The Structure of Echovirus Type 12 Bound to a Two-domain Fragment of Its Cellular Attachment Protein Decay-accelerating Factor (CD 55)
2004; Elsevier BV; Volume: 279; Issue: 9 Linguagem: Inglês
10.1074/jbc.m311334200
ISSN1083-351X
AutoresDavid Bhella, Ian Goodfellow, Pietro Roversi, David M. Pettigrew, Yasmin Chaudhry, David J. Evans, Susan M. Lea,
Tópico(s)Viral gastroenteritis research and epidemiology
ResumoEchovirus type 12 (EV12), an Enterovirus of the Picornaviridae family, uses the complement regulator decay-accelerating factor (DAF, CD55) as a cellular receptor. We have calculated a three-dimensional reconstruction of EV12 bound to a fragment of DAF consisting of short consensus repeat domains 3 and 4 from cryo-negative stain electron microscopy data (EMD code 1057). This shows that, as for an earlier reconstruction of the related echovirus type 7 bound to DAF, attachment is not within the viral canyon but occurs close to the 2-fold symmetry axes. Despite this general similarity our reconstruction reveals a receptor interaction that is quite different from that observed for EV7. Fitting of the crystallographic co-ordinates for DAF34 and EV11 into the reconstruction shows a close agreement between the crystal structure of the receptor fragment and the density for the virus-bound receptor, allowing unambiguous positioning of the receptor with respect to the virion (PDB code 1UPN). Our finding that the mode of virus-receptor interaction in EV12 is distinct from that seen for EV7 raises interesting questions regarding the evolution and biological significance of the DAF binding phenotype in these viruses. Echovirus type 12 (EV12), an Enterovirus of the Picornaviridae family, uses the complement regulator decay-accelerating factor (DAF, CD55) as a cellular receptor. We have calculated a three-dimensional reconstruction of EV12 bound to a fragment of DAF consisting of short consensus repeat domains 3 and 4 from cryo-negative stain electron microscopy data (EMD code 1057). This shows that, as for an earlier reconstruction of the related echovirus type 7 bound to DAF, attachment is not within the viral canyon but occurs close to the 2-fold symmetry axes. Despite this general similarity our reconstruction reveals a receptor interaction that is quite different from that observed for EV7. Fitting of the crystallographic co-ordinates for DAF34 and EV11 into the reconstruction shows a close agreement between the crystal structure of the receptor fragment and the density for the virus-bound receptor, allowing unambiguous positioning of the receptor with respect to the virion (PDB code 1UPN). Our finding that the mode of virus-receptor interaction in EV12 is distinct from that seen for EV7 raises interesting questions regarding the evolution and biological significance of the DAF binding phenotype in these viruses. Echoviruses along with the coxsackieviruses and polioviruses comprise the Enterovirus genus within the Picornaviridae family. Echovirus infection is usually mild, although these viruses are sometimes associated with severe disease such as aseptic meningitis, encephalitis, hemorrhagic conjunctivitis, and myocarditis. The Picornaviridae family also includes a number of other important human and animal pathogens including rhinoviruses and foot and mouth disease virus. Picornaviruses are small (∼300 Å in diameter), non-enveloped icosahedral viruses that have a single-stranded positive-sense RNA genome of between 7.0 and 8.5 kilobases. The genome encodes a single polyprotein, which is co- and post-translationally processed by viral proteases to yield the capsid and non-structural proteins required for virus replication. The capsid assembles as a pseudo T = 3 icosahedral shell from four protein species, VP1-VP4. VP1-3 occupy the three quasi-equivalent positions in the icosahedral lattice, whereas VP4 is located at the inner surface of the capsid (1Hogle J.M. Chow M. Filman D.J. Science. 1985; 229: 1358-1365Crossref PubMed Scopus (890) Google Scholar). The rhinoviruses and enteroviruses share a common and distinctive morphology consisting of a raised, star-shaped platform at the pentameric apices of the capsid. Surrounding this is a narrow cleft, termed the "canyon" (2Rossmann M.G. Arnold E. Erickson J.W. Frankenberger E.A. Griffith J.P. Hecht H.J. Johnson J.E. Kamer G. Luo M. Mosser A.G. Rueckert R.R. Sherry B. Vriend G. Nature. 