Structures of Unliganded and ATP-Bound States of the Escherichia coli Chaperonin GroEL by Cryoelectron Microscopy Alan M. Roseman,* ,1 Neil A. Ranson,* Brent Gowen,† ,2 Stephen D. Fuller,† ,2 and Helen R. Saibil* ,3 *Department of Crystallography, Birkbeck College London, Malet Street, London, WC1E 7HX, United Kingdom; and †The Structural Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Received March 5, 2001, and in revised form April 23, 2001; published online August 2, 2001 We have developed an angular refinement proce- dure incorporating correction for the microscope contrast transfer function, to determine cryoelec- tron microscopy (cryo-EM) structures of the Esche- richia coli chaperonin GroEL in its apo and ATP- bound forms. This image reconstruction procedure is verified to 13-Å resolution by comparison of the cryo-EM structure of unliganded GroEL with the crystal structure. Binding, encapsulation, and re- lease of nonnative proteins by GroEL and its co- chaperone GroES are controlled by the binding and hydrolysis of ATP. Seven ATP molecules bind coop- eratively to one heptameric ring of GroEL. This binding causes long-range conformational changes that determine the orientations of remote sub- strate-binding sites, and it also determines the con- formation of subunits in the opposite ring of GroEL, in a negatively cooperative mechanism. The confor- mation of GroEL–ATP was determined at 15-Å res- olution. In one ring of GroEL–ATP, the apical (sub- strate-binding) domains are extremely disordered, consistent with the high mobility needed for them to achieve the 60° elevation and 90° twist of the GroES-bound state. Unexpectedly, ATP binding also increases the separation between the two rings, although the interring contacts are present in the density map. © 2001 Academic Press Key Words: cryoelectron microscopy; 3D recon- struction; molecular chaperone; chaperonin; ATP binding. INTRODUCTION The Escherichia coli molecular chaperones GroEL and GroES are the most widely studied members of the chaperonin family. They are absolutely required for the folding of a subset of E. coli proteins and thus are essential for cell viability at all growth temper- atures (Fayet et al., 1989). In vitro, they can increase the yield (and in some cases the rate) of folding of some substrate proteins in an ATP-dependent man- ner (Hartl, 1996; Bukau and Horwich, 1998). GroEL is a homo-tetradecamer of 58-kDa sub- units, which form two back-to-back stacked hep- tameric rings (Sigler et al., 1998; Ranson et al., 1998). The crystal structure shows that each GroEL subunit is divided into three domains (Braig et al., 1994). An equatorial domain in the central layer of the complex forms all inter-ring as well as most of the intra-ring contacts and contains the ATP-bind- ing site (Boisvert et al., 1996). An apical domain forms the ends of the chaperonin complex and con- tains the binding sites for nonnative substrate pro- teins and a coprotein, GroES (Fenton et al., 1994). Linking the equatorial and apical domains is a small intermediate domain connected via two flexible hinge regions that allows a wide range of apical domain conformations to be adopted. This hierarchical oligomeric structure allows the binding of ATP to GroEL to be regulated by a com- plex system of cooperativity. Binding of ATP occurs to one ring of the GroEL complex with high affinity and strong positive cooperativity (Gray and Fersht, 1991; Jackson et al., 1993). However, this binding event reduces the affinity of the second ring for ATP in a negatively cooperative manner (Yifrach and Horovitz, 1994, 1995; Burston et al., 1995). As a result, the two GroEL rings adopt different confor- mations and functional states throughout the reac- tion cycle. Implicit in these cooperative binding phe- nomena is the fact that allosteric signals must be sent around the GroEL ring, and between GroEL 1 Present address: Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK. 2 Present address: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roos- evelt Drive, Oxford, OX3 7BN, UK. 3 To whom correspondence should be addressed. E-mail: h.saibil@mail.cryst.bbk.ac.uk. Journal of Structural Biology 135, 115–125 (2001) doi:10.1006/jsbi.2001.4374, available online at http://www.idealibrary.com on 115 1047-8477/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.