+Open data
-Basic information
Entry | Database: PDB / ID: 8a2t | |||||||||||||||
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Title | Cryo-EM structure of F-actin in the Mg2+-ADP nucleotide state. | |||||||||||||||
Components | Actin, alpha skeletal muscle | |||||||||||||||
Keywords | STRUCTURAL PROTEIN / actin / cytoskeleton / filament / nucleotide state | |||||||||||||||
Function / homology | Function and homology information cytoskeletal motor activator activity / tropomyosin binding / mesenchyme migration / troponin I binding / myosin heavy chain binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle assembly ...cytoskeletal motor activator activity / tropomyosin binding / mesenchyme migration / troponin I binding / myosin heavy chain binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle assembly / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / lamellipodium / cell body / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein binding / cytoplasm Similarity search - Function | |||||||||||||||
Biological species | Oryctolagus cuniculus (rabbit) | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.24 Å | |||||||||||||||
Authors | Oosterheert, W. / Klink, B.U. / Belyy, A. / Pospich, S. / Raunser, S. | |||||||||||||||
Funding support | European Union, Germany, 4items
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Citation | Journal: Nature / Year: 2022 Title: Structural basis of actin filament assembly and aging. Authors: Wout Oosterheert / Björn U Klink / Alexander Belyy / Sabrina Pospich / Stefan Raunser / Abstract: The dynamic turnover of actin filaments (F-actin) controls cellular motility in eukaryotes and is coupled to changes in the F-actin nucleotide state. It remains unclear how F-actin hydrolyses ATP and ...The dynamic turnover of actin filaments (F-actin) controls cellular motility in eukaryotes and is coupled to changes in the F-actin nucleotide state. It remains unclear how F-actin hydrolyses ATP and subsequently undergoes subtle conformational rearrangements that ultimately lead to filament depolymerization by actin-binding proteins. Here we present cryo-electron microscopy structures of F-actin in all nucleotide states, polymerized in the presence of Mg or Ca at approximately 2.2 Å resolution. The structures show that actin polymerization induces the relocation of water molecules in the nucleotide-binding pocket, activating one of them for the nucleophilic attack of ATP. Unexpectedly, the back door for the subsequent release of inorganic phosphate (P) is closed in all structures, indicating that P release occurs transiently. The small changes in the nucleotide-binding pocket after ATP hydrolysis and P release are sensed by a key amino acid, amplified and transmitted to the filament periphery. Furthermore, differences in the positions of water molecules in the nucleotide-binding pocket explain why Ca-actin shows slower polymerization rates than Mg-actin. Our work elucidates the solvent-driven rearrangements that govern actin filament assembly and aging and lays the foundation for the rational design of drugs and small molecules for imaging and therapeutic applications. | |||||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8a2t.cif.gz | 396.4 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8a2t.ent.gz | 274.4 KB | Display | PDB format |
PDBx/mmJSON format | 8a2t.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8a2t_validation.pdf.gz | 1.3 MB | Display | wwPDB validaton report |
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Full document | 8a2t_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 8a2t_validation.xml.gz | 64 KB | Display | |
Data in CIF | 8a2t_validation.cif.gz | 96.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/a2/8a2t ftp://data.pdbj.org/pub/pdb/validation_reports/a2/8a2t | HTTPS FTP |
-Related structure data
Related structure data | 15106MC 8a2rC 8a2sC 8a2uC 8a2yC 8a2zC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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Noncrystallographic symmetry (NCS) | NCS domain:
NCS domain segments:
NCS oper:
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-Components
#1: Protein | Mass: 41875.633 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / Organ: skeletal muscle / References: UniProt: P68135 #2: Chemical | ChemComp-ADP / #3: Chemical | ChemComp-MG / #4: Water | ChemComp-HOH / | Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: rabbit skeletal alpha-actin in the filamentous state. / Type: COMPLEX Details: The helical rise of F-actin is 27.5 Angstrom, with a helical twist of ~166.5 degrees. Entity ID: #1 / Source: NATURAL | ||||||||||||||||||||||||||||||
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Molecular weight | Value: 15.2 kDa/nm / Experimental value: NO | ||||||||||||||||||||||||||||||
Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / Organ: skeletal muscle / Tissue: purified from muscle acetone powder | ||||||||||||||||||||||||||||||
Buffer solution | pH: 7.5 Details: F-buffer: 5 mM Tris pH 7.5, 100 mM KCl, 2 mM MgCl2, 2 mM NaN3, 1 mM DTT | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 286 K Details: The Vitrobot was operated at 13 degrees celsius and the samples were blotted for 9 seconds with a blot force of -25. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 3 sec. / Electron dose: 76.4 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 9842 / Details: Images were collected in supperresolution mode. |
EM imaging optics | Energyfilter slit width: 15 eV |
Image scans | Width: 5760 / Height: 4092 |
-Processing
Software |
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EM software |
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Image processing | Details: superresolution mode | ||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1296776 Details: We picked helical segments with a box distance of 40 pixels or 27.8 Angstrom and a minimum number of six boxes per filament. | ||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1114051 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL Details: The structures were then refined through a similar protocol of iterative cycles in Coot and phenix real-space refine. All solvent molecules (ions, waters) were placed manually in Coot in the ...Details: The structures were then refined through a similar protocol of iterative cycles in Coot and phenix real-space refine. All solvent molecules (ions, waters) were placed manually in Coot in the central actin subunit, and were then placed in the other subunits using NCS. Because the local resolution of each F-actin reconstruction is highest in the center and lower at the periphery of the map, we inspected all waters in each structure manually before the final phenix refinement; water molecular with poor corresponding cryo-EM density were removed. | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 7AHN | ||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 37.79 Å2 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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Refine LS restraints NCS |
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