3.88 A structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy.

Cytoplasmic polyhedrosis virus (CPV) is unique within the Reoviridae family in having a turreted single-layer capsid contained within polyhedrin inclusion bodies, yet being fully capable of cell entry and endogenous RNA transcription. Biochemical data have shown that the amino-terminal 79 residues of the CPV turret protein (TP) is sufficient to bring CPV or engineered proteins into the polyhedrin matrix for micro-encapsulation. Here we report the three-dimensional structure of CPV at 3.88 A resolution using single-particle cryo-electron microscopy. Our map clearly shows the turns and deep grooves of alpha-helices, the strand separation in beta-sheets, and densities for loops and many bulky side chains; thus permitting atomic model-building effort from cryo-electron microscopy maps. We observed a helix-to-beta-hairpin conformational change between the two conformational states of the capsid shell protein in the region directly interacting with genomic RNA. We have also discovered a messenger RNA release hole coupled with the mRNA capping machinery unique to CPV. Furthermore, we have identified the polyhedrin-binding domain, a structure that has potential in nanobiotechnology applications.

Structural studies of proteinase entrapment by human alpha 2-macroglobulin using 3-D electron microscopy

Human a2 -macroglobulin ( a2 M; homotetramer, Mr 720 kDa) is an essential scavenger of proteinases in the serum. Each of its four subunits has a ‘bait region’, with cleavage sequences for almost all endo-proteinases, an unusual thiol ester moiety and a receptor-binding domain (RBD). Bait region cleavage in native a2 M ( a2 M-N) by a proteinase results in rapid thiol ester breakage, with a large-scale structural transformation, in which a2 M uniquely entraps the proteinase in a cage-like structure and exposes receptor-binding domains for rapid endocytosis. Transformed a2 M ( a2 M-TR) contains up to two proteinases, which remain active to small substrates. 3-D electron microscopy is optimally suited to study this unusual structural change at resolutions near (1/30) Å−1. ^ The structural importance of the thiol esters was demonstrated by a genetically-engineered a2 M, with the cysteines involved in thiol ester formation mutated to serines, which appeared structurally homologous to a2 M-TR. This demonstrates that the four highly labile thiol esters alone maintain the a2 M-N structure, while the ‘closed trap’ formed by a2 M-TR is a more stable structural form. ^ Half-transformed a2 M ( a2 M-HT), with cleaved bait regions and thiol esters in only two of its four subunits, provides an important structural link between a2 M-N and a2 M-TR. A comparison with a2 M-N showed the two proteinase-entrapping domains were above and below the plane bisecting the long axis. Both a2 M-N and a2 M-TR consist of two dense, oppositely twisted strands with significant interconnections, indicating that the structural change involves a rotation of these strands. In a2 M-HT these strands were partially untwisted with large central openings, revealing the manner in which the proteinase enters the internal cavity of a2 M. ^ In reconstructions of a2 M-N, a2 M-HT and a2 M-TR labeled with a monoclonal Fab, the Fabs were located on distal ends of each constitutive strand, demonstrating an anti-parallel arrangement of the subunits. Separation between the top and bottom pairs of Fabs was nearly the same on all structures, but the pairs were rotated about the long axis. Taken together, these results indicate that upon proteinase cleavage the two strands in a2 M-N separate. The proteinase enters the structure, while the strands re-twist to encage it. In a2 M-TR, which displays receptor-binding arms, more than two subunits are transformed as strands in the transformed half of a2 M-HT were not separated. ^