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Yorodumi- EMDB-16889: Cryo-EM structure of ADP-bound, filamentous beta-actin harboring ... -
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-Basic information
Entry | Database: EMDB / ID: EMD-16889 | ||||||||||||
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Title | Cryo-EM structure of ADP-bound, filamentous beta-actin harboring the N111S mutation | ||||||||||||
Map data | Sharpened, local-resolution filtered cryo-EM density map of filamentous beta-actin harboring the N111S mutation. | ||||||||||||
Sample |
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Keywords | Actin filament / cytoskeletal protein / ATPase / STRUCTURAL PROTEIN | ||||||||||||
Function / homology | Function and homology information positive regulation of norepinephrine uptake / cellular response to cytochalasin B / bBAF complex / npBAF complex / postsynaptic actin cytoskeleton organization / regulation of transepithelial transport / brahma complex / nBAF complex / structural constituent of postsynaptic actin cytoskeleton / morphogenesis of a polarized epithelium ...positive regulation of norepinephrine uptake / cellular response to cytochalasin B / bBAF complex / npBAF complex / postsynaptic actin cytoskeleton organization / regulation of transepithelial transport / brahma complex / nBAF complex / structural constituent of postsynaptic actin cytoskeleton / morphogenesis of a polarized epithelium / Formation of annular gap junctions / GBAF complex / Gap junction degradation / postsynaptic actin cytoskeleton / protein localization to adherens junction / regulation of G0 to G1 transition / dense body / Cell-extracellular matrix interactions / Tat protein binding / Folding of actin by CCT/TriC / regulation of double-strand break repair / regulation of nucleotide-excision repair / RSC-type complex / apical protein localization / Prefoldin mediated transfer of substrate to CCT/TriC / adherens junction assembly / RHOF GTPase cycle / Adherens junctions interactions / tight junction / Sensory processing of sound by outer hair cells of the cochlea / SWI/SNF complex / Interaction between L1 and Ankyrins / Sensory processing of sound by inner hair cells of the cochlea / regulation of mitotic metaphase/anaphase transition / regulation of norepinephrine uptake / positive regulation of double-strand break repair / positive regulation of T cell differentiation / NuA4 histone acetyltransferase complex / regulation of synaptic vesicle endocytosis / apical junction complex / maintenance of blood-brain barrier / establishment or maintenance of cell polarity / cortical cytoskeleton / positive regulation of double-strand break repair via homologous recombination / positive regulation of stem cell population maintenance / nitric-oxide synthase binding / Recycling pathway of L1 / regulation of cyclin-dependent protein serine/threonine kinase activity / regulation of G1/S transition of mitotic cell cycle / negative regulation of cell differentiation / brush border / kinesin binding / calyx of Held / EPH-ephrin mediated repulsion of cells / RHO GTPases Activate WASPs and WAVEs / RHO GTPases activate IQGAPs / positive regulation of myoblast differentiation / regulation of protein localization to plasma membrane / EPHB-mediated forward signaling / substantia nigra development / axonogenesis / negative regulation of protein binding / actin filament / cell motility / RHO GTPases Activate Formins / Translocation of SLC2A4 (GLUT4) to the plasma membrane / regulation of transmembrane transporter activity / positive regulation of cell differentiation / FCGR3A-mediated phagocytosis / adherens junction / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / DNA Damage Recognition in GG-NER / tau protein binding / Signaling by high-kinase activity BRAF mutants / Schaffer collateral - CA1 synapse / MAP2K and MAPK activation / B-WICH complex positively regulates rRNA expression / structural constituent of cytoskeleton / cytoplasmic ribonucleoprotein granule / kinetochore / Regulation of actin dynamics for phagocytic cup formation / platelet aggregation / nuclear matrix / VEGFA-VEGFR2 Pathway / UCH proteinases / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / nucleosome / cell-cell junction / Signaling by BRAF and RAF1 fusions / actin cytoskeleton / presynapse / lamellipodium / Clathrin-mediated endocytosis / Factors involved in megakaryocyte development and platelet production / HATs acetylate histones / blood microparticle / regulation of apoptotic process Similarity search - Function | ||||||||||||
Biological species | Homo sapiens (human) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.3 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_16889.map.gz | 129.5 MB | EMDB map data format | |
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Header (meta data) | emd-16889-v30.xml emd-16889.xml | 25.6 KB 25.6 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_16889_fsc.xml | 13.6 KB | Display | FSC data file |
Images | emd_16889.png | 89.7 KB | ||
Masks | emd_16889_msk_1.map | 216 MB | Mask map | |
Filedesc metadata | emd-16889.cif.gz | 7.8 KB | ||
Others | emd_16889_additional_1.map.gz emd_16889_half_map_1.map.gz emd_16889_half_map_2.map.gz | 168.6 MB 170.5 MB 170.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-16889 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-16889 | HTTPS FTP |
-Related structure data
Related structure data | 8oidMC 8oi6C 8oi8C C: citing same article (ref.) M: atomic model generated by this map |
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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_16889.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Sharpened, local-resolution filtered cryo-EM density map of filamentous beta-actin harboring the N111S mutation. | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.695 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_16889_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 filamentous...
