Animation of the Dynamical Events of the Elongation Cycle Based on Cryoelectron Microscopy of Functional Complexes of the Ribosome Joachim Frank,* , † , ‡ Amy B. Heagle,* , † and Rajendra K. Agrawal* *Wadsworth Center, †Howard Hughes Medical Institute, and ‡Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, New York, 12201-0509 Received May 3, 1999 Using three-dimensional cryoelectron micros- copy, the binding positions of tRNA and elongation factors EF-G and EF-Tu (the latter complexed with aminoacyl tRNA and GTP) on the ribosome were determined in previous studies. On the basis of these studies, the dynamical events that take place in the course of the elongation cycle of protein synthesis have been animated. The resulting 3-min movie is accessible on the website of this journal (http://www.idealibrary.com). The following article provides a brief annotation of those frames of the movie for which experimental support is avail- able.  1999 Academic Press Key Words: protein biosynthesis; elongation cycle. INTRODUCTION The ribosome, a complex macromolecular ma- chine, is the site of protein synthesis in all organ- isms. The genetic message, in the form of base triplets (codons) of the messenger RNA (mRNA), is translated into a colinear sequence of amino acids, which are strung together to form a polypeptide chain. The translation is accomplished by the bind- ing, to the ribosome, of transfer RNA (tRNA) mol- ecules that bring amino acids to the ribosome in a codon-specific manner. The bacterial ribosome is composed of a small (30S) and a large (50S) subunit. While decoding of the mRNA by the tRNA takes place on the small subunit, the catalysis of peptide bond formation takes place on the large subunit. The elongation of the polypeptide chain by addition of one amino acid is a cyclic process (elongation cycle) and involves the coordinated movements of tRNA and mRNA through the ribosome. Cryoelectron microscopy has provided the first detailed three-dimensional maps of the Escherichia coli 70S ribosome (Frank et al., 1995a,b; Stark et al., 1995; Malhotra et al., 1998). While the resolution is still limited to the range of 15–20 Å (see review by Agrawal and Frank, 1999), it has been sufficient to allow the visualization and localization of tRNA and protein factors that enable the insertion and translo- cation of tRNA, respectively, at various stages of the elongation cycle. On the basis of several ligand binding experiments (Agrawal et al., 1996, 1998a,b, 1999a,b; Malhotra et al., 1998; R. K. Agrawal, R. A. Grassucci, N. Burkhardt, K. H. Nierhaus, and J. Frank, unpublished results) in which the ribosome, programmed with mRNA, is at different, defined stages of the cycle, the three-dimensional trajecto- ries of the tRNA and elongation factors have been animated to show their movements through the ribosome. While the actual time spent and the precise paths between successive binding positions are not well understood, the movie nevertheless captures the main sequence of events and should be valuable as a teaching tool. Within the movie, we identify a series of ‘‘anchor frames’’ showing ligand positions that are based on experimental evidence obtained from one or several cryo-EM reconstructions. Thus, the main body of this article describes a gallery of anchor frames (Fig. 1) that are annotated and linked to the supporting literature. METHODS Design of the Movie The movie was designed using 3D modeling/animation software (Lightwave 3D, version 5.5, NewTek Inc., San Antonio, TX) that allows for the experimentally determined binding positions of various ligands on the ribosome to be recorded as a set of individual ‘‘keyframes.’’ Intermediate frames between the speci- fied keyframes are calculated by interpolation to produce a smooth transition from one position to the next. In this way, we demonstrate the movements of each ligand as an animated sequence of events. The original cryo-EM density of the ribosome was visualized as a surface rendering using IRIS Explorer (version 3.5, The Numeri- cal Algorithms Group., Inc., Downers Grove, IL) and converted to Inventor format. The models of tRNAs and elongation factors were derived from their X-ray structures that were fitted into the cryo-EM densities. Upon conversion of the 3D models from Inventor format to Lightwave 3D format (using Interchange, version 4.5, Viewpoint Data Labs International, Inc., Orem, UT), Journal of Structural Biology 128, 15–18 (1999) Article ID jsbi.1999.4138, available online at http://www.idealibrary.com on 15 1047-8477/99 $30.00 Copyright  1999 by Academic Press All rights of reproduction in any form reserved.