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- PDB-6d8c: Cryo-EM structure of FLNaABD E254K bound to phalloidin-stabilized... -

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Basic information

Entry
Database: PDB / ID: 6d8c
TitleCryo-EM structure of FLNaABD E254K bound to phalloidin-stabilized F-actin
Components
  • Actin, alpha skeletal muscle
  • Filamin-A
  • Phalloidin
KeywordsSTRUCTURAL 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 / adenylate cyclase-inhibiting dopamine receptor signaling pathway / formation of radial glial scaffolds / positive regulation of integrin-mediated signaling pathway / actin crosslink formation / tubulin deacetylation ...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 / adenylate cyclase-inhibiting dopamine receptor signaling pathway / formation of radial glial scaffolds / positive regulation of integrin-mediated signaling pathway / actin crosslink formation / tubulin deacetylation / blood coagulation, intrinsic pathway / protein localization to bicellular tight junction / OAS antiviral response / Striated Muscle Contraction / positive regulation of actin filament bundle assembly / positive regulation of neuron migration / Cell-extracellular matrix interactions / early endosome to late endosome transport / Fc-gamma receptor I complex binding / positive regulation of potassium ion transmembrane transport / apical dendrite / 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 / podosome / negative regulation of transcription by RNA polymerase I / megakaryocyte development / GP1b-IX-V activation signalling / receptor clustering / positive regulation of axon regeneration / cortical cytoskeleton / SMAD binding / RHO GTPases activate PAKs / actin filament bundle / brush border / semaphorin-plexin signaling pathway / striated muscle thin filament / skeletal muscle thin filament assembly / blood vessel remodeling / cilium assembly / mitotic spindle assembly / epithelial to mesenchymal transition / potassium channel regulator activity / axonal growth cone / heart morphogenesis / stress fiber / skeletal muscle fiber development / release of sequestered calcium ion into cytosol / positive regulation of substrate adhesion-dependent cell spreading / protein sequestering activity / regulation of cell migration / dendritic shaft / protein kinase C binding / protein localization to plasma membrane / actin filament / negative regulation of DNA-binding transcription factor activity / trans-Golgi network / mRNA transcription by RNA polymerase II / establishment of protein localization / G protein-coupled receptor binding / synapse organization / negative regulation of protein catabolic process / cerebral cortex development / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / small GTPase binding / Z disc / platelet aggregation / kinase binding / positive regulation of protein import into nucleus / actin filament binding / cell-cell junction / Platelet degranulation / actin cytoskeleton / negative regulation of neuron projection development / actin cytoskeleton organization / toxin activity / GTPase binding / angiogenesis / DNA-binding transcription factor binding / perikaryon / transmembrane transporter binding / positive regulation of canonical NF-kappaB signal transduction / hydrolase activity / postsynapse / protein stabilization / cadherin binding / focal adhesion / negative regulation of apoptotic process / nucleolus / perinuclear region of cytoplasm / glutamatergic synapse / protein homodimerization activity / RNA binding / extracellular exosome / extracellular region / ATP binding / nucleus / membrane
Similarity search - Function
Amanitin/phalloidin toxin / Actin; Chain A, domain 2 / Actin; Chain A, domain 2 / Filamin family / Calponin-like domain / Filamin/ABP280 repeat / Filamin-type immunoglobulin domains / Actin-binding Protein, T-fimbrin; domain 1 / Filamin/ABP280 repeat / Filamin/ABP280 repeat profile. ...Amanitin/phalloidin toxin / Actin; Chain A, domain 2 / Actin; Chain A, domain 2 / Filamin family / Calponin-like domain / Filamin/ABP280 repeat / Filamin-type immunoglobulin domains / Actin-binding Protein, T-fimbrin; domain 1 / Filamin/ABP280 repeat / Filamin/ABP280 repeat profile. / Filamin/ABP280 repeat-like / Actinin-type actin-binding domain signature 1. / Actinin-type actin-binding domain signature 2. / Actinin-type actin-binding domain, conserved site / Calponin homology domain / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / ATPase, substrate binding domain, subdomain 4 / Actin; Chain A, domain 4 / ATPase, nucleotide binding domain / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain / Nucleotidyltransferase; domain 5 / Immunoglobulin E-set / Roll / Immunoglobulin-like fold / Alpha-Beta Complex / 2-Layer Sandwich / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Phalloidin / ADENOSINE-5'-DIPHOSPHATE / Phalloidin proprotein / Filamin-A / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesHomo sapiens (human)
Gallus gallus (chicken)
Amanita phalloides (death cap)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsIwamoto, D.V. / Huehn, A.R. / Simon, B. / Huet-Calderwood, C. / Baldassarre, M. / Sindelar, C.V. / Calderwood, D.A.
CitationJournal: 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
DepositionApr 26, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 19, 2018Provider: repository / Type: Initial release
Revision 1.1Oct 3, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Oct 17, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 2.0Nov 15, 2023Group: Atomic model / Data collection ...Atomic model / Data collection / Database references / Derived calculations / Refinement description
Category: atom_site / chem_comp_atom ...atom_site / chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_conn / struct_conn_type
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_atom_id / _atom_site.label_atom_id / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn_type.id

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
D: Filamin-A
H: Actin, alpha skeletal muscle
A: Filamin-A
J: Actin, alpha skeletal muscle
B: Filamin-A
K: Actin, alpha skeletal muscle
C: Filamin-A
L: Actin, alpha skeletal muscle
E: Filamin-A
M: Actin, alpha skeletal muscle
N: Phalloidin
O: Phalloidin
P: Phalloidin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)386,37923
Polymers384,12113
Non-polymers2,25810
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, Fluorescent microscopy co-localization studies, actin co-sedimentation assays, etc
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area36060 Å2
ΔGint-267 kcal/mol
Surface area94080 Å2

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Components

#1: Protein
Filamin-A / FLN-A / Actin-binding protein 280 / ABP-280 / Alpha-filamin / Endothelial actin-binding protein / ...FLN-A / Actin-binding protein 280 / ABP-280 / Alpha-filamin / Endothelial actin-binding protein / Filamin-1 / Non-muscle filamin


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
Actin, alpha skeletal muscle / Alpha-actin-1


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 Phalloidin


Type: Cyclic peptide / Class: Toxin / Mass: 808.899 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Amanita phalloides (death cap) / References: Phalloidin, UniProt: P0CU65*PLUS
#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Helical complex of FLNaABD E254K bound to phalloidin-stabilized F-actin
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Rosetta
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: 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 holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1EMAN2particle selection
2SerialEMimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
13RELION2.0.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.73 ° / Axial rise/subunit: 27.54 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 67000
3D reconstructionResolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 67000 / Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-IDPdb chain residue range
16C1D

6c1d

B6C1D11-375
23HOC

3hoc

A3HOC239-153

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