, were they are retained untilbudding occurs (8). Most enveloped viruses bud from the plasmamembrane into the extracellular space; however, bunyavirus virionsform by budding into the Golgi (9). Bunyaviruses do not have amatrix protein facilitating the interaction between the membraneand RNPs, as is seen in most negative-sense RNA viruses (10).Instead, the RNPs interact directly with the cytoplasmic tails of GNor GC. The GN cytoplasmic tail has been shown to interact withRNPs and to mediate genome packaging (11), and the cytoplasmictails of both GN and GC contain motifs essential for virus assembly(12, 13). From the Golgi, large vesicles filled with numerous virusesare transported to the plasma membrane, and the viruses arereleased (14).

Uukuniemi virus (UUKV; genus Phlebovirus) is a particularlyconvenient model virus for studying bunyaviral packaging, budding,and structure. Firstly, UUKV is not a human pathogen (15), andvirions can be grown to high titers in cell culture and studied inlow-biosafety-level laboratories. In addition, virus-like particles canbe produced in different compositions, for example, with variableamounts of genome segments, for structural studies (16). Thistick-borne virus was isolated in Uukuniemi, Finland, in 1964 (17).The UUKV glycoproteins, GN (75 kDa) and GC (65 kDa) (18), areresponsible for the structural stability of the virus and have beenshown to be sufficient for formation of virus-like particles that aremorphologically similar to wild-type UUKV (11). The UUKVglycoproteins have been depicted clustered as hollow cylindricalmorphological units (19). The lateral organization of the spikes hasbeen postulated to be icosahedrally symmetric, with hexameric andpentameric clusters creating a specific so-called ‘‘T � 12’’ arrange-ment (19), but the exact organization and structure of the spikeshave remained obscure. In general, bunyavirus virion structureconstitutes largely uncharted territory because no crystal structureof the two glycoproteins—and no 3D structures of the virions—exist for any of the 350 members of the family. Many familymembers have pleomorphic virion structures (20) and thus have notbeen amenable to structural characterization using the averagingtechniques commonly used in the study of icosahedrally symmetricvirions (21).

In this study, we analyzed the structure of extracellular, purified,unstained UUKV particles by using electron cryotomography. Thetechnique allows the 3D density distribution for a biological objectto be resolved in a near-native, vitreous state (22, 23). The 3D

Author contributions: A.K.O., R.F.P., and J.T.H. designed research; A.K.O. and J.T.H. per-formed research; K.G. and J.T.H. analyzed data; and A.K.O., K.G., and J.T.H. wrote thepaper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

‡Present address: Department of Virology, Institute for Medical Microbiology and Hygiene,Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany.

�To whom correspondence should be addressed. E-mail: huiskone@biochem.mpg.de.

This article contains supporting information online at www.pnas.org/cgi/content/full/0708738105/DC1.

© 2008 by The National Academy of Sciences of the USA

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structure of the UUK virion was solved to 5.9 nm resolution,providing a structural model for a bunyavirus and insight intoconformational changes in the glycoproteins during entry.

ResultsUUKV Particles Display a Variable Degree of Pleiomorphy. We col-lected tomographic tilt series of vitrified UUKV samples, and fromthese image series we calculated 3D density maps (tomograms).Three types of samples, which differed in their pH and fixation byglutaraldehyde (19), were used for tomography: (i) pH6, a virussuspension buffered to pH 6 and fixed with glutaraldehyde, (ii)pH7, a virus suspension buffered to pH 7 and fixed with glutaral-dehyde, and (iii) pH7*, a virus suspension without buffering, fixedwith glutaraldehyde (Table 1). Although particles in the pH7 (Fig.1a) and pH7* (not depicted) samples were distributed evenly acrossthe electron microscopy grids, particles in the pH6 sample (Fig. 1b)formed large aggregates.

A slice through one of the pH7 tomograms is shown in Fig. 1c toillustrate general UUKV particle morphology. A variable degree ofpleiomorphy is present in the particles. Some particles are roundand regular, whereas other particles are distorted (Fig. 1, arrows),possibly as a result of the purification procedure. The outer rim ofthe particles is occupied by spikes (Fig. 1, arrowheads), which weassign to viral membrane glycoproteins. Beneath the spike layer isa continuous layer, which we assign to the lipid bilayer. Most of theanalyzed particles (pH7, n � 165, 92%) were uniform in size, witha maximal outer diameter of 125 � 6 nm (Fig. 2).

