title: Determination of molecular structures of HIV envelope glycoproteins using cryo-electron tomography and automated sub-tomogram averaging.
abstract: Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) (2). Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor (5,9). The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine. The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at ˜20 Å resolution (9,14). This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH (http://biowulf.nih.gov). This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, USA.
authors.forename: Joel R
authors.initials: JR
authors.lastname: Meyerson
authors.forename: Tommi A
authors.initials: TA
authors.lastname: White
authors.forename: Donald
authors.initials: D
authors.lastname: Bliss
authors.forename: Amy
authors.initials: A
authors.lastname: Moran
authors.forename: Alberto
authors.initials: A
authors.lastname: Bartesaghi
authors.forename: Mario J
authors.initials: MJ
authors.lastname: Borgnia
authors.forename: M Jason V
authors.initials: MJ
authors.lastname: de la Cruz
authors.forename: David
authors.initials: D
authors.lastname: Schauder
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Hartnell
authors.forename: Rachna
authors.initials: R
authors.lastname: Nandwani
authors.forename: Moez
authors.initials: M
authors.lastname: Dawood
authors.forename: Brianna
authors.initials: B
authors.lastname: Kim
authors.forename: Jun Hong
authors.initials: JH
authors.lastname: Kim
authors.forename: John
authors.initials: J
authors.lastname: Sununu
authors.forename: Lisa
authors.initials: L
authors.lastname: Yang
authors.forename: Siddhant
authors.initials: S
authors.lastname: Bhatia
authors.forename: Carolyn
authors.initials: C
authors.lastname: Subramaniam
authors.forename: Darrell E
authors.initials: DE
authors.lastname: Hurt
authors.forename: Laurent
authors.initials: L
authors.lastname: Gaudreault
authors.forename: Sriram
authors.initials: S
authors.lastname: Subramaniam
mesh_terms: D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D055032:Electron Microscope Tomography / Q000295:instrumentation / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry*; D015497:HIV-1 / Q000737:chemistry* / Q000648:ultrastructure; D006801:Humans; D008958:Models, Molecular; D014759:Viral Envelope Proteins / Q000737:chemistry*
chemical_list: D006023:Glycoproteins; D014759:Viral Envelope Proteins