The trimeric Sec61/SecY complex is a protein-conducting channel (PCC) for secretory and membrane proteins. Although Sec complexes can form oligomers, it has been suggested that a single copy may serve as an active PCC. We determined sub-nanometer resolution cryo-electron microscopy structures of eukaryotic ribosome-Sec61 complexes. In combination with biochemical data we found that in both idle and active states, the Sec complex is not oligomeric and interacts mainly via two cytoplasmic loops with the universal ribosomal adaptor site. In the active state the ribosomal tunnel and a central pore of the monomeric PCC were occupied by the nascent chain contacting loop 6 of the Sec complex. This provides a structural basis for the activity of a solitary Sec complex in cotranslational protein translocation.The protein-conducting channel (PCC) of the canonical secretory pathway is formed in all cells by the Sec61/SecY complex. It engages in the post-and co-translational translocation of secretory proteins across, and the insertion of integral membrane proteins into the membrane of the endoplasmic reticulum (ER) in eukaryotes and the plasma membrane of bacteria (1,2).# To whom correspondence should be addressed. beckmann@lmb.uni-muenchen.de; elisabet.mandon@umassmed.edu. In the co-translational translocation mode the ribosome with an emerging signal sequence is targeted to the membrane by the signal recognition particle (SRP) and its receptor (3). Here, the Sec complex acts as a receptor for the ribosome via its cytosolic loops (4). The alignment of the ribosomal tunnel with a central pore of the PCC allows direct movement of the nascent chain from the ribosomal tunnel exit across or into the membrane (5,6).The PCC-forming heterotrimeric Sec complex consists of one large subunit (Sec61α in Mammalia, Sec61p/Ssh1p in yeast, SecY in Bacteria) and two small subunits (Sec61β, γ in eukaryotes and SecE, G in Bacteria). Conflicting models have been presented as to how many of these heterotrimers are necessary to build an active PCC and what the actual path of the polypeptide chain is. The Escherichia coli SecYEG complex forms back-to-back dimers in two-dimensional crystals (7), and low resolution single particle electron microscopic (EM) data revealed a pentagonal ringlike morphology of the PCC interpreted as oligomers (5,6,(8)(9)(10)(11)(12). The monomeric crystal structure of an archaeal SecYEß complex (13), in combination with chemical cross-linking data (14), led to the interpretation that a single copy of the Sec complex is sufficient to serve as an active PCC, even when assembled into a dimer for posttranslational translocation (15) Cryo-EM and 3D reconstructionFor structure determination by cryo-EM we used digitonin-solubilized purified Ssh1 complex (Sec sixty-one homologue 1 from the yeast Saccharomyces cerevisiae) containing Ssh1p, Sbh2p and Sss1p (19). This complex is active in the co-translational translocation mode only, i.e. when ribosome-bound (20,21).We reconstituted the Ssh1 complex with in vitro progra...

Three programs have been developed to aid in the classification and visualization of RNA structure. BPViewer provides a web interface for displaying three-dimensional (3D) coordinates of individual base pairs or base pair collections. A web server, RNAview, automatically identifies and classifies the types of base pairs that are formed in nucleic acid structures by various combinations of the three edges, Watson-Crick, Hoogsteen and the Sugar edge. RNAView produces two-dimensional (2D) diagrams of secondary and tertiary structure in either Postscript, VRML or RNAML formats. The application RNAMLview can be used to rearrange various parts of the RNAView 2D diagram to generate a standard representation (like the cloverleaf structure of tRNAs) or any layout desired by the user. A 2D diagram can be rapidly reformatted using RNAMLview since all the parts of RNA (like helices and single strands) are dynamically linked while moving the selected parts. With the base pair annotation and the 2D graphic display, RNA motifs are rapidly identified and classified. A survey has been carried out for 41 unique structures selected from the NDB database. The statistics for the occurrence of each edge and of each of the 12 bp families are given for the combinations of the four bases: A, G, U and C. The program also allows for visualization of the base pair interactions by using a symbolic convention previously proposed for base pairs. The web servers for BPViewer and RNAview are available at http://ndbserver.rutgers.edu/services/. The application RNAMLview can also be downloaded from this site. The 2D diagrams produced by RNAview are available for RNA structures in the Nucleic Acid Database (NDB) at http://ndbserver.rutgers.edu/atlas/.