title: Model Building and Refinement of a Natively Glycosylated HIV-1 Env Protein by High-Resolution Cryoelectron Microscopy.
abstract: Secretory and membrane proteins from mammalian cells undergo post-translational modifications, including N-linked glycosylation, which can result in a large number of possible glycoforms. This sample heterogeneity can be problematic for structural studies, particularly X-ray crystallography. Thus, crystal structures of heavily glycosylated proteins such as the HIV-1 Env viral spike protein have been determined by removing the majority of glycans. This step is most frequently carried out using Endoglycosidase H (EndoH) and requires that all expressed glycans be in the high-mannose form, which is often not the native glycoform. With significantly improved technologies in single-particle cryoelectron microscopy, we demonstrate that it is now possible to refine and build natively glycosylated HIV-1 Env structures in solution to 4.36 Å resolution. At this resolution we can now analyze the complete epitope of a broadly neutralizing antibody (bnAb), PGT128, in the context of the trimer expressed with native glycans.
authors.affiliation: Department of Integrative Structural and Computational Biology, Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; International AIDS Vaccine Initiative (IAVI) Neutralizing Antibody Center and Collaboration for AIDS Vaccine Discovery (CAVD), The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Jeong Hyun
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Integrative Structural and Computational Biology, Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; International AIDS Vaccine Initiative (IAVI) Neutralizing Antibody Center and Collaboration for AIDS Vaccine Discovery (CAVD), The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Natalia
authors.initials: N
authors.lastname: de Val
authors.affiliation: Laboratory of Genetics and Helmsley Center for Genomic Medicine, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Lyumkis
authors.affiliation: Department of Integrative Structural and Computational Biology, Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; International AIDS Vaccine Initiative (IAVI) Neutralizing Antibody Center and Collaboration for AIDS Vaccine Discovery (CAVD), The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address: abward@scripps.edu.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Ward
mesh_terms: D057134:Antibodies, Neutralizing / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D000939:Epitopes / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D015483:HIV Antibodies / Q000737:chemistry* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D057134:Antibodies, Neutralizing; D000939:Epitopes; D015483:HIV Antibodies; D011994:Recombinant Proteins; D054299:env Gene Products, Human Immunodeficiency Virus