The Ebola virus (EBOV) GP gene encodes two glycoproteins. The major product is a soluble, dimeric glycoprotein (sGP) that is secreted abundantly. Despite the abundance of sGP during infection, little is known regarding its structure or functional role. A minor product, resulting from transcriptional editing, is the transmembrane-anchored, trimeric viral surface glycoprotein (GP). GP mediates attachment to and entry into host cells, and is the intended target of antibody therapeutics. Because large portions of sequence are shared between GP and sGP, it has been hypothesized that sGP may potentially subvert the immune response or may contribute to pathogenicity. In this study, we present cryo-electron microscopy structures of GP and sGP in complex with GP-specific and GP/sGP cross-reactive antibodies undergoing human clinical trials. The structure of the sGP dimer presented here, in complex with both an sGP-specific antibody and a GP/sGP cross-reactive antibody, permits us to unambiguously assign the oligomeric arrangement of sGP and compare its structure and epitope presentation to those of GP. We also provide biophysical evaluation of naturally occurring GP/sGP mutations that fall within the footprints identified by our high-resolution structures. Taken together, our data provide a detailed and more complete picture of the accessible Ebolavirus glycoprotein landscape and a structural basis to evaluate patient and vaccine antibody responses towards differently structured products of the GP gene.

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  1. 1.

    et al. Efficacy and effectiveness of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomised trial. Lancet 386, 857–866 (2015).

  2. 2.

    et al. Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge. Nature Med. 20, 1126–1129 (2014).

  3. 3.

    et al. Use of ChAd3-EBO-Z Ebola virus vaccine in Malian and US adults, and boosting of Malian adults with MVA-BN-Filo: a phase 1, single-blind, randomised trial, a phase 1b, open-label and double-blind, dose-escalation trial, and a nested, randomised, double-blind, placebo-controlled trial. Lancet Infect. Dis. 16, 31–42 (2016).

  4. 4.

    et al. Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature 454, 177–182 (2008).

  5. 5.

    & Ebolavirus glycoprotein structure and mechanism of entry. Future Virol. 4, 621–635 (2009).

  6. 6.

    , , , & The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing. Proc. Natl Acad. Sci. USA 93, 3602–3607 (1996).

  7. 7.

    , , & The nonstructural small glycoprotein sGP of Ebola virus is secreted as an antiparallel-orientated homodimer. Virology 250, 408–414 (1998).

  8. 8.

    et al. Biochemical analysis of the secreted and virion glycoproteins of Ebola virus. J. Virol. 72, 6442–6447 (1998).

  9. 9.

    et al. Cross-reactive and potent neutralizing antibody responses in human survivors of natural ebolavirus infection. Cell 164, 392–405 (2016).

  10. 10.

    , , & The multiple roles of sGP in Ebola pathogenesis. Viral Immunol. 28, 3–9 (2015).

  11. 11.

    et al. Characterization of Zaire ebolavirus glycoprotein-specific monoclonal antibodies. Clin. Immunol. 141, 218–227 (2011).

  12. 12.

    et al. Successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies. Sci. Transl. Med. 4, 138ra81 (2012).

  13. 13.

    et al. Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques. Proc. Natl Acad. Sci. USA 109, 18030–18035 (2012).

  14. 14.

    et al. Epitopes involved in antibody-mediated protection from Ebola virus. Science 287, 1664–1666 (2000).

  15. 15.

    et al. Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature 514, 47–53 (2014).

  16. 16.

    et al. Structures of protective antibodies reveal sites of vulnerability on Ebola virus. Proc. Natl Acad. Sci. USA 111, 17182–17187 (2014).

  17. 17.

    et al. Mapping of Ebolavirus neutralization by monoclonal antibodies in the ZMapp cocktail using cryo-electron tomography and studies of cellular entry. J. Virol. (2016).

  18. 18.

    et al. Mechanism of binding to Ebola virus glycoprotein by the ZMapp, ZMAb, and MB-003 cocktail antibodies. J. Virol. 89, 10982–10992 (2015).

  19. 19.

    et al. Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein. Acta Crystallogr. D 65, 1162–1180 (2009).

  20. 20.

    et al. A shared structural solution for neutralizing ebolaviruses. Nature Struct. Mol. Biol. 18, 1424–1427 (2011).

  21. 21.

    et al. Host-primed Ebola virus GP exposes a hydrophobic NPC1 receptor-binding pocket, revealing a target for broadly neutralizing antibodies. mBio 7, e02154-15 (2016).

  22. 22.

    et al. Cathepsin cleavage potentiates the Ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change. J. Virol. 86, 364–372 (2012).

  23. 23.

    et al. Structural and molecular basis for Ebola virus neutralization by protective human antibodies. Science 351, 1343–1346 (2016).

  24. 24.

    et al. Neutralizing antibody fails to impact the course of Ebola virus infection in monkeys. PLoS Pathogens 3, e9 (2007).

  25. 25.

    et al. Fabs enable single particle cryoEM studies of small proteins. Structure 20, 582–592 (2012).

  26. 26.

    , , & Disulfide bond assignment of the Ebola virus secreted glycoprotein SGP. Biochem. Biophys. Res. Commun. 323, 696–702 (2004).

  27. 27.

    et al. Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak. Science 345, 1369–1372 (2014).

  28. 28.

    , , , & Genomic RNA editing and its impact on Ebola virus adaptation during serial passages in cell culture and infection of guinea pigs. J. Infect. Dis. 204 (Suppl 3), S941–S946 (2011).

  29. 29.

    et al. Type I and type II Fc receptors regulate innate and adaptive immunity. Nature Immunol. 15, 707–716 (2014).

  30. 30.

    et al. Monitoring of Ebola virus Makona evolution through establishment of advanced genomic capability in Liberia. Emerg. Infect. Dis. 21, 1135–1143 (2015).