1985; 317: 145-153Crossref PubMed Scopus (1020) Google Scholar). For many picornaviruses (the polioviruses, the major receptor group rhinoviruses, Coxsackie A virus type 21. and Coxsackie B virus type 3) it has been shown that the interaction with their cellular receptors (all of which are members of the Ig-superfamily of proteins) occurs in the canyon (3He Y.N. Mueller S. Chipman P.R. Bator C.M. Peng X.Z. Bowman V.D. Mukhopadhyay S. Wimmer E. Kuhn R.J. Rossmann M.G. J. 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Binding of these viruses to soluble monomeric receptor protein in vitro triggers an irreversible conformational change in the virus particle that manifests as a change in the sedimentation characteristics of the virus (from the 160 S mature virion to the 135 S A-particle) (8Hoover-Litty H. Greve J.M. J. Virol. 1993; 67: 390-397Crossref PubMed Google Scholar). Similar changes in sedimentation coefficient are seen when the virus interacts with the receptor at the cell surface, and such transformations are thought to be essential for successful entry and infection (9Fenwick M.L. Cooper P.D. Virology. 1962; 18: 212-223Crossref PubMed Scopus (67) Google Scholar, 10Guttman N. Baltimore D. Virology. 1977; 82: 25-36Crossref PubMed Scopus (53) Google Scholar). The structural rearrangements that lead to this change in sedimentation include loss of VP4 and externalization of the N terminus of VP1, which is normally located at the inner surface of the capsid surrounding the pentameric apices in the mature virion. The extruded terminus of VP1, which is hydrophobic, becomes membrane-associated and is thought to form a pore through which the genome enters the cell cytoplasm (11Fricks C.E. Hogle J.M. J. Virol. 1990; 64: 1934-1945Crossref PubMed Google Scholar). Within the Rhinovirus and Enterovirus genera other types of receptor interactions have also been identified. The minor receptor group rhinoviruses, for example, bind the very low density lipoprotein receptor (12Hofer F. Gruenberger M. Kowalski H. Machat H. Huettinger M. Kuechler E. Blass D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1839-1842Crossref PubMed Scopus (362) Google Scholar). This interaction does not lead to the irreversible conformational changes outlined above, and recent studies demonstrate that this receptor binds to the virion around the 5-fold symmetry axes, not in the canyon (13Hewat E.A. Neumann E. Conway J.F. Moser R. Ronacher B. Marlovits T.C. Blaas D. EMBO J. 2000; 19: 6317-6325Crossref PubMed Google Scholar). The lack of virus uncoating upon receptor binding suggests the existence of another, as yet unknown receptor or co-factor that induces uncoating. Many enteroviruses have been shown to bind decay-accelerating factor (DAF 1The abbreviations used are: DAF, decay-accelerating factor; SCR, short consensus repeat; EV, echovirus; ENV, enterovirus. ; CD55), a member of the regulator of complement activity protein family (14Bergelson J.M. Chan M. Solomon K.R. Stjohn N.F. Lin H.M. Finberg R.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6245-6248Crossref PubMed Scopus (273) Google Scholar, 15Karnauchow T.M. Tolson D.L. Harrison B.A. Altman E. 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DAF has four such domains, and these are linked to the C-terminal glycosylphosphatidylinositol anchor by a heavily O-glycosylated serine/threonine/proline-rich region (19Lublin D.M. Atkinson J.P. Annu. Rev. Immunol. 1989; 7: 35-58Crossref PubMed Scopus (400) Google Scholar). Complement control, Enterovirus binding, and other known interactions, including binding of the cellular ligand CD97 and bacterial adhesins, only involve the SCR domains (14Bergelson J.M. Chan M. Solomon K.R. Stjohn N.F. Lin H.M. Finberg R.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6245-6248Crossref PubMed Scopus (273) Google Scholar, 20Brodbeck W.G. Liu D. Sperry J. Mold C. Medof M.E. J. Immunol. 1996; 156: 2528-2533PubMed Google Scholar, 21Lin H.H. Stacey M. Saxby C. Knott V. Chaudhry Y. Evans D. Gordon S. McKnight A.J. Handford P. Lea S. J. Biol. Chem. 2001; 276: 24160-24169Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 22Nowicki B. Hart A. Coyne K.E. Lublin D.M. Nowicki S. J. Exp. 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Enterovirus binding to soluble monomeric DAF does not, however, lead to the conformational changes normally associated with uncoating that these viruses are observed to undergo at the cell surface (25Powell R.M. Ward T. Evans D.J. Almond J.W. J. Virol. 1997; 71: 9306-9312Crossref PubMed Google Scholar). The status of DAF as a sole cellular receptor has, therefore, been subject to question. Although other molecules have been implicated in cell binding and entry by DAF binding enteroviruses (26Spiller O.B. Goodfellow I.G. Evans D.J. Hinchliffe S.J. Morgan B.P. J. Gen. Virol. 