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- PDB-8ru0: Structure of the undecorated barbed end of F-actin. -

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Basic information

Entry
Database: PDB / ID: 8ru0
TitleStructure of the undecorated barbed end of F-actin.
ComponentsActin, alpha skeletal muscle
KeywordsSTRUCTURAL PROTEIN / actin / actin end / barbed end / actin assembly
Function / homology
Function and homology information


cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament ...cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / lamellipodium / cell body / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein binding / 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 / ADENOSINE-5'-TRIPHOSPHATE / PHOSPHATE ION / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsOosterheert, W. / Boiero Sanders, M. / Funk, J. / Prumbaum, D. / Raunser, S. / Bieling, P.
Funding support Germany, European Union, 3items
OrganizationGrant numberCountry
Alexander von Humboldt Foundation Germany
German Research Foundation (DFG)BI 1998/2-1 Germany
European Research Council (ERC)856118European Union
CitationJournal: Science / Year: 2024
Title: Molecular mechanism of actin filament elongation by formins.
Authors: Wout Oosterheert / Micaela Boiero Sanders / Johanna Funk / Daniel Prumbaum / Stefan Raunser / Peter Bieling /
Abstract: Formins control the assembly of actin filaments (F-actin) that drive cell morphogenesis and motility in eukaryotes. However, their molecular interaction with F-actin and their mechanism of action ...Formins control the assembly of actin filaments (F-actin) that drive cell morphogenesis and motility in eukaryotes. However, their molecular interaction with F-actin and their mechanism of action remain unclear. In this work, we present high-resolution cryo-electron microscopy structures of F-actin barbed ends bound by three distinct formins, revealing a common asymmetric formin conformation imposed by the filament. Formation of new intersubunit contacts during actin polymerization sterically displaces formin and triggers its translocation. This "undock-and-lock" mechanism explains how actin-filament growth is coordinated with formin movement. Filament elongation speeds are controlled by the positioning and stability of actin-formin interfaces, which distinguish fast and slow formins. Furthermore, we provide a structure of the actin-formin-profilin ring complex, which resolves how profilin is rapidly released from the barbed end during filament elongation.
History
DepositionJan 29, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 10, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2024Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Actin, alpha skeletal muscle
A: Actin, alpha skeletal muscle
C: Actin, alpha skeletal muscle
D: Actin, alpha skeletal muscle
hetero molecules


Theoretical massNumber of molelcules
Total (without water)169,67315
Polymers167,5034
Non-polymers2,17111
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12160 Å2
ΔGint-139 kcal/mol
Surface area58630 Å2
MethodPISA

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Components

#1: Protein
Actin, alpha skeletal muscle / / Alpha-actin-1


Mass: 41875.633 Da / Num. of mol.: 4 / Source method: isolated from a natural source
Details: Rabbit skeletal alpha actin purified from frozen rabbit muscle acetone powder.
Source: (natural) Oryctolagus cuniculus (rabbit) / Tissue: Skeletal muscle / References: UniProt: P68135
#2: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: PO4
#5: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
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
1Complex of the actin subunits that form the barbed end of actin filaments.COMPLEXAlpha actin was purified from rabbit skeletal muscle. Actin filaments were polymerized in the presence of the formin INF2, which was purified separetely and added during polymerization prior to cryo-EM grid preparation.#10NATURAL
2Actin filamentMicrofilamentCOMPLEX#11NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Organ: Skeletal muscle
Buffer solutionpH: 7.1
Details: 12 mM HEPES pH 7.1, 100 mM KCl, 2.1 mM MgCl2, 1 mM EGTA, 1 mM TCEP, 0.2 mM ATP
Buffer component
IDConc.NameFormulaBuffer-ID
112 mMHEPES1
2100 mMsodium chlorideNaClSodium chloride1
32.1 mMmagnesium chlorideMgCl21
41 mMEGTA1
51 mMTCEP1
60.2 mMATPAdenosine triphosphate1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 286 K / Details: 3 seconds, force 0.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: 300 kV Titan Krios G3 microscope (Thermo Fisher Scientific).
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2900 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 20305
EM imaging opticsEnergyfilter name: GIF Bioquantum / Details: Gatan energy filter. / Energyfilter slit width: 15 eV
Spherical aberration corrector: 300 kV Titan Krios G3 microscope (Thermo Fisher Scientific).

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Processing

EM software
IDNameVersionCategory
1crYOLO1.8particle selection
2EPUimage acquisition
4CTFFIND4.13CTF correction
7UCSF ChimeraX1.5model fitting
8Coot0.9.8.1model fitting
10cryoSPARCv4.1.0initial Euler assignment
11cryoSPARCv4.1.1final Euler assignment
12cryoSPARCv4.1.1classification
13cryoSPARCv4.2.13D reconstruction
14Coot0.9.8.1model refinement
15PHENIX1.21rc1_5015model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1935707 / Details: Particles picked using SPHIRE-crYOLO.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 150174 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Details: Real Space Refinement in Phenix.
Atomic model buildingPDB-ID: 8A2S

8a2s


Accession code: 8A2S / Details: actin model / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 86.64 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001611912
ELECTRON MICROSCOPYf_angle_d0.463416172
ELECTRON MICROSCOPYf_chiral_restr0.04111794
ELECTRON MICROSCOPYf_plane_restr0.00342063
ELECTRON MICROSCOPYf_dihedral_angle_d7.37071649

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