Structural basis for cofilin binding and actin filament disassembly.

Protein preparation

Actin was extracted from chicken skeletal muscle (purchased from a slaughterhouse as chicken meat) acetone powder, purified by polymerization and depolymerization steps 47 , and further purified by gel filtration chromatography on a Sephacryl S-200 h column (GE Healthcare). Purified actin was stored on ice until use. Chicken cofilin was expressed in Escherichia coli (BL21(DE3), Thermo Fisher) transformed with an expression vector of wild type chicken cofilin, kindly provided by H. Abe, Chiba University, Japan 27 , 48 . The Escherichia coli ( E. coli ) cells were harvested by centrifugation and then re-suspended in a buffer containing 50 mM Tris-HCl, pH 8.0, 20 mM EDTA, 1 mM DTT, and cOmplete Protease Inhibitor Cocktail (Roche). To lyse the cells, Bug Buster Protein Extract Reagent (Novagen) and 1 mg/ml lysozyme (from Egg White, Wako) were added to the cell suspension and incubated for 15 min, followed by sonication using the UD-201 sonicator (TOMY). The cell lysate was clarified by ultra-centrifugation at 165,000 × g . Cofilin was fractionated from the clarified cell lysate by ammonium sulfate fractionation (60–80% saturation) and purified by cation exchange chromatography using a HiTrapSP HP column (GE Healthcare) with a start buffer containing 20 mM MES (pH 6.2) and 5 mM EDTA and an elution buffer containing 20 mM MES (pH 6.2), 5 mM EDTA, and 1 M NaCl. The eluted sample was further purified by gel filtration chromatography on a Superdex 75 pg column (GE Healthcare) with a buffer containing 5 mM PIPES (pH 6.6), 2 mM MgCl 2 and 50 mM KCl. After addition of 1 mM DTT, purified cofilin was frozen using liquid nitrogen and stored at –80 °C until use. Cryo-EM sample preparation Actin (12 μM) was polymerized in F-buffer (15 mM PIPES-NaOH (pH 6.6), 2 mM MgCl 2 , 50 mM KCl, 0.1 mM EGTA, 0.2 mM ATP, and 1 mM DTT) for 90 min at room temperature. The acidic pH (6.6) stabilizes the cofilactin filament 49 , 50 . An equal volume of 50 μM cofilin in F-buffer without ATP was added to the F-actin solution and then incubated on ice for >30 min. The mixed solution (2.1–2.5 μL) was applied onto a grid with a holey carbon support film (Quantifoil R 1.2/1.3 Molybdenum) and plunge-frozen in liquid ethane by a Vitrobot Mark IV (FEI). Before use, the grids were cleaned by acetone, as previously described 51 then soaked in pure water overnight. Cleaned grids were glow-discharged with a 5 mA current for 60 s just before sample application. The parameters for plunge-freezing were as follows: blot time 2 s, humidity 100%, temperature 4 °C. After plunge-freezing, residual liquid ethane on the grid was thoroughly blotted off with filter paper. The best conditions for making cryo grids were screened by FEI Polara at Nagoya University.

Image acquisition

Samples were imaged using a Titan Krios microscope (FEI) operated at 300 kV installed with EPU software (FEI) at Osaka University. The imaging parameters were: nominal magnification 75,000, illuminated diameter 0.67 μm, objective aperture 100 µm, actual defocus 1.0–3.0 µm, dose rate 45 e − /Å 2 /s, exposure time 1 s, three image acquisitions per hole. Images were recorded with a FalconII detector (FEI) at a pixel size of 1.1 Å/pixel and a frame rate of 17 frames/s. For each image, the first two frames were discarded and the successive seven frames (accumulated dose of 24 e − /Å 2 ) were saved and used for image processing (Supplementary Fig. 1a ).

Image processing

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 52 , 53 . After motion correction with MotionCorr 54 and CTF estimation with Gctf 55 , 1,093 images were selected for further image processing. Filaments were manually picked with e2helixboxer 56 , 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) 24 with sxpdb2em 57 . 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 Δ ϕ = 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. 1b ). Model building The initial atomic model consisted of five actin and three cofilin molecules that were prepared from an atomic model of G-actin (PDB 1J6Z) 32 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) 23 . Moreover, the ADP-associated Ca 2+ and methylated-histidine (residue 73) were replaced with Mg 2+ 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 58 , 59 on NAMD 2.12 software 60 . The calculation was performed under harmonic helical symmetry restraints 61 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 62 . The resulting model was iteratively refined using COOT 63 , MDFF and phenix.real_space_refine. After convergence, histidine 73 was replaced by a methylated-histidine. Rigid body search The IDs (residues 138–336) of the following eight actin structures were aligned with each other: G-actin modified with TMR (1J6Z), G-actin bound with a tropomodulin actin-binding domain and gelsolin domain 1 (4PKH), actin bound with twinfilin C-terminal domain (3DAW), F-actin structure determined by cryo-EM (5JLF), the F-form actin structure in our recent crystal structure (Takeda et al., unpublished results), actin in the actin-profilin-VASP peptide complex (2PAV), the actin portion of actin-thymosin beta 4 hybrid protein (4PL7) and the actin structure in the cofilactin. The boundary between the inner domain and subdomain 1 was assumed at the hinge position (Supplementary Fig. 3a–d ). The standard deviation of each Cα position was calculated, and residues with