2002; 83: 45-52Crossref PubMed Scopus (20) Google Scholar, 27Goodfellow I.G. Powell R.M. Ward T. Spiller O.B. Almond J.W. Evans D.J. J. Gen. Virol. 2000; 81: 1393-1401Crossref PubMed Scopus (18) Google Scholar, 28Goodfellow I.G. Sioofy A.B. Powell R.M. Evans D.J. J. Virol. 2001; 75: 4918-4921Crossref PubMed Scopus (70) Google Scholar), candidates that induce irreversible conformational changes in the virion have yet to be identified. Structural analysis of variants of echovirus type 11 that exhibit different cell tropism has, however, provided evidence for an as yet undiscovered canyon binding receptor while implicating a number of residues in the binding interaction with DAF both around the 5-fold symmetry axis and in the EF loop of VP2 (29Stuart A.D. McKee T.A. Williams P.A. Harley C. Shen S. Stuart D.I. Brown T.D.K. Lea S.M. J. Virol. 2002; 76: 7694-7704Crossref PubMed Scopus (47) Google Scholar). The recent analysis of DAF binding in echovirus type 7 by cryomicroscopy and image reconstruction has shown that DAF does not bind in the canyon; rather, it binds to the hyper-variable region of VP2 just outside of the "south" rim of the canyon and also to another hyper-variable region of VP3 (30He Y.N. Lin F. Chipman P.R. Bator C.M. Baker T.S. Shoham M. Kuhn R.J. Medof M.E. Rossmann M.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10325-10329Crossref PubMed Scopus (59) Google Scholar). In related viruses these regions have been shown to be antigenic and capable of eliciting a neutralizing antibody response, suggesting that the paradigm of segregation of receptor binding regions from those parts of the capsid surface subject to immune surveillance is not strictly adhered to in the enteroviruses. Similarly, receptor binding to hyper-variable epitopes has been demonstrated for the minor group rhinoviruses and foot and mouth disease virus. In these viruses a strategy is employed whereby conserved receptor binding domains are embedded in hyper-variable regions capable of mutation to escape the host immune response (13Hewat E.A. Neumann E. Conway J.F. Moser R. Ronacher B. Marlovits T.C. Blaas D. EMBO J. 2000; 19: 6317-6325Crossref PubMed Google Scholar, 31Acharya R. Fry E. Stuart D. Fox G. Rowlands D. Brown F. Nature. 1989; 337: 709-716Crossref PubMed Scopus (673) Google Scholar). In the EV7-receptor complex, bound DAF is located close to and lies across the 2-fold symmetry axes such that symmetry-related molecules are in steric collision (30He Y.N. Lin F. Chipman P.R. Bator C.M. Baker T.S. Shoham M. Kuhn R.J. Medof M.E. Rossmann M.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10325-10329Crossref PubMed Scopus (59) Google Scholar). This complicates the interpretation of this reconstruction in terms of the known atomic structures for DAF (32Williams P. Chaudhry Y. Goodfellow I.G. Billington J. Powell R. Spiller O.B. Evans D.J. Lea S. J. Biol. Chem. 2003; 278: 10691-10696Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 33Uhrinova S. Lin F. Ball G. Bromek K. Uhrin D. Medof M.E. Barlow P.N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4718-4723Crossref PubMed Scopus (40) Google Scholar, 34Lukacik P. Roversi P. White J. Esser D. Smith G.P. Billington J. Willliams P.A. Rudd P.M. Wormald M.R. Harvey D.J. Crispin M.D.M. Radcliffe C.M. Dwek R.A. Evans D.J. Morgan B.P. Smith R.A.G. Lea S.M. Proc. Natl. Acad. Sci. U. S. A. 2003; (in press)Google Scholar). There are, however, substantial data regarding specific residues both on the virus capsid and in the SCR 3 and 4 domains of DAF that are involved in receptor binding in the echoviruses (29Stuart A.D. McKee T.A. Williams P.A. Harley C. Shen S. Stuart D.I. Brown T.D.K. Lea S.M. J. Virol. 2002; 76: 7694-7704Crossref PubMed Scopus (47) Google Scholar, 32Williams P. Chaudhry Y. Goodfellow I.G. Billington J. Powell R. Spiller O.B. Evans D.J. Lea S. J. Biol. Chem. 2003; 278: 10691-10696Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Echovirus type 12 (EV12) exhibits a distinct interaction with DAF, which predominantly involves SCR domains 3 and 4, a feature shared only with echovirus type 29 (16Powell R.M. Ward T. Goodfellow I. Almond J.W. Evans D.J. J. Gen. Virol. 