The 3D Arrangement of the 23 S and 5 S rRNA in the Escherichia coli 50 S Ribosomal Subunit Based on a Cryo-electron Microscopic Reconstruction at 7.5 A Ê Resolution Florian Mueller 1 , Ingolf Sommer 1 , Pavel Baranov 2 , Rishi Matadeen 3 Matthias Stoldt 4 , Jens Wo È hnert 4 , Matthias Go È rlach 4 , Marin van Heel 3 and Richard Brimacombe 1 * 1 Max-Planck-Institut fu Èr Molekulare Genetik, Ihnestrasse 73, 14195 Berlin, Germany 2 A.N. Belozerksy Institute of Physico-Chemical Biology & Department of Chemistry Moscow State University Moscow, 119899, Russia 3 Imperial College of Science Medicine and Technology Department of Biochemistry London, SW7 2AY, UK 4 Abteilung Molekulare Biophysik/NMR-Spektroskopie Institut fu È r Molekulare Bio- technologie, Postfach 100813 07708, Jena, Germany The Escherichia coli 23 S and 5 S rRNA molecules have been ®tted helix by helix to a cryo-electron microscopic (EM) reconstruction of the 50 S ribosomal subunit, using an un®ltered version of the recently published 50 S reconstruction at 7.5 A Ê resolution. At this resolution, the EM density shows a well-de®ned network of ®ne structural elements, in which the major and minor grooves of the rRNA helices can be discerned at many locations. The 3D folding of the rRNA molecules within this EM density is constrained by their well-established secondary structures, and further constraints are provided by intra and inter-rRNA crosslinking data, as well as by tertiary interactions and pseudoknots. RNA-protein cross-link and foot-print sites on the 23 S and 5 S rRNA were used to position the rRNA elements concerned in relation to the known arrangement of the ribosomal proteins as determined by immuno-electron microscopy. The published X-ray or NMR structures of seven 50 S ribosomal proteins or RNA-protein complexes were incorporated into the EM density. The 3D locations of cross-link and foot-print sites to the 23 S rRNA from tRNA bound to the ribosomal A, P or E sites were correlated with the positions of the tRNA molecules directly observed in earlier reconstructions of the 70 S ribosome at 13 A Ê or 20 A Ê . Similarly, the positions of cross-link sites within the peptidyl transferase ring of the 23 S rRNA from the aminoacyl residue of tRNA were correlated with the locations of the CCA ends of the A and P site tRNA. Sites on the 23 S rRNA that are cross-linked to the N termini of peptides of different lengths were all found to lie within or close to the internal tunnel connecting the peptidyl transferase region with the presumed peptide exit site on the solvent side of the 50 S sub- unit. The post-transcriptionally modi®ed bases in the 23 S rRNA form a cluster close to the peptidyl transferase area. The minimum conserved core elements of the secondary structure of the 23 S rRNA form a com- pact block within the 3D structure and, conversely, the points corre- sponding to the locations of expansion segments in 28 S rRNA all lie on the outside of the structure. # 2000 Academic Press Keywords: cryo-electron microscopy; computer modelling; cross-links and foot-prints; functional sites on rRNA; atomic structures of ribosomal components *Corresponding author Introduction The ribosome is a highly complex macromolecu- lar machine, and the elucidation of its structure has been the object of intensive research for several Present address: P. Baranov, Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA. Abbreviations used: EM, electron microscopy; IEM, immuno-EM; PIP, principal interface protrusion. doi:10.1006/jmbi.2000.3635 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 298, 35±59 0022-2836/00/010035±25 $35.00/0 # 2000 Academic Press