  31. 31.

    et al. Molecular characterization of the monoclonal antibodies composing ZMAb: a protective cocktail against Ebola virus. Sci. Rep. 4, 6881 (2014).

  32. 32.

    et al. Sustained protection against Ebola virus infection following treatment of infected nonhuman primates with ZMAb. Sci. Rep. 3, 3365 (2013).

  33. 33.

    et al. Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant. Proc. Natl Acad. Sci. USA 108, 20690–20694 (2011).

  34. 34.

    et al. Mechanism of human antibody-mediated neutralization of Marburg virus. Cell 160, 893–903 (2015).

  35. 35.

    et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nature Methods 10, 584–590 (2013).

  36. 36.

    & Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

  37. 37.

    , , , & DoG Picker and TiltPicker: software tools to facilitate particle selection in single particle electron microscopy. J. Struct. Biol. 166, 205–213 (2009).

  38. 38.

    RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

  39. 39.

    & Prevention of overfitting in cryo-EM structure determination. Nature Methods 9, 853–854 (2012).

  40. 40.

    et al. Serverification of molecular modeling applications: the Rosetta Online Server that Includes Everyone (ROSIE). PLoS ONE 8, e63906 (2013).

  41. 41.

    , , & Toward high-resolution homology modeling of antibody Fv regions and application to antibody-antigen docking. Proteins 74, 497–514 (2009).

  42. 42.

    et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

  43. 43.

    & Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinformatics 47, 5.6.1–5.6.32 (2014).

  44. 44.

    et al. Atomic-accuracy models from 4.5-Å cryo-electron microscopy data with density-guided iterative local refinement. Nature Methods 12, 361–365 (2015).

  45. 45.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

  46. 46.

    , , , & Refinement of protein structures into low-resolution density maps using rosetta. J. Mol. Biol. 392, 181–190 (2009).

  47. 47.

    et al. EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nature Methods 12, 943–946 (2015).

  48. 48.

    et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).

  49. 49.

    & GlyProt: in silico glycosylation of proteins. Nucleic Acids Res. 33, W214–W219 (2005).

  50. 50.

    et al. Privateer: software for the conformational validation of carbohydrate structures. Nature Struct. Mol. Biol. 22, 833–834 (2015).

  51. 51.

    , & Carbohydrate Structure Suite (CSS): analysis of carbohydrate 3D structures derived from the PDB. Nucleic Acids Res. 33, D242–D246 (2005).

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The authors thank K. Swope and J. Morton of Kentucky Bioprocessing for preparing and sending all ZMapp IgGs. The authors thank T. Nieusma and B. Anderson of TSRI for assistance with loading cryo samples, data collection and running the microscopy facility, and B. Barad for providing the scripts for EMRinger. This work was supported by R01 AI067927 ‘Ebola Viral Glycoproteins: Structural Analysis’, the NIH/National Institute of Allergy and Infectious Diseases Center for Excellence in Translational Research grant no. U19AI109762 ‘Consortium for Immunotherapeutics Against Viral Hemorrhagic Fevers’, and U19AI109711 ‘Advancement of Treatments for Ebola and Marburg Virus Infections’. C.D.M. was supported by a predoctoral fellowship from the National Science Foundation. This is manuscript no. 29260 from The Scripps Research Institute.

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Author notes

    • Jesper Pallesen
    •  & Charles D. Murin

    These authors contributed equally to this work.


  1. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California 92037, USA

    • Jesper Pallesen
    • , Charles D. Murin
    • , Natalia de Val
    • , Christopher A. Cottrell
    • , Hannah L. Turner
    • , Kristian G. Andersen
    •  & Andrew B. Ward
  2. Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA

    • Charles D. Murin
    • , Kathryn M. Hastie
    • , Marnie L. Fusco
    • , Kristian G. Andersen
    •  & Erica Ollmann Saphire
  3. Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, Tennessee 37232, USA

    • Andrew I. Flyak
    •  & James E. Crowe Jr
  4. Mapp Biopharmaceutical, San Diego, California 92121, USA

    • Larry Zeitlin
  5. Department of Pediatrics, Vanderbilt University, Nashville, Tennessee 37232, USA

    • James E. Crowe Jr
  6. Vanderbilt Vaccine Center, Vanderbilt University, Nashville, Tennessee 37232, USA

    • James E. Crowe Jr
  7. Scripps Translational Institute, La Jolla, California 92122, USA

    • Kristian G. Andersen
  8. The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA

    • Erica Ollmann Saphire


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J.P., C.D.M., E.O.S. and A.B.W. designed the research plan. M.L.F. designed the protein expression constructs for GP and sGP. A.I.F. and J.E.C. isolated and purified BDBV91. L.Z. provided ZMapp antibodies. C.D.M. and H.L.T. expressed and purified proteins. K.M.H. and C.D.M. performed biophysical experiments. J.P., C.D.M. and H.L.T. prepared protein complexes. J.P., C.D.M and N.d.-V. froze grids. J.P. and N.d.-V. collected cryo data. J.P. processed the cryo data. C.D.M. collected and processed negative stain data. C.A.C. performed structure modelling and refinement. J.P., C.D.M., C.A.C., K.M.H., M.L.F., E.O.S. and A.B.W. analysed data. J.P., C.D.M., C.A.C., E.O.S. and A.B.W. wrote the manuscript. K.G.A. provided Makona outbreak sequence information.

Competing interests

Larry Zeitlin is a co-owner of Mapp Biopharmaceutical, Inc. The other authors declare no competing financial interests.

Corresponding authors

Correspondence to Erica Ollmann Saphire or Andrew B. Ward.

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    Supplementary Table 5

    2014 EBOV outbreak GP mutations.

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