1999; 80: 3145-3152Crossref PubMed Scopus (40) Google Scholar). A recent mutagenic analysis of DAF structure and function demonstrated that EV12 binding involves a different face of the receptor than that bound by other echoviruses (32Williams P. Chaudhry Y. Goodfellow I.G. Billington J. Powell R. Spiller O.B. Evans D.J. Lea S. J. Biol. Chem. 2003; 278: 10691-10696Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We have, therefore, conducted a structural investigation of the virus-receptor complex and present here a reconstruction of echovirus type 12 bound to SCR domains 3 and 4 of DAF, determined to 16-Å resolution by cryo-negative stain transmission electron microscopy and image reconstruction. Similarly to echovirus type 7, DAF binds to this virus outside the south rim of the canyon close to (but not over) the 2-fold symmetry axes. The distribution of density in our reconstruction is quite different, however, supporting the previous findings that there are at least three distinct virus-DAF interactions within the enteroviruses. By labeling the virus with only two domains of DAF we have been able to determine an unambiguous density for the receptor that is consistent with the crystallographic co-ordinates for DAF34. Docking of these data into the reconstructed protein envelope reveals that DAF binds to EV12 mainly via interactions between SCR 3 and VP2. The orientation determined for the DAF34 fragment allows us to superimpose the crystallographic data from a DAF fragment comprising all four SCR domains (DAF1234) onto our model. We find that 60 copies of this protein may be modeled onto the surface of the EV12 capsid without any significant molecular clashes between symmetry-related molecules or the virion itself. This further highlights the differences between the complex seen here and that seen in the earlier investigation of the EV7-DAF interaction. Virus and DAF34Preparation—Echovirus type 12 was routinely passaged in rhabdomyosarcoma cells. Ten 1750-cm2 roller bottles (Corning Glass) of confluent rhabdomyosarcoma cells were infected with EV12 at high multiplicity (5 or higher), and the infection was allowed to proceed for 24 h at 37 °C. Cell-associated virus was released by 2 cycles of freeze thawing, and the resultant supernatant was clarified by low speed centrifugation (2000 × g for 10 min) and filtered (0.2 μm) to remove particulates. Sodium chloride was added to a final concentration of 2.3% (w/v) followed by 7% polyethylene glycol 8000, and the preparation was stirred at 4 °C overnight. The precipitated virus was collected by centrifugation (5000 × g for 30 min) and resuspended in 10 ml of Dulbecco's modified Eagle's medium using an 18-gauge needle and syringe. Large particulate material was removed by low speed centrifugation, and the remaining virus was pelleted through a 30% sucrose cushion by centrifugation at 40,000 rpm for 6 h (TH641, Sorval) and resuspended in 1 ml of Dulbecco's modified Eagle's medium using an 18-gauge needle and syringe. The partially purified virus was subsequently layered onto a 10-25% sucrose gradient and centrifuged at 40,000 rpm for 1 h (TH641, Sorval); 160 S virus was extracted from the gradient and pelleted by centrifugation for 6 h at 40,000 rpm (TLS 55). Purified virus was resuspended in 200 μl of phosphate-buffered saline and stored at -20 °C. The purity of the virus preparations was assessed by SDS-PAGE. The two SCR domain fragment of decay-accelerating factor (DAF34) was expressed in Pichia pastoris and purified as previously described (25Powell R.M. Ward T. Evans D.J. Almond J.W. J. Virol. 1997; 71: 9306-9312Crossref PubMed Google Scholar, 35Powell R.M. Schmitt V. Ward T. Goodfellow I. Evans D.J. Almond J.W. J. Gen. Virol. 1998; 79: 1707-1713Crossref PubMed Scopus (59) Google Scholar). Electron Microscopy—EV12 preparations were labeled with DAF34 by incubation overnight at 4 °C. Unlabeled and labeled virus was then prepared for electron microscopy using the cryo-negative stain technique (36Adrian M. Dubochet J. Fuller S.D. Harris J.R. Micron. 1998; 29: 145-160Crossref PubMed Scopus (138) Google Scholar). In brief, a 5-μl droplet of virus or labeled virus was loaded onto a freshly glow-discharged Quantifoil holey carbon support film (Quantifoil Micro Tools GmbH, Jena, Germany) for 30 s. Grids were then transferred to a droplet of ∼20% (w/v) ammonium molybdate solution (pH 7.4) for 10 s. Finally, grids were blotted and plunged into liquid nitrogen-cooled ethane slush. Vitrified virus preparations were then imaged in a JEOL 1200 EX II transmission electron microscope equipped with an Oxford instruments cryo-transfer stage. Focal-pair images were recorded at a nominal magnification of 30,000× and defocus of between 300 and 2,000 nm on