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Yorodumi- PDB-8oi6: Cryo-EM structure of the undecorated barbed end of filamentous be... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8oi6 | ||||||||||||
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Title | Cryo-EM structure of the undecorated barbed end of filamentous beta/gamma actin | ||||||||||||
Components |
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Keywords | STRUCTURAL PROTEIN / Actin filament / cytoskeletal protein / ATPase | ||||||||||||
Function / homology | Function and homology information Adherens junctions interactions / Cell-extracellular matrix interactions / RHOBTB2 GTPase cycle / Gap junction degradation / Formation of annular gap junctions / MAP2K and MAPK activation / EPHB-mediated forward signaling / Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / RHO GTPases activate IQGAPs ...Adherens junctions interactions / Cell-extracellular matrix interactions / RHOBTB2 GTPase cycle / Gap junction degradation / Formation of annular gap junctions / MAP2K and MAPK activation / EPHB-mediated forward signaling / Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / structural constituent of postsynaptic actin cytoskeleton / VEGFA-VEGFR2 Pathway / dense body / Clathrin-mediated endocytosis / NuA4 histone acetyltransferase complex / axonogenesis / actin filament / cell motility / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / actin cytoskeleton / cytoskeleton / hydrolase activity / axon / focal adhesion / synapse / protein kinase binding / protein-containing complex / ATP binding / membrane / nucleus / plasma membrane / cytosol / cytoplasm Similarity search - Function | ||||||||||||
Biological species | Bos taurus (cattle) Amanita phalloides (death cap) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.59 Å | ||||||||||||
Authors | Oosterheert, W. / Blanc, F.E.C. / Roy, A. / Belyy, A. / Hofnagel, O. / Hummer, G. / Bieling, P. / Raunser, S. | ||||||||||||
Funding support | Germany, European Union, 3items
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Citation | Journal: Nat Struct Mol Biol / Year: 2023 Title: Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments. Authors: Wout Oosterheert / Florian E C Blanc / Ankit Roy / Alexander Belyy / Micaela Boiero Sanders / Oliver Hofnagel / Gerhard Hummer / Peter Bieling / Stefan Raunser / Abstract: The release of inorganic phosphate (P) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. The mechanisms underlying P release ...The release of inorganic phosphate (P) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. The mechanisms underlying P release from the core and barbed end of actin filaments remain unclear. Here, using human and bovine actin isoforms, we combine cryo-EM with molecular-dynamics simulations and in vitro reconstitution to demonstrate how actin releases P through a 'molecular backdoor'. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and opens only transiently through concerted amino acid rearrangements. This explains why P escapes rapidly from the filament end but slowly from internal subunits. In a nemaline-myopathy-associated actin variant, the backdoor is predominantly open in filament-core subunits, resulting in accelerated P release and filaments with drastically shortened ADP-P caps. Our results provide the molecular basis for P release from actin and exemplify how a disease-linked mutation distorts the nucleotide-state distribution and atomic structure of the filament. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8oi6.cif.gz | 311.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8oi6.ent.gz | 211.9 KB | Display | PDB format |
PDBx/mmJSON format | 8oi6.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/oi/8oi6 ftp://data.pdbj.org/pub/pdb/validation_reports/oi/8oi6 | HTTPS FTP |
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-Related structure data
Related structure data | 16887MC 8oi8C 8oidC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 41795.680 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Plasmid details: slaughterhouse / Tissue: thymus / References: UniProt: P60712 #2: Protein/peptide | Type: Peptide-like / Class: Toxin / Mass: 808.899 Da / Num. of mol.: 3 / Source method: obtained synthetically / Source: (synth.) Amanita phalloides (death cap) / References: BIRD: PRD_002366 #3: Chemical | ChemComp-ADP / #4: Chemical | ChemComp-MG / Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Molecular weight | Experimental value: NO | ||||||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) | Organism: synthetic construct (others) | ||||||||||||||||||||||||||||
Buffer solution | pH: 7.1 Details: 10 mM imidazole pH 7.1, 100 mM KCl, 2 mM MgCl2 and 1 mM EGTA | ||||||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Sample was directly collected from size-exclusion chromatography fractions. | ||||||||||||||||||||||||||||
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-PROPANE / Humidity: 100 % / Chamber temperature: 286 K |
-Electron microscopy imaging
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA Details: Microsope alignment was performed using SHERPA (Thermo Fisher) |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 120000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 1200 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: 4 sec. / Electron dose: 56 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1316 |
-Processing
Software |
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EM software |
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Image processing | Details: Falcon III operated in linear mode. | ||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 252982 / Details: CrYOLO was trained to pick filament ends. | ||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43618 Details: Performed in CryoSPARC. All barbed end particles were isolated from from other particles that represented pointed end and filament core. Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL / Details: Real space refinement in phenix. | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 8A2T Pdb chain-ID: c / Accession code: 8A2T / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 76.44 Å2 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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