Structural Studies of HIV-1 Proteins by Solid State NMR

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Progress in FY2021 was in the following area: STRUCTURAL CHARACTERIZATION OF SARS COV-2 NUCLEOCAPSID PROTEIN/RNA COMPLEXES: Within mature coronaviruses, the viral RNA is associated in some way with the nucleocapsid (N) protein. The molecular structures of RNA-binding and dimerization domains are well-known from crystallographic and NMR studies. These two domains are connected by a flexible linker segment. Due to the flexibility of the linker segment, the flexibility of RNA, and the absence of experimental structural data for full-length N protein and for complexes of full-length N, the structure of such complexes remains unknown. Models in which N protein/RNA complexes are filamentous, helical assemblies have been proposed. It has also been proposed that these assemblies have a beads-on-a-string morphology, with little detail about the molecular structural nature of the beads. To address this problem, we are attempting to form N protein/RNA complexes in vitro, using synthetic RNA and full-length N protein expressed in bacteria, and determine their molecular structures by cryo-EM. After multiple attempts, we have found conditions under which filamentous assemblies can be created reproducibly with high yield. We have recorded cryo-EM images and preliminary analyses. These image analyses lead to good-quality 2D class images (i.e., averages of images of multiple filament segments that show clear structural features). However, the 2D class images indicate that our N protein/RNA filaments adhere to cryo-EM grids in a single preferred orientation. Since full 3D density reconstruction requires that all orientations be sampled in the cryo-EM images, we have not yet been able to determine a 3D density map. As an alternative, we have recorded tomographic data sets (cryo-EM images with multiple tilt angles) and are working towards determination of the 3D density from these tomograms. We hope to complete this work within the next several months.