Graduate School of Frontier Biosciences, Osaka University

Japanese

Insight into the dynamics of protein machinery by near-atomic cryo-EM: DGNNV virus and RNA polymerase II
Dr. Wei-hau Chang (Associate Research Fellow, Institute of Chemistry, Academia Sinica, Taipei, Taiwan)

Date/Time

Nov 16, 2016 (Wed), 11:00-

Place

3F Seminar room, Nanobiology Building

Speaker

Dr. Wei-hau Chang (Associate Research Fellow, Institute of Chemistry, Academia Sinica, Taipei, Taiwan)

Title

Insight into the dynamics of protein machinery by near-atomic cryo-EM: DGNNV virus and RNA polymerase II

Abstract

The first part of the talk is on a DGNNV virus where we use an old model 200 kV cryo-EM equipped with direct electron camera to reach about 3 Angstrom resolution. Nervous necrosis viruses (NNV) cause severe loss in fish aquaculture as they target more than 40 species of fish worldwide. NNV is a simple virus whose proteins contain an RNA polymerase and a capsid protein, comprised of a shell and a protrusion domain. NNV can be internalized into the host cell by endocytosis pathways, but the structural mechanism of genomic RNAs releasing has remained unknown. Here, we survey the cryo-EM structure of DGNNV particles in pH 8, 6.5, and 5 conditions, which mimic the environments of sea water, early endosome and late endosome, respectively. Strikingly, as pH approaches pH 5, massive structural re-modeling occurs both at the shell and the protrusion en bloc. The 5-fold pore opens up to 12 Å, sufficiently large for the passage of single strand RNA; the protrusion becomes enlarged with increased hydrophobic surface, moves toward the shell by 5 Å, and corkscrews clockwise further by 10 degrees. These findings suggest that the protrusion domain together with the hinge of the capsid protein is a foldable cramponthat can respond to the lowering of pH and pull up towards or insert itself into the membrane, thereby facilitating the delivery of the genome into the cytosol. The second part of the talk is on RNA polymerase II where we started the collaboration on phase plate imaging with Nagayama and Murata since 2012. By using RELION algorithm, we were able to separate 60,000 RNA polymerase II phase plate images into 8 classes, each of them has reached 9 Angstrom resolution. This work has helped demonstrate that the phase plate is powerful in recording information sensitive to conformation.

Host

Keiichi Namba