Glycoprotein Spikes Show Two Distinct pH-Dependent Conformations.The study provided direct evidence regarding conformationalchanges in the glycoprotein spikes and revealed that these changesare pH-dependent (Fig. 3). We analyzed the structures of UUKVparticles extracted from the tomograms of the three samples (Table1). Depending on pH, the spikes appeared as either ‘‘flat’’ (�8 nmin height), with a barrel-like appearance, or ‘‘tall’’ (�13 nm inheight), with a more pointed appearance. Virions fixed with glu-taraldehyde at constant pH 7.0 revealed tall spikes in 34 of 40 cases(85%) (Fig. 3a, pH7). In contrast, virions fixed at constant pH 6.0revealed flat spikes in 7 of 9 cases (78%) (Fig. 3a, pH6). The thirdsample (pH7*) was fixed with glutaraldehyde without buffering thepH. This treatment caused a drop in pH from 7 to 6, presumablyconcomitant to fixation, and thus allowed us to capture a spectrumof conformations between the two extremes (pH7*; Fig. 3a).Comparison of particles with and without imposed icosahedralsymmetry (Fig. 3b left and right halves, respectively) verified thepresence of icosahedral symmetry in the unsymmetrized particles.The comparison also verified the observed spike conformations inthe unprocessed, although much noisier, data. Both tall and flatconformations 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).

Glycoprotein Spikes Organize on a T � 12 Icosahedral Lattice. In themost regular particles of the three samples (pH7, pH7*, and pH6),we observed 122 glycoprotein spikes on the positions of a T � 12icosahedral lattice. In this lattice type, three hexavalent positionsnot related to each other by icosahedral symmetry (Fig. 4, positionsnumbered 1–3) are located between the pentavalent positions (Fig.4, pentagons). Position 2 is on an icosahedral 2-fold axis ofsymmetry, and position 3 is on an icosahedral 3-fold axis ofsymmetry. No other lattice types were observed in any of the 416analyzed UUKV particles. The spikes at the pentavalent positionsare slightly smaller (�12 nm in diameter) than spikes at otherpositions (�15 nm in diameter). Center-to-center spacing betweenthe spikes is �17 nm, and an �3-nm-wide gap is present betweenthe external parts of the spikes. Thin densities bridge this gap at allof the spike-to-spike interfaces, creating the major interspikecontacts throughout the T � 12 lattice (Fig. 4, arrows). The spikesat hexavalent positions 1 and 2 were very similar in their structures.Because these positions are not related by icosahedral symmetry,the observed structural similarity between them proves that theapplied symmetry is valid.

RNP Interacts with the Membrane. The tomograms revealed notonly the surface structures but also the UUKV lipid bilayer andinner RNP densities (Figs. 3b and 5). The lipid bilayer appearedas a continuous �5-nm thick layer at the average radii of 45 nmin the pH7 sample and 43 nm in the pH6 sample (Fig. 5). This

Table 1. The number of 3D tomographic reconstructions(tomograms), and particles extracted from them, for thethree Uukuniemi virion samples

SampleNo. of

tomogramsNo. of

particles

pH7 20 180pH7* 6 196pH6 16 40

Total 42 416

* *

a

b

c

Fig. 1. UUKV particle morphology. (a and b) Low-magnification electroncryomicroscopy images of UUKV samples at pH 7 (a) and pH 6 (b). Both imagesshow a representative area of the carbon support film with four 2-�m holesand vitrified virus sample. One virion is indicated with an arrowhead in a. AtpH 7, the virions were distributed evenly, but at pH 6 they aggregated heavily.(Scale bars, 1 �m.) (c) A 5-nm-thick slice through a 3D tomogram of the pH7sample is shown. The interior of UUK virions is visible, revealing the membraneand RNP density (black asterisk). Similar-appearing density, possibly leakedfrom the particles, was also identified (white asterisk). A few representativespikes around the UUKV particles are indicated (arrowheads). Some particlesshow deformations from a spherical shape, and often the membrane has anegative curvature (arrows). (Scale bar, 100 nm.)

Fig. 2. Size histogram for UUKV particles. The maximum diameter of parti-cles from the pH7 sample (n � 165, 92%) followed a normal distribution(mean � 125 nm, � � 6 nm).

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difference in the position of the membrane indicates a changenot only in the conformation of the external part of the glyco-protein spikes but also in the shape of the membrane when thepH is lowered from 7 to 6.

