Computational protocol: Structural basis for cofilin binding and actin filament disassembly

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Protocol publication

[…] A total of 1,111 images were collected in two microscope sessions (414 and 697 micrographs, respectively). Image processing was performed with RELION 2.0 software,. After motion correction with MotionCorr and CTF estimation with Gctf, 1,093 images were selected for further image processing. Filaments were manually picked with e2helixboxer, after which 245,236 particles were extracted at a box size of 320 × 320 pixels. After 2D classification, 202,217 particles were selected. The initial 3D reference was prepared from the Galkin model of cofilactin (PDB 3J0S) with sxpdb2em. The initial 3D reference was low-pass filtered to 25 Å, and 3D refinement was performed with a helical symmetry search without solvent flattening. Helical symmetry was converged to –162.1° twist/27.6 Å rise along the left-handed helix (corresponding to Δϕ = 35.8° in the main body of the text along the actin right-handed helical strand) and the resolution reached 4.3 Å. Additional 3D refinement was performed with fixed helical symmetry with solvent flattening, refining the resolution to 4.0 Å. RELION movie refinement, particle polishing and final 3D refinement were performed against the images collected in the second microscope session only (697 micrographs, 86,388 particles). The final resolution reached 3.8 Å (Supplementary Fig. ). [...] The initial atomic model consisted of five actin and three cofilin molecules that were prepared from an atomic model of G-actin (PDB 1J6Z) and chicken cofilin (PDB 1TVJ). Actin was modified so that its N-terminus (residue 1–9), C-terminus (361–375) and subdomain 2 (36–67) were replaced with those of the atomic model of F-actin (PDB 2ZWH). Moreover, the ADP-associated Ca2+ and methylated-histidine (residue 73) were replaced with Mg2+ and unmodified histidine, respectively, and the N-terminal amine was acetylated. The initial atomic model was flexibly fitted to a cryo-EM density map with the cascade-MDFF protocol, on NAMD 2.12 software. The calculation was performed under harmonic helical symmetry restraints on all atoms except hydrogens, and the solvent effect was modeled with Generalized Born implicit solvent. After the cascade-MDFF calculation converged, Ramachandran outliers were fixed and refined with the phenix.real_space_refine program. The resulting model was iteratively refined using COOT, MDFF and phenix.real_space_refine. After convergence, histidine 73 was replaced by a methylated-histidine. [...] The rotation axes of domain movement were obtained by the DomainSelect program (DynDom) based on the two rigid bodies shown in Supplementary Table  and Fig. . […]

Pipeline specifications

Software tools RELION, MotionCorr, Gctf, NAMD, PHENIX, Coot, DynDom
Applications cryo-EM, Protein structure analysis
Chemicals Adenosine Diphosphate