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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 / homologyFilamin A / Actinin-type actin-binding domain signature 1. / Immunoglobulin E-set / Immunoglobulin-like fold / CH domain superfamily / Actin, conserved site / Actin family / Actin / Calponin homology (CH) domain / Calponin homology domain ...Filamin A / Actinin-type actin-binding domain signature 1. / Immunoglobulin E-set / Immunoglobulin-like fold / CH domain superfamily / Actin, conserved site / Actin family / Actin / Calponin homology (CH) domain / Calponin homology domain / Filamin/ABP280 repeat / Actinin-type actin-binding domain, conserved site / Actinin-type actin-binding domain signature 2. / Filamin/ABP280 repeat-like / Actins signature 1. / Actins signature 2. / Filamin/ABP280 repeat / Actins and actin-related proteins signature. / Calponin homology (CH) domain profile. / Filamin/ABP280 repeat profile. / Platelet degranulation / GP1b-IX-V activation signalling / Cell-extracellular matrix interactions / RHO GTPases activate PAKs / OAS antiviral response / Actin/actin-like conserved site / Fc-gamma receptor I complex binding / regulation of membrane repolarization during atrial cardiac muscle cell action potential / regulation of membrane repolarization during cardiac muscle cell action potential / negative regulation of transcription by RNA polymerase I / positive regulation of integrin-mediated signaling pathway / adenylate cyclase-inhibiting dopamine receptor signaling pathway / Myb complex / formation of radial glial scaffolds / positive regulation of neuron migration / establishment of Sertoli cell barrier / Ral GTPase binding / cytoplasmic sequestering of protein / mu-type opioid receptor binding / protein localization to bicellular tight junction / positive regulation of potassium ion transmembrane transport / positive regulation of neural precursor cell proliferation / small GTPase binding / actin crosslink formation / positive regulation of actin filament bundle assembly / apical dendrite / Rho GTPase binding / potassium channel regulator activity / mRNA transcription by RNA polymerase II / protein localization to cell surface / cortical cytoskeleton / wound healing, spreading of cells / cell junction assembly / semaphorin-plexin signaling pathway / cilium assembly / skeletal muscle thin filament assembly / receptor clustering / striated muscle thin filament / skeletal muscle fiber development / Rac GTPase binding / SMAD binding / G protein-coupled receptor binding / mitotic spindle assembly / stress fiber / positive regulation of substrate adhesion-dependent cell spreading / cerebral cortex development / regulation of cell migration / GTPase binding / actin cytoskeleton reorganization / positive regulation of protein import into nucleus / protein localization to plasma membrane / dendritic shaft / actin filament / platelet aggregation / Z disc / kinase binding / negative regulation of protein catabolic process / postsynapse / actin cytoskeleton / establishment of protein localization / cell-cell junction / negative regulation of DNA-binding transcription factor activity / defense response to virus / platelet degranulation / actin filament binding / platelet activation / ion channel binding / protein stabilization / positive regulation of I-kappaB kinase/NF-kappaB signaling / cadherin binding / glutamatergic synapse / focal adhesion / transcription factor binding / neuronal cell body / nucleolus / negative regulation of apoptotic process / perinuclear region of cytoplasm / protein homodimerization activity / RNA binding / extracellular exosome
Function and homology information
Specimen sourceHomo sapiens (human)
Gallus gallus (chicken)
Amanita phalloides (death cap)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / 3.54 Å resolution
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
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Apr 26, 2018 / Release: Sep 19, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Sep 19, 2018Structure modelrepositoryInitial release
1.1Oct 3, 2018Structure modelData collection / Database referencescitation / citation_author_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
1.2Oct 17, 2018Structure modelData collection / Database referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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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
Polyers384,12113
Non-polymers2,25810
Water0
1


  • idetical with deposited unit
  • defined by author
  • Evidence: microscopy, Fluorescent microscopy co-localization studies, actin co-sedimentation assays, etc
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)36060
ΔGint (kcal/M)-267
Surface area (Å2)94080

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Components

#1: Protein/peptide
Filamin-A / 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: (gene. exp.) Homo sapiens (human) / Gene: FLNA, FLN, FLN1 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta / References: UniProt: P21333
#2: Protein/peptide
Actin, alpha skeletal muscle / / Alpha-actin-1


Mass: 41862.613 Da / Num. of mol.: 5 / Source: (natural) Gallus gallus (chicken) / Tissue: Breast / References: UniProt: P68139
#3: Protein/peptide Phalloidin


Mass: 808.899 Da / Num. of mol.: 3 / Source: (natural) Amanita phalloides (death cap)

about BIRD dictionary

PRD-IDPRD_002307
ClassToxin
NamePhalloidin
TypeCyclic peptide
#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Formula: C10H15N5O10P2 / Adenosine diphosphate / Comment: ADP (energy-carrying molecule) *YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Formula: Mg / Magnesium

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 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: 1,2,3 / 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
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 / Calibrated magnification: 37500 / Nominal defocus max: 2900 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 microns / Alignment procedure: COMA FREE
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 12 sec. / Electron dose: 50 e/Å2
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%).
Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 2 / Number of real images: 2140
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 deg. / Axial rise/subunit: 27.54 Å / Axial symmetry: C1
Particle selectionNumber of particles selected: 67000
3D reconstructionResolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 67000 / Symmetry type: HELICAL
Atomic model buildingRef protocol: RIGID BODY FIT
Atomic model building
IDPDB-IDPdb chain ID 3D fitting IDPdb chain residue range
16C1DB11-375
23HOCA139-153

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