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Yorodumi- PDB-6d8c: Cryo-EM structure of FLNaABD E254K bound to phalloidin-stabilized... -
+Open data
-Basic information
Entry | Database: PDB / ID: 6d8c | |||||||||
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Title | Cryo-EM structure of FLNaABD E254K bound to phalloidin-stabilized F-actin | |||||||||
Components |
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Keywords | STRUCTURAL PROTEIN / Actin-binding domain / Actin crosslinker | |||||||||
Function / homology | Function and homology information regulation of membrane repolarization during atrial cardiac muscle cell action potential / regulation of membrane repolarization during cardiac muscle cell action potential / establishment of Sertoli cell barrier / Myb complex / glycoprotein Ib-IX-V complex / formation of radial glial scaffolds / adenylate cyclase-inhibiting dopamine receptor signaling pathway / positive regulation of integrin-mediated signaling pathway / tubulin deacetylation / cytoplasmic sequestering of protein ...regulation of membrane repolarization during atrial cardiac muscle cell action potential / regulation of membrane repolarization during cardiac muscle cell action potential / establishment of Sertoli cell barrier / Myb complex / glycoprotein Ib-IX-V complex / formation of radial glial scaffolds / adenylate cyclase-inhibiting dopamine receptor signaling pathway / positive regulation of integrin-mediated signaling pathway / tubulin deacetylation / cytoplasmic sequestering of protein / actin crosslink formation / blood coagulation, intrinsic pathway / Striated Muscle Contraction / protein localization to bicellular tight junction / OAS antiviral response / positive regulation of actin filament bundle assembly / positive regulation of neuron migration / positive regulation of potassium ion transmembrane transport / Cell-extracellular matrix interactions / early endosome to late endosome transport / apical dendrite / Fc-gamma receptor I complex binding / cell-cell junction organization / positive regulation of neural precursor cell proliferation / positive regulation of platelet activation / protein localization to cell surface / wound healing, spreading of cells / negative regulation of transcription by RNA polymerase I / megakaryocyte development / GP1b-IX-V activation signalling / cortical cytoskeleton / positive regulation of axon regeneration / receptor clustering / SMAD binding / actin filament bundle / RHO GTPases activate PAKs / brush border / skeletal muscle thin filament assembly / striated muscle thin filament / semaphorin-plexin signaling pathway / mitotic spindle assembly / cilium assembly / potassium channel regulator activity / epithelial to mesenchymal transition / blood vessel remodeling / skeletal muscle fiber development / axonal growth cone / heart morphogenesis / stress fiber / positive regulation of substrate adhesion-dependent cell spreading / release of sequestered calcium ion into cytosol / regulation of cell migration / dendritic shaft / protein localization to plasma membrane / mRNA transcription by RNA polymerase II / negative regulation of protein catabolic process / G protein-coupled receptor binding / actin filament / protein kinase C binding / kinase binding / synapse organization / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / trans-Golgi network / establishment of protein localization / negative regulation of DNA-binding transcription factor activity / cerebral cortex development / platelet aggregation / small GTPase binding / Z disc / actin filament binding / positive regulation of protein import into nucleus / cell-cell junction / actin cytoskeleton / negative regulation of neuron projection development / Platelet degranulation / GTPase binding / positive regulation of canonical NF-kappaB signal transduction / actin cytoskeleton organization / DNA-binding transcription factor binding / perikaryon / angiogenesis / toxin activity / transmembrane transporter binding / postsynapse / protein stabilization / cadherin binding / hydrolase activity / focal adhesion / glutamatergic synapse / negative regulation of apoptotic process / nucleolus / perinuclear region of cytoplasm / protein homodimerization activity / RNA binding / extracellular exosome / extracellular region / ATP binding / membrane / nucleus / plasma membrane Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) Gallus gallus (chicken) Amanita phalloides (death cap) | |||||||||
Method | ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.54 Å | |||||||||
Authors | Iwamoto, D.V. / Huehn, A.R. / Simon, B. / Huet-Calderwood, C. / Baldassarre, M. / Sindelar, C.V. / Calderwood, D.A. | |||||||||
Citation | Journal: Nat Struct Mol Biol / Year: 2018 Title: Structural basis of the filamin A actin-binding domain interaction with F-actin. Authors: Daniel V Iwamoto / Andrew Huehn / Bertrand Simon / Clotilde Huet-Calderwood / Massimiliano Baldassarre / Charles V Sindelar / David A Calderwood / Abstract: Actin-cross-linking proteins assemble actin filaments into higher-order structures essential for orchestrating cell shape, adhesion, and motility. Missense mutations in the tandem calponin homology ...Actin-cross-linking proteins assemble actin filaments into higher-order structures essential for orchestrating cell shape, adhesion, and motility. Missense mutations in the tandem calponin homology domains of their actin-binding domains (ABDs) underlie numerous genetic diseases, but a molecular understanding of these pathologies is hampered by the lack of high-resolution structures of any actin-cross-linking protein bound to F-actin. Here, taking advantage of a high-affinity, disease-associated mutant of the human filamin A (FLNa) ABD, we combine cryo-electron microscopy and functional studies to reveal at near-atomic resolution how the first calponin homology domain (CH1) and residues immediately N-terminal to it engage actin. We further show that reorientation of CH2 relative to CH1 is required to avoid clashes with actin and to expose F-actin-binding residues on CH1. Our data explain localization of disease-associated loss-of-function mutations to FLNaCH1 and gain-of-function mutations to the regulatory FLNaCH2. Sequence conservation argues that this provides a general model for ABD-F-actin binding. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6d8c.cif.gz | 854.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6d8c.ent.gz | 711.6 KB | Display | PDB format |
PDBx/mmJSON format | 6d8c.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6d8c_validation.pdf.gz | 1.4 MB | Display | wwPDB validaton report |
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Full document | 6d8c_full_validation.pdf.gz | 1.4 MB | Display | |
Data in XML | 6d8c_validation.xml.gz | 76.6 KB | Display | |
Data in CIF | 6d8c_validation.cif.gz | 114.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/d8/6d8c ftp://data.pdbj.org/pub/pdb/validation_reports/d8/6d8c | HTTPS FTP |
-Related structure data
Related structure data | 7831MC 7832C 7833C 8918C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 34476.250 Da / Num. of mol.: 5 / Fragment: UNP residues 1-278 / Mutation: E254K Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: FLNA, FLN, FLN1 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta / References: UniProt: P21333 #2: Protein | Mass: 41862.613 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken) / Tissue: Breast / References: UniProt: P68139 #3: Protein/peptide | #4: Chemical | ChemComp-ADP / #5: Chemical | ChemComp-MG / |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction |
-Sample preparation
Component | Name: Helical complex of FLNaABD E254K bound to phalloidin-stabilized F-actin Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES |
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Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: Escherichia coli (E. coli) / Strain: Rosetta |
Buffer solution | pH: 8 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE |
-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: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 37500 X / Nominal defocus max: 2900 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 12 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 2140 Details: Micrographs from only one of the grids were used in the reconstruction. From that grid, only micrographs where Gctf detected signal at resolutions better than 4A were used in the reconstruction (~15%). |
Image scans | Movie frames/image: 40 / Used frames/image: 1-40 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||
Helical symmerty | Angular rotation/subunit: -166.73 ° / Axial rise/subunit: 27.54 Å / Axial symmetry: C1 | ||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 67000 | ||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 67000 / Symmetry type: HELICAL | ||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT | ||||||||||||||||||||||||||||
Atomic model building | 3D fitting-ID: 1 / Source name: PDB / Type: experimental model
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