The volume enclosed by the bilayer is �3 � 105 nm3. RNPsoccupy this volume as densely packed, thread-like structures, fillingthe interior volume and often closely following the curvature of themembrane. At times, several globular densities were evident, but noseparation into three classes of RNPs—corresponding to the threedifferent lengths of genome segments—was detected. Although theUUKV RNP density is not organized on regular shells, in the mostordered particles it located preferentially at radii 34, 19, and 9 nmin the pH7 particles and at radii 32, 18, and 10 nm in the pH6particles (Fig. 5). Thus, in both samples, the preferred radius of theRNP density proximal to the membrane was 11 nm from the

membrane center. This spacing likely reflects the extensive contactsseen between the membrane and the RNP (Fig. 3b, arrows).

DiscussionUUKV presents a unique enveloped virus architecture with glyco-protein spikes at T � 12 positions (Fig. 6). Because the nucleopro-tein did not appear particularly organized under the membrane,and because UUKV lacks a matrix protein, the interspike contactslikely have a major role in determining virion shape. However,because the virion structure seems fairly flexible, the structurecould only be captured by using electron cryotomography. Incontrast, in all previously studied enveloped viruses, such as theflaviviruses, icosahedral organization of the continuous outer cap-sid results in a well defined structure and thus nearly identicalvirions (24). Therefore, UUKV appears to represent a borderlinecase between strictly icosahedrally symmetric and truly pleiomor-phic viruses. Before the development of electron cryotomographictechniques, pleomorphic viruses were not amenable to structural

Fig. 3. pH-dependent conformational differencesin the glycoprotein spikes. (a) Individual particleswith icosahedral symmetry applied are shown alongthe icosahedral 2-fold axis of symmetry. Representa-tive particles from three different types of samplesare shown, as indicated above the columns: pH7 wasbuffered at pH 7 and fixed with glutaraldehyde;pH7* was fixed with glutaraldehyde without buffer-ing; pH6 was buffered at pH 6 before fixation. Eachcolumn 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 ina. Within each panel, the icosahedrally symmetrizedversion of the particle is shown on the left, with theunsymmetrized, denoised particle on the right forcomparison. The top row shows the central section.Arrows indicate RNP interactions with the mem-brane. (Scale bar, 100 nm.) (c) Close-ups from b (cen-tral section, top row) show the conformation of thespike in more detail. A cavity at the base of the spikeis indicated with an arrow in pH7 and pH6 particles.(Scale bar, 50 nm.)

132

132

ba

Fig. 4. Glycoprotein spike organization. Shown are virions with spikes in tallconformation (a) and in flat conformation (b). Insets show close-ups of thearea indicated with dashed lines. Flat spikes have a barrel-like appearance,whereas taller spikes have a more pointed appearance. The relationshipbetween the five-coordinated positions (indicated with a pentagon) andsix-coordinated positions (numbered 1–3) is consistent with T � 12 triangula-tion (43). Bridging densities (indicated by arrows) are present between thespikes at every position and in both conformations. Isosurfaces were renderedat 1.5� above the mean density. (Scale bar, 100 nm.)

Fig. 5. Radial density distribution. The radii for the RNPs, membrane, andspike protein layer are indicated for pH6 and pH7 samples. The RNP is pref-erentially located at three radii, as indicated by the three density maxima.

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analysis at this level of detail. However, only after more virusfamilies are studied by using tomographic techniques will it beknown whether this combination of pleomorphism and order is asurprising anomaly.

Although glycoproteins form heterodimers during transportfrom the endoplasmic reticulum to the Golgi (25), and formhomodimers in solution when purified from virions (26), thebiochemical composition of the glycoprotein spikes in thevirion remains unknown. In the icosahedral lattice, highermultimers would be expected to be present, creating theobserved pentavalent and hexavalent structures; however, nooligomers of glycoproteins higher than a dimer have beencharacterized. On the other hand, hexameric glycoproteins athexavalent positions are not strictly required, contrary to whathas been previously suggested (19). In fact, pseudohexamericdimers or trimers could occupy these positions—a phenome-non seen in many other viruses (27). For instance, dimers ofglycoproteins at hexavalent positions 1 and 2 (Fig. 4), or spikeswith different biochemical compositions at different positions,would give rise to a so-called ‘‘pseudo-T � 12’’ triangulation.