File | emd_16889_additional_1.map | ||||||||||||
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Annotation | 3D refined, unsharpened cryo-EM density map of filamentous beta-actin harboring the N111S mutation. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 1 of the refinement of filamentous...
File | emd_16889_half_map_1.map | ||||||||||||
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Annotation | Half map 1 of the refinement of filamentous beta-actin harboring the N111S mutation. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 2 of the refinement of filamentous...
File | emd_16889_half_map_2.map | ||||||||||||
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Annotation | Half map 2 of the refinement of filamentous beta-actin harboring the N111S mutation. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Actin filament harboring the N111S mutation.
Entire | Name: Actin filament harboring the N111S mutation. |
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Components |
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-Supramolecule #1: Actin filament harboring the N111S mutation.
Supramolecule | Name: Actin filament harboring the N111S mutation. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 Details: Beta-actin was expressed as fusion protein, with thymosin beta4 and a deca-His-tag fused to the actin C-terminus. During the purification, thymosin beta-4 and the deca-His-tag were removed. ...Details: Beta-actin was expressed as fusion protein, with thymosin beta4 and a deca-His-tag fused to the actin C-terminus. During the purification, thymosin beta-4 and the deca-His-tag were removed. Actin was purified as monomer from insect cells. It was then polymerized into a filament in vitro. |
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Source (natural) | Organism: Homo sapiens (human) |
-Macromolecule #1: Actin, cytoplasmic 1, N-terminally processed
Macromolecule | Name: Actin, cytoplasmic 1, N-terminally processed / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 41.73659 KDa |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) |
Sequence | String: MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GIV TNW DDMEKIWHHT FYNELRVAPE EHPVLLTEAP LSPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSG DG VTHTVPIYEG ...String: MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GIV TNW DDMEKIWHHT FYNELRVAPE EHPVLLTEAP LSPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSG DG VTHTVPIYEG YALPHAILRL DLAGRDLTDY LMKILTERGY SFTTTAEREI VRDIKEKLCY VALDFEQEMA TAASSSSL E KSYELPDGQV ITIGNERFRC PEALFQPSFL GMESAGIHET TFNSIMKCDV DIRKDLYANT VLSGGTTMYP GIADRMQKE ITALAPSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ISKQEYDESG PSIVHRKCF UniProtKB: Actin, cytoplasmic 1 |
-Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 5 / Formula: ADP |
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Molecular weight | Theoretical: 427.201 Da |
Chemical component information | ChemComp-ADP: |
-Macromolecule #3: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 5 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #4: water
Macromolecule | Name: water / type: ligand / ID: 4 / Number of copies: 616 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | filament |
-Sample preparation
Concentration | 2.5 mg/mL | ||||||||||||||||||
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Buffer | pH: 7.1 Component:
Details: 1x KMEH (10 mM HEPES pH 7.1, 100 mM KCl, 2 mM MgCl2, 1 mM EGTA) supplemented with 0.02% Tween20 (v/v) | ||||||||||||||||||
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 | Actin filaments were reconstituted by adding salt to monomeric actin. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 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: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000 |
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 15 eV |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Details | Titan Krios G3 microscope was aligned using Sherpa (FEI). Data collected in superresolution mode. |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 9516 / Average electron dose: 70.0 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Details | chain C of pdb 8A2T (including all water molecules) was fit in the central actin subunit of the density map. After substitution of all alpha-actin specific amino-acids to the corresponding beta-actin residues, introducing the N111S mutation, and further manual model building in Coot, the resulting model was fitted in four more actin subunits (chains A, B, D, E) in the density map. The filament was modeled as a pentamer to capture the full interaction interface of the central subunit with its four neighboring subunits. All water molecules were first manually built, inspected and adjusted in the central subunit, and were then copied to the other chains with non-crystallographic symmetry (NCS). Because the local resolution was worse at the periphery of the reconstruction, we removed water molecules that displayed poor corresponding cryo-EM density in the non-central actin chains. The model was refined in Phenix real-space refine with NCS restraints but without Ramachandran and rotamer restraints. |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | PDB-8oid: |