Tubular Crystals and Helical Arrays: Structural Determination of HIV-1 Capsid Assemblies Using Iterative Helical Real-Space Reconstruction

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 955)

Abstract

Helical structures are important in many different life forms and are well-suited for structural studies by cryo-EM. A unique feature of helical objects is that a single projection image contains all the views needed to perform a three-dimensional (3D) crystallographic reconstruction. Here, we use HIV-1 capsid assemblies to illustrate the detailed approaches to obtain 3D density maps from helical objects. Mature HIV-1 particles contain a conical- or tubular-shaped capsid that encloses the viral RNA genome and performs essential functions in the virus life cycle. The capsid is composed of capsid protein (CA) oligomers which are helically arranged on the surface. The N-terminal domain (NTD) of CA is connected to its C-terminal domain (CTD) through a flexible hinge. Structural analysis of two- and three-dimensional crystals provided molecular models of the capsid protein (CA) and its oligomer forms. We determined the 3D density map of helically assembled HIV-1 CA hexamers at 16 Å resolution using an iterative helical real-space reconstruction method. Docking of atomic models of CA-NTD and CA-CTD dimer into the electron density map indicated that the CTD dimer interface is retained in the assembled CA. Furthermore, molecular docking revealed an additional, novel CTD trimer interface.

Key words

Cryo-EM Tubular crystals Helical reconstruction Real space Docking HIV-1 capsid 

Notes

Acknowledgments

The authors would like to thank Dr. Jinwoo Ahn and Danxia Ke for technical support, and Dr. Teresa Brosenitsch for critical reading of the manuscript. This work was supported by GM082251 and GM085043.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Department of Structural BiologyUniversity of Pittsburgh School of MedicinePittsburghUSA

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