Structure and size determination of bacteriophage P2 and P4 procapsids: function of size responsiveness mutations.

Bacteriophage P4 is dependent on structural proteins supplied by a helper phage, P2, to assemble infectious virions. Bacteriophage P2 normally forms an icosahedral capsid with T=7 symmetry from the gpN capsid protein, the gpO scaffolding protein and the gpQ portal protein. In the presence of P4, however, the same structural proteins are assembled into a smaller capsid with T=4 symmetry. This size determination is effected by the P4-encoded protein Sid, which forms an external scaffold around the small P4 procapsids. Size responsiveness (sir) mutants in gpN fail to assemble small capsids even in the presence of Sid. We have produced large and small procapsids by co-expression of gpN with gpO and Sid, respectively, and applied cryo-electron microscopy and three-dimensional reconstruction methods to visualize these procapsids. gpN has an HK97-like fold and interacts with Sid in an exposed loop where the sir mutations are clustered. The T=7 lattice of P2 has dextro handedness, unlike the laevo lattices of other phages with this fold observed so far.

Validation of the orthogonal tilt reconstruction method with a biological test sample.

Electron microscopy of frozen-hydrated samples (cryo-EM) can yield high resolution structures of macromolecular complexes by accurately determining the orientation of large numbers of experimental views of the sample relative to an existing 3D model. The "initial model problem", the challenge of obtaining these orientations ab initio, remains a major bottleneck in determining the structure of novel macromolecules, chiefly those lacking internal symmetry. We previously proposed a method for the generation of initial models--orthogonal tilt reconstruction (OTR)--that bypasses limitations inherent to the other two existing methods, random conical tilt (RCT) and angular reconstitution (AR). Here we present a validation of OTR with a biological test sample whose structure was previously solved by RCT: the complex between the yeast exosome and the subunit Rrp44. We show that, as originally demonstrated with synthetic data, OTR generates initial models that do not exhibit the "missing cone" artifacts associated with RCT and show an isotropic distribution of information when compared with the known structure. This eliminates the need for further user intervention to solve these artifacts and makes OTR ideal for automation and the analysis of heterogeneous samples. With the former in mind, we propose a set of simple quantitative criteria that can be used, in combination, to select from a large set of initial reconstructions a subset that can be used as reliable references for refinement to higher resolution.

Structure of the mammalian 80S ribosome at 8.7 A resolution.

In this paper, we present a structure of the mammalian ribosome determined at approximately 8.7 A resolution by electron cryomicroscopy and single-particle methods. A model of the ribosome was created by docking homology models of subunit rRNAs and conserved proteins into the density map. We then modeled expansion segments in the subunit rRNAs and found unclaimed density for approximately 20 proteins. In general, many conserved proteins and novel proteins interact with expansion segments to form an integrated framework that may stabilize the mature ribosome. Our structure provides a snapshot of the mammalian ribosome at the beginning of translation and lends support to current models in which large movements of the small subunit and L1 stalk occur during tRNA translocation. Finally, details are presented for intersubunit bridges that are specific to the eukaryotic ribosome. We suggest that these bridges may help reset the conformation of the ribosome to prepare for the next cycle of chain elongation.

Architecture of the ribosome-channel complex derived from native membranes.

The mammalian Sec61 complex forms a protein translocation channel whose function depends upon its interaction with the ribosome and with membrane proteins of the endoplasmic reticulum (ER). To study these interactions, we determined structures of "native" ribosome-channel complexes derived from ER membranes. We find that the ribosome is linked to the channel by seven connections, but the junction may still provide a path for domains of nascent membrane proteins to move into the cytoplasm. In addition, the native channel is significantly larger than a channel formed by the Sec61 complex, due to the presence of a second membrane protein. We identified this component as TRAP, the translocon-associated protein complex. TRAP interacts with Sec61 through its transmembrane domain and has a prominent lumenal domain. The presence of TRAP in the native channel indicates that it may play a general role in translocation. Crystal structures of two Sec61 homologues were used to model the channel. This analysis indicates that there are four Sec61 complexes and two TRAP molecules in each native channel. Thus, we suggest that a single Sec61 complex may form a conduit for translocating polypeptides, while three copies of Sec61 play a structural role or recruit accessory factors such as TRAP.

Cleavage leads to expansion of bacteriophage P4 procapsids in vitro.

Proteolytic cleavage of the structural proteins is an important part of the maturation process for most bacteriophages and other viruses. In the double-stranded DNA bacteriophages this cleavage is associated with DNA packaging, capsid expansion, and scaffold removal. To understand the role of protein cleavage in the expansion of bacteriophages P2 and P4, we have experimentally cleaved P4 procapsids produced by overexpression of the capsid and scaffolding proteins. The cleavage leads to particle expansion and scaffold removal in vitro. The resulting expanded capsid has a thin-shelled structure similar, but not identical, to that of mature virions.