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- PDB-8oi6: Cryo-EM structure of the undecorated barbed end of filamentous be... -

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Basic information

Entry
Database: PDB / ID: 8oi6
TitleCryo-EM structure of the undecorated barbed end of filamentous beta/gamma actin
Components
  • Actin, cytoplasmic 1
  • PHALLOIDIN
KeywordsSTRUCTURAL PROTEIN / Actin filament / cytoskeletal protein / ATPase
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
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
Similarity search - Domain/homology
: / ADENOSINE-5'-DIPHOSPHATE / Actin, cytoplasmic 1
Similarity search - Component
Biological speciesBos taurus (cattle)
Amanita phalloides (death cap)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.59 Å
AuthorsOosterheert, W. / Blanc, F.E.C. / Roy, A. / Belyy, A. / Hofnagel, O. / Hummer, G. / Bieling, P. / Raunser, S.
Funding support Germany, European Union, 3items
OrganizationGrant numberCountry
Max Planck Society Germany
Alexander von Humboldt Foundation Germany
European Research Council (ERC)856118European Union
CitationJournal: 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
DepositionMar 22, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 9, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 16, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.year
Revision 1.2Sep 27, 2023Group: Database references / Refinement description / Category: citation / citation_author / em_3d_fitting_list
Item: _citation.pdbx_database_id_DOI / _em_3d_fitting_list.initial_refinement_model_id
Revision 1.3Oct 4, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.4Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2
Revision 1.5Nov 22, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Actin, cytoplasmic 1
B: Actin, cytoplasmic 1
C: Actin, cytoplasmic 1
D: Actin, cytoplasmic 1
H: PHALLOIDIN
I: PHALLOIDIN
J: PHALLOIDIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)171,41515
Polymers169,6097
Non-polymers1,8068
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Actin, cytoplasmic 1 / / Beta-actin


Mass: 41795.680 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Plasmid details: slaughterhouse / Tissue: thymus / References: UniProt: P60712
#2: Protein/peptide PHALLOIDIN / /


Type: Peptide-like / Class: Toxin / Mass: 808.899 Da / Num. of mol.: 3 / Source method: obtained synthetically / Source: (synth.) Amanita phalloides (death cap) / References: BIRD: PRD_002366
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1actin-phalloidin complexCOMPLEXActin was purified as monomer from bovine thymus. Short filaments were reconstituted in vitro to obtain the barbed end structure.#10MULTIPLE SOURCES
2cytosolic beta-gamma actinCOMPLEX1NATURAL
3phalloidinCOMPLEX1RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular locationTissue
22Bos taurus (cattle)9913cytoplasmthymus
33Amanita phalloides (death cap)67723
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 7.1
Details: 10 mM imidazole pH 7.1, 100 mM KCl, 2 mM MgCl2 and 1 mM EGTA
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMimidazole1
2100 mMpotassium chlorideKCl1
32 mMmagnesium clorideMgCl21
41 mMethylene glycol-bis(beta-aminoethyl ether)-N,N,N,N-tetraacetic acidEGTA1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was directly collected from size-exclusion chromatography fractions.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 286 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Details: Microsope alignment was performed using SHERPA (Thermo Fisher)
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 120000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 56 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1316

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategoryDetails
1crYOLO1.8particle selection
2EPUimage acquisition
4CTFFIND4.13CTF correction
7Coot0.9.8.1model fittingon Mac
9PHENIX1.20.1-4487-000model refinement
10cryoSPARC3.3.2initial Euler assignment
11cryoSPARC3.3.2final Euler assignment
12cryoSPARC3.3.2classification
13cryoSPARC3.3.23D reconstruction
Image processingDetails: Falcon III operated in linear mode.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 252982 / Details: CrYOLO was trained to pick filament ends.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43618
Details: Performed in CryoSPARC. All barbed end particles were isolated from from other particles that represented pointed end and filament core.
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Details: Real space refinement in phenix.
Atomic model buildingPDB-ID: 8A2T

8a2t


Pdb chain-ID: c / Accession code: 8A2T / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 76.44 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002111825
ELECTRON MICROSCOPYf_angle_d0.560616054
ELECTRON MICROSCOPYf_chiral_restr0.04171795
ELECTRON MICROSCOPYf_plane_restr0.00332034
ELECTRON MICROSCOPYf_dihedral_angle_d5.30481638

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