The tomograms also revealed RNP densities in the interior of thevirions. Although globular densities were sometimes evident, theRNP seemed mainly organized as a thread-like structure and waslocated preferentially at three radii. Very dense packing of theRNP, and limited resolution in the tomograms, hampered tracingof the continuity in the RNP densities. However, the contactsobserved between the RNP and the membrane (Fig. 3b), and aconstant distance between them (Fig. 5), likely correspond to theinteraction between the cytoplasmic tails of the GN glycoproteinand the N protein in the RNP (11). Because the RNP segments didnot reveal segregation into separate densities, their organizationmay be principally different from that of influenza virus, in whichseven rod-like segments seem to surround the eighth segment in themiddle (28, 29). The number of each genome segment, and thepackaging mechanism in UUKV, remain to be determined.

The different pH-dependent conformations of the glycopro-tein spikes—tall vs. f lat (Fig. 3)—likely reflect a pH-dependentconformational change, primarily in the GC glycoprotein, aputative class II fusion protein (7, 30–32). Both GN and GC formhomodimers, but they differ in their pH sensitivity, as shown for

proteins purified from the virions (26). GN exists in a purelydimeric form, independent of pH, whereas GC exists as bothdimers and monomers at pH �6.2 or above but will monomerizeif pH is decreased below 6.0. The monomerization is thought tobe caused by a pH-dependent conformational change. In addi-tion, we observed aggregation of the virions at pH 6 (Fig. 1b),probably due to exposure of hydrophobic areas of the glyco-proteins. This observation agrees with a previous study (33) inwhich another bunyavirus, La Crosse virus, aggregated at pH 6.2and below. It is also known that Uukuniemi virus entry ispH-dependent and occurs under conditions similar to those forSemliki Forest virus (at pH 6 or below) (26, 34). We propose thatthe conformational change induced by low pH exposes hydro-phobic parts of the spike glycoproteins, possibly in the GCprotein. Furthermore, this conformational change would facil-itate fusion between the viral and the host endosomal mem-brane, similar to many other membrane-containing virusesentering via an endocytic pathway (35).

Our UUKV structural model (Fig. 6) is directly applicable for thephleboviruses in the family Bunyaviridae. Phleboviruses includeseveral human pathogens, such as Rift Valley fever virus, sandflyfever virus, and Punta Toro virus (2, 3). The surface morphologyof these viruses is very similar to UUKV, as shown by electronmicroscopy of negative-stained samples (20). However, at the levelof the whole Bunyaviridae family, a great degree of plasticity seemsto exist in the glycoprotein lattices. Although phleboviruses appearto be the most regular members of the family, having a constant sizeand being only mildly pleiomorphic, other members vary in size andshape and have nonicosahedral glycoprotein lattice organizations.For example, the glycoprotein lattice in Hantaan virus (Hantavirus)has revealed a grid-like pattern, and the virions are usually elon-gated (20). A electron cryomicroscopy study on La Crosse virus hasshown that the majority of the particles are round but heteroge-neous in size (36). Further electron cryomicroscopy studies, incombination with tomographic reconstruction, will be required inorder to determine the level of structural variation in the membersof family Bunyaviridae. Together with x-ray crystallographic struc-ture determination of the viral proteins, these studies will form thebasis for understanding the virus–host interactions on cell entry andfor a rational drug design against bunyaviral diseases.

Materials and MethodsCell Culture and Virus Purification. BHK-21 cells (American Type Culture Collec-tion) were grown in MEM, supplemented with 5% FCS, 5% tryptose phosphatebroth, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 �g/ml streptomycin (Invitro-gen). The cells were infected with UUKV at an infectious virus-per-cell ratio(‘‘multiplicity of infection’’) of 3. The stock virus, diluted in 5 ml PBS with 0.02%BSA, was incubated for 1 h in a T75 flask (37°C, 5% CO2). Unadsorbed virus wasremoved, and 10 ml of cell culture medium (with 2% FCS and antibiotics) wasadded. The supernatant containing UUKV was collected 48 h after infection andclarified by low-speed centrifugation (1,000 � g for 10 min). The pH of thesupernatant was set by adding phosphate buffer to give pH 6.0 or pH 7.0 (50 �Mfinal concentration). No phosphate buffer was added to the pH7* sample. Topreserve the particle surface morphology at different pH, acidic glutaraldehydewas added to the medium to a final concentration of 0.5% (19). The virussuspension was concentrated through a sucrose cushion [20% sucrose in TNbuffer (0.05 M Tris�HCl, 0.1 M NaCl, pH 6 or 7)] at 100,000 � g for 1 h. The pelletwas washed once in TN buffer before resuspending in the same buffer.

