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Yorodumi- EMDB-16887: Cryo-EM structure of the undecorated barbed end of filamentous be... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-16887 | ||||||||||||
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Title | Cryo-EM structure of the undecorated barbed end of filamentous beta/gamma actin | ||||||||||||
Map data | 3D-refined, sharpened cryo-EM density map of the undecorated barbed end of F-actin. | ||||||||||||
Sample |
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Keywords | Actin filament / cytoskeletal protein / ATPase / STRUCTURAL PROTEIN | ||||||||||||
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 | single particle reconstruction / cryo EM / Resolution: 3.59 Å | ||||||||||||
Authors | Oosterheert W / Blanc FEC / Roy A / Belyy A / Hofnagel O / Hummer G / Bieling P / Raunser S | ||||||||||||
Funding support | Germany, European Union, 3 items
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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
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_16887.map.gz | 203.8 MB | EMDB map data format | |
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Header (meta data) | emd-16887-v30.xml emd-16887.xml | 24.2 KB 24.2 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_16887_fsc.xml | 12.6 KB | Display | FSC data file |
Images | emd_16887.png | 90.9 KB | ||
Masks | emd_16887_msk_1.map | 216 MB | Mask map | |
Filedesc metadata | emd-16887.cif.gz | 7.2 KB | ||
Others | emd_16887_additional_1.map.gz emd_16887_half_map_1.map.gz emd_16887_half_map_2.map.gz | 108.1 MB 200.4 MB 200.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-16887 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-16887 | HTTPS FTP |
-Related structure data
Related structure data | 8oi6MC 8oi8C 8oidC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_16887.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | 3D-refined, sharpened cryo-EM density map of the undecorated barbed end of F-actin. | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.21 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_16887_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: 3D-refined, unsharpened cryo-EM density map of the undecorated...
File | emd_16887_additional_1.map | ||||||||||||
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Annotation | 3D-refined, unsharpened cryo-EM density map of the undecorated barbed end of F-actin. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 2 of the refinement of the...
File | emd_16887_half_map_1.map | ||||||||||||
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Annotation | Half map 2 of the refinement of the undecorated barbed end of F-actin. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 1 of the refinement of the...
File | emd_16887_half_map_2.map | ||||||||||||
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Annotation | Half map 1 of the refinement of the undecorated barbed end of F-actin. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : actin-phalloidin complex
Entire | Name: actin-phalloidin complex |
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Components |
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-Supramolecule #1: actin-phalloidin complex
Supramolecule | Name: actin-phalloidin complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 Details: Actin was purified as monomer from bovine thymus. Short filaments were reconstituted in vitro to obtain the barbed end structure. |
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-Supramolecule #2: cytosolic beta-gamma actin
Supramolecule | Name: cytosolic beta-gamma actin / type: complex / ID: 2 / Parent: 1 |
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Source (natural) | Organism: Bos taurus (cattle) / Tissue: thymus / Location in cell: cytoplasm |
-Supramolecule #3: phalloidin
Supramolecule | Name: phalloidin / type: complex / ID: 3 / Parent: 1 |
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Source (natural) | Organism: Amanita phalloides (death cap) |
-Macromolecule #1: Actin, cytoplasmic 1
Macromolecule | Name: Actin, cytoplasmic 1 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: Bos taurus (cattle) / Tissue: thymus |
Molecular weight | Theoretical: 41.79568 KDa |
Sequence | String: MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GIV TNW DDMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSG DG VTHTVPIYEG ...String: MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GIV TNW DDMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSG DG VTHTVPIYEG YALPHAILRL DLAGRDLTDY LMKILTERGY SFTTTAEREI VRDIKEKLCY VALDFEQEMA TAASSSSL E KSYELPDGQV ITIGNERFRC PEALFQPSFL GMESCGIHET TFNSIMKCDV DIRKDLYANT VLSGGTTMYP GIADRMQKE ITALAPSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ISKQEYDESG PSIVHRKCF UniProtKB: Actin, cytoplasmic 1 |
-Macromolecule #2: PHALLOIDIN
Macromolecule | Name: PHALLOIDIN / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Amanita phalloides (death cap) |
Molecular weight | Theoretical: 808.899 Da |
Sequence | String: W(EEP)A(DTH)C(HYP)A |
-Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 4 / Formula: ADP |
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Molecular weight | Theoretical: 427.201 Da |
Chemical component information | ChemComp-ADP: |
-Macromolecule #4: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.1 Component:
Details: 10 mM imidazole pH 7.1, 100 mM KCl, 2 mM MgCl2 and 1 mM EGTA | |||||||||||||||
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Grid | Model: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. | |||||||||||||||
Vitrification | Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 286 K / Instrument: FEI VITROBOT MARK IV | |||||||||||||||
Details | Sample was directly collected from size-exclusion chromatography fractions. |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 120000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Details | Microsope alignment was performed using SHERPA (Thermo Fisher) |
Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 1316 / Average exposure time: 4.0 sec. / Average electron dose: 56.0 e/Å2 |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
-Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: c / Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Details | Real space refinement in phenix. |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | PDB-8oi6: |