Kodak SO-163 film under low electron dose conditions. Image Reconstruction—In total 14 focal pairs of unlabeled EV12 and 8 focal pairs of DAF34-labeled EV12 were selected for processing. Micrographs were digitized on a Dunvegan Hi-Scan drum scanner (Dunvegan SA, Lausanne, Switzerland) at a raster step size corresponding to 3.4 Å/pixel in the specimen. Particles were selected from each pair of micrographs using the program X3d, and deconvolution of the contrast transfer function was accomplished using the CTFMIX program, at which point the focal pairs were merged (37Conway J.F. Steven A.C. J. Struct. Biol. 1999; 128: 106-118Crossref PubMed Scopus (146) Google Scholar). Orientations and origins were determined for the unlabeled EV12 data set using the polar Fourier transform method (PFT) (38Baker T.S. Cheng R.H. J. Struct. Biol. 1996; 116: 120-130Crossref PubMed Scopus (324) Google Scholar) using a starting model that was generated from the crystal structure of echovirus type 1 (39Filman D.J. Wien M.W. Cunningham J.A. Bergelson J.M. Hogle J.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 1261-1272Crossref PubMed Google Scholar) by the EMAN PDB2MRC program (40Ludtke S.J. Baldwin P.R. Chiu W. J. Struct. Biol. 1999; 128: 82-97Crossref PubMed Scopus (2102) Google Scholar). Subsequent iterations of three-dimensional reconstruction (41Crowther R.A. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1971; 261: 221-230Crossref PubMed Scopus (333) Google Scholar) and polar Fourier transform refinement led to the calculation of a 20-Å resolution reconstruction that was used to determine initial origins and orientations for the DAF34 labeled data set. In both cases origins and orientations were further refined until they ceased changing significantly. Final reconstructions were calculated to 14 Å, and in both cases all inverse eigenvalues were less than 0.01, indicating that the data adequately filled reciprocal space to this resolution. Final resolutions for each reconstruction were estimated by randomly dividing the data sets into two subsets and calculating independent reconstructions, for which a number of measures of agreement were calculated, including the Fourier-shell correlation and the spectral signal to noise ratio. Reconstructions were deposited in the EM data bank (accession numbers are EMD 1057 for the DAF34-labeled virus and EMD 1058 for the unlabeled virus). Isosurfaces calculated from three-dimensional reconstructions were visualized in Iris Explorer (Numerical Algorithms Group, Oxford, UK) using a radial depth cueing module (42Dougherty M. Chiu W. Microsc. Microanal. 2000; 6: 282-283Crossref Google Scholar). To draw a direct comparison between our reconstructions and the previously determined model for EV7 bound to DAF1234, a density map was calculated at 14-Å resolution using the EMAN PDB2MRC program from the deposited co-ordinates of He et al. (30He Y.N. Lin F. Chipman P.R. Bator C.M. Baker T.S. Shoham M. Kuhn R.J. Medof M.E. Rossmann M.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10325-10329Crossref PubMed Scopus (59) Google Scholar) (PDB code 1M11). Fitting of the EV11 and DAF34Crystal Structures to the Cryo-negative Stain Reconstruction—Visual inspection of an EV11 virus model (PDB code 1H8T) overlapped with the EV12-DAF34 density map confirmed the correctness of the original hand assignment for the reconstruction. The reconstructed volume was then placed at the origin of a cubic cell with a cell edge a = 599.375 Å, oriented so that its 3- and 2-fold axes coincided with the symmetry axes through the origin of space group P23, and was rendered periodic with P23 symmetry. The x-ray model for a pentameric asymmetric unit of EV11 was oriented and placed around the origin in the same cell. The overall correlation coefficients in real space between the experimental and model density was 0.76. The x-ray model for DAF domains 3 and 4 (PDB code 1H03) with all B factors set to 95 was manually placed in the EM density in an initial orientation with domain 3 closer to the virus (placement A). A second placement of the model was also generated by rotating the first so as to swap the positions of domains 3 and 4 (placement B). Alternative orientations of both placements A and B were sampled by rotation of each model around its major axis (defined by the midpoints of the 4 disulfide bonds) in steps of 30 degrees. Fig. 3 shows the real space correlation coefficients for the main chain atoms in each of the rotated models. Placement A in its original orientation gave the