Electron Cryotomography and Image Processing. For electron cryomicroscopy, a3-�l aliquotofvirus suspensionwaspipettedonaglow-discharged,holeycarbon-coated EM grid (37). Excess suspension was blotted with filter paper, and the gridwas vitrified by plunging into liquid ethane, before transfer into the microscopeunder liquid nitrogen temperature. Electron cryotomography was performedusing a 300-keV electron microscope (Polara; FEI) with an energy filter (GIF 2002;Gatan) operated at zero-energy-loss mode with a slit width of 10–20 eV. Forty-two single-axis tomographic tilt series (Table 1) were collected, covering theangular range of approximately �66° to �66° at 2° increments, using TOMacquisition software (38) or serialEM (39) under low-dose conditions, giving atotal dose of �65–140 electrons per square angstrom per series. The images were

Fig. 6. Composite model of the UUK virion in a native state. During entry tothe host cell via the endocytotic pathway, the glycoprotein spikes (blue, tallconformation) are thought to change their conformation to facilitate fusionbetween the viral membrane (yellow) and a host endosomal membrane and,further, to release the RNPs (brown) into host cell cytoplasm. A part of theglycoprotein shell, and a slightly smaller part of the membrane, are removedto reveal the virion interior. The glycoprotein spikes and the membrane arerendered at 1.5� and the RNP at 1.0� above the mean density. The resolutionof the reconstruction is 5.9 nm. For visualization, the RNP density wasdenoised.

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acquired by a CCD camera (2,048 � 2,048 pixels; Gatan) at �7 �m defocus and afinal magnification of �37,000, giving a pixel size of 0.81 nm. The data werecomputationally down-sampled to give a final pixel size of 1.6 nanometers perpixel. A low-pass filter to 1/3.6-nm spatial frequency was imposed during recon-struction, to account for the contrast reversals due to the contrast transferfunction of the microscope at higher frequencies. Gold particles (10-nm diame-ter), added to the sample as fiducial markers, were used to align the tilt imageswith respect to each other, and reconstructions were calculated in IMOD (40).

Further imageprocessingwascarriedout inBsoft (41),unless statedotherwise.If indicated, noise was computationally reduced in the tomograms by usingnonlinear anisotropic diffusion in a combined edge-enhancing diffusion /coher-ence-enhancing diffusion mode for 10 iterations (42). A total of 416 smallervolumes, each containing one virus particle, were extracted from 42 tomograms.Particles were centered iteratively against their own 3D radial density profile,independent of each other and the presence or absence of any symmetry. Theposition at which the radially averaged density dropped to the level of back-ground density determined the maximal particle radius. A histogram of diame-ters was calculated, and a Gaussian function was fitted to give an averagediameter and standard deviation (Fig. 2). T � 12 lattice was identified by using atemplate-matching approach, as described in supporting information (SI) Mate-

rials and Methods. Briefly, an iterative approach was used in which a computa-tionally created model for the T � 12 lattice served as an initial template. In thesubsequent iterations, care was taken to avoid model bias. The particle orienta-tions were refined to 3° accuracy. Radial density profiles (Fig. 5) were calculatedfor the averages of the most ordered particles (34 particles from the pH7 sampleand 7 particles from the pH6 sample).

The visualized particles (Fig. 3) were subjected to resolution assessment. Twohalf-maps were calculated for each particle, using one half, or the other half, ofthe 60 icosahedral symmetry operators. The spatial frequency at which thecalculated Fourier shell correlation coefficient between the two half-mapsdropped below 0.5 determined the resolution of the particle. The analyzedparticles were 5.9–7.8 nm in resolution. This measure reflected not only theresolution of the original tomogram but also the degree of icosahedral symmetryin each particle and is thus a lower estimate for the overall resolution of thetomograms.

ACKNOWLEDGMENTS. This work was supported by Academy of Finland Post-doctoral Researcher’s Grant 114649 (to J.T.H.); European Molecular Biology Or-ganization Long-Term Fellowship ALTF 820-2006 (to J.T.H.); and Deutsche For-schungsgemeinschaft Grants GR1990/1-2 and 2-1 (to K.G.).

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