Molecular dynamics of protein complexes from four-dimensional cryo-electron microscopy J. Bernard Heymann, a,1 James F. Conway, b and Alasdair C. Steven a, * a Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA b Institut de Biologie Structurale J.-P. Ebel, 38027 Grenoble, France Received 22 December 2003, and in revised form 4 February 2004 Available online 14 March 2004 Abstract Cryo-electron microscopy of single particles offers a unique opportunity to detect and quantify conformational variation of protein complexes. Different conformers may, in principle, be distinguished by classification of individual projections in which image differences arising from viewing geometry are disentangled from variability in the underlying structures by ‘‘multiple particle analysis’’—MPA. If the various conformers represent dynamically related states of the same complex, MPA has the potential to visualize transition states, and eventually to yield movies of the dynamic process. Ordering the various conformers into a time series is facilitated if cryo-EM data are taken at successive times from a system that is known to be developing in time. Virus maturation represents a relatively tractable dynamic process because the changes are large and irreversible and the rate of the natural process may be conveniently slowed in vitro by adjusting the environmental conditions. We describe the strategy employed in a recent analysis of herpes simplex virus procapsid maturation (Nat. Struct. Biol. 10 (2003) 334–341), compare it with previous work on the maturation of bacteriophage HK97 procapsid, and discuss various factors that impinge on the feasibility of performing similar experimental analyses of molecular dynamics in the general case. Published by Elsevier Inc. Keywords: Conformational change; Molecular movie; Molecular dynamics; Image classification; Single particle analysis; Multiple particle analysis 1. Introduction It is now increasingly appreciated that most funda- mental biological processes are carried out by large macromolecular assemblies (or ÔmachinesÕ) (Alberts, 1998; Sali et al., 2003), and that these assemblies are often dynamic systems that follow elaborate duty cycles while undergoing large structural changes. Cryo-elec- tron microscopy in combination with single particle analysis (SPA) has emerged as a powerful approach for visualizing assemblies in three dimensions (Baumeister and Steven, 2000; Frank, 2002; Frank et al., 2002; Ruprecht and Nield, 2001). This approach has pro- gressively improved in resolution—now better than 10  A in a growing number of cases; is relatively parsimonious in terms of the amounts of material required; and has the advantage of depicting the solution structure of the specimen, unconstrained by crystal contacts. The basic principle underlying SPA is that each member of a large set of two-dimensional cryo-EM images represents a projection of the same three-dimensional object, and after identifying as precisely as possible the viewing geometry of each projection, these data are used to reconstruct its three-dimensional structure. Although cryo-EM has been used to characterize a substantial number of conformational changes in the sense of comparing ÔbeforeÕ and ÔafterÕ states (e.g. Jontes et al., 1995), it has for the most part remained a static visualization technique. It does, however, have the po- tential to capture functional intermediates or transition states according to the following scenario. If in vitro * Corresponding author: Fax: 1-301-480-7629. E-mail address: Alasdair_Steven@nih.gov (A.C. Steven). 1 Present address: Division of Biology, California Institute of Technology, 1200 East California Blvd., Pasadena, CA 91125, USA. 1047-8477/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.jsb.2004.02.006 Journal of Structural Biology 147 (2004) 291–301 Journal of Structural Biology www.elsevier.com/locate/yjsbi