best correlation coefficients, suggesting that both the domain assignment and the DAF34 face assignments are correct. Maximum-likelihood rigid body refinement of the domain 4 model (residues 189-251) targeting the phases computed from the reconstruction (program CNS (43Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar)) further improved the main chain atom correlation coefficients (Fig. 3) and yielded the final model (Fig. 4). The Cα-Cα distance for the domain 3-4 junction is only 0.5 Å away from the distance given by ideal geometry, a reasonable level of accuracy for a fit at 16-Å resolution. DAF1234 was overlaid on the membrane-proximal fragment using the CCP4 program LSQkab (44Kabsch W. Acta Crystallogr. Sect. A. 1976; 32: 922-923Crossref Scopus (2358) Google Scholar, 45Bailey S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (42) Google Scholar). Molecular models were visualized using PyMol (DeLano Scientific, San Carlos, CA), Molscript (46Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), and Raster3D (47Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3875) Google Scholar). Co-ordinates for virus receptor complexes were deposited in the protein data bank (PDB code 1UPN). Cryo-negative Stain Imaging and Three-dimensional Reconstruction of EV12 Labeled with DAF34—The cryo-negative stain approach yielded high contrast images of virus preparations in a frozen hydrated state (Fig. 1). Analysis of the incoherently averaged power spectra, calculated from particle images cut from focal pair micrographs, showed that Thon rings were clearly visible out to 10 Å in these data. In total, 720 focal pair images of unlabeled EV12 virions and 1590 paired images of DAF34-labeled EV12 were corrected for the effects of the microscope contrast transfer function and processed to calculate three-dimensional image reconstructions. Final reconstructions were calculated from 617 unlabeled particle images and 903 DAF34-labeled images (Fig. 2). The resolutions of these reconstructions were determined to be 18 and 16 Å, respectively.Fig. 2Stereo pairs of surface rendered three-dimensional reconstructions of unlabeled EV12 virions (A) and DAF34-labeled virions (B). Isosurfaces of these reconstructions are merged and rendered in their respective color schemes to highlight the differences in density attributed to the two SCR domain fragment of DAF (C). A low resolution representation of EV7 bound to DAF1234, derived from PDB code 1M11 (30He Y.N. Lin F. Chipman P.R. Bator C.M. Baker T.S. Shoham M. Kuhn R.J. Medof M.E. Rossmann M.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10325-10329Crossref PubMed Scopus (59) Google Scholar), highlights the differently oriented densities in these two complexes (D). In this model the densities of two copies of DAF1234 are superimposed, laying across the virion 2-fold symmetry axes, giving rise to a hybrid density representing the two possible positions for the molecule. A radial depth-cue color scheme is used to indicate distance from the center of the virion (see the key).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The reconstruction of the DAF34-bound virion has clear regions of contiguous density decorating the exterior of the capsid that can be attributed to the DAF fragment. This density is linear in appearance and consists of two readily defined domains. The density attributed to the DAF34 molecule is much weaker than that of the capsid itself, suggesting a low level of receptor occupancy. A low density threshold setting approximately equal to the average density of the reconstructed volume was, therefore, necessary to calculate an isosurface that encompassed the correct molecular volume for the DAF component of this reconstruction. Fortunately the high contrast in the reconstruction, brought about by the use of the cryo-negative stain technique, prevented the expansion of the virion density or incorporation of noise in the final representation of the volume. The apparent low occupancy in the virus-receptor complex is consistent with biochemical data and previously published reconstructions (16Powell R.M. Ward T. Goodfellow I. Almond J.W. Evans D.J. J. Gen. Virol. 1999; 80: 3145-3152Crossref PubMed Scopus (40) Google Scholar, 30He Y.N. Lin F. Chipman P.R. Bator C.M. Baker T.S. Shoham M. Kuhn R.J. Medof M.E. Rossmann M.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10325-10329Crossref PubMed Scopus (59) Google Scholar, 48Lea S.M. Powell R.M. McKee T. Evans D.J. Brown D. 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