D-loop Dynamics and Near-Atomic-Resolution Cryo-EM Structure of Phalloidin-Bound F-Actin

Structure. 2020 May 5;28(5):586-593.e3. doi: 10.1016/j.str.2020.04.004. Epub 2020 Apr 28.


Detailed molecular information on G-actin assembly into filaments (F-actin), and their structure, dynamics, and interactions, is essential for understanding their cellular functions. Previous studies indicate that a flexible DNase I binding loop (D-loop, residues 40-50) plays a major role in actin's conformational dynamics. Phalloidin, a "gold standard" for actin filament staining, stabilizes them and affects the D-loop. Using disulfide crosslinking in yeast actin D-loop mutant Q41C/V45C, light-scattering measurements, and cryoelectron microscopy reconstructions, we probed the constraints of D-loop dynamics and its contribution to F-actin formation/stability. Our data support a model of residues 41-45 distances that facilitate G- to F-actin transition. We report also a 3.3-Å resolution structure of phalloidin-bound F-actin in the ADP-Pi-like (ADP-BeFx) state. This shows the phalloidin-binding site on F-actin and how the relative movement between its two protofilaments is restricted by it. Together, our results provide molecular details of F-actin structure and D-loop dynamics.

Keywords: D-loop dynamics; F-actin structure; chemical crosslinking; high-resolution cryo-EM.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / chemistry*
  • Actins / genetics
  • Actins / metabolism*
  • Cross-Linking Reagents / chemistry
  • Cryoelectron Microscopy / methods
  • Deoxyribonuclease I / metabolism
  • Disulfides / chemistry
  • Models, Molecular
  • Mutation
  • Phalloidine / chemistry*
  • Phalloidine / metabolism*
  • Saccharomyces cerevisiae / genetics


  • Actins
  • Cross-Linking Reagents
  • Disulfides
  • Phalloidine
  • Deoxyribonuclease I