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- PDB-8f8r: Cryo-EM structure of the free barbed end of F-actin -

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Basic information

Entry
Database: PDB / ID: 8f8r
TitleCryo-EM structure of the free barbed end of F-actin
ComponentsActin, alpha skeletal muscle
KeywordsSTRUCTURAL PROTEIN / Actin cytoskeleton / filament / F-actin / barbed-end
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 / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsCarman, P.J. / Barrie, K.R. / Dominguez, R.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM073791 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM136511 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL156431 United States
CitationJournal: Science / Year: 2023
Title: Structures of the free and capped ends of the actin filament.
Authors: Peter J Carman / Kyle R Barrie / Grzegorz Rebowski / Roberto Dominguez /
Abstract: The barbed and pointed ends of the actin filament (F-actin) are the sites of growth and shrinkage and the targets of capping proteins that block subunit exchange, including CapZ at the barbed end and ...The barbed and pointed ends of the actin filament (F-actin) are the sites of growth and shrinkage and the targets of capping proteins that block subunit exchange, including CapZ at the barbed end and tropomodulin at the pointed end. We describe cryo-electron microscopy structures of the free and capped ends of F-actin. Terminal subunits at the free barbed end adopt a "flat" F-actin conformation. CapZ binds with minor changes to the barbed end but with major changes to itself. By contrast, subunits at the free pointed end adopt a "twisted" monomeric actin (G-actin) conformation. Tropomodulin binding forces the second subunit into an F-actin conformation. The structures reveal how the ends differ from the middle in F-actin and how these differences control subunit addition, dissociation, capping, and interactions with end-binding proteins.
History
DepositionNov 22, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 7, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 5, 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

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)297,93021
Polymers294,7707
Non-polymers3,16114
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 42109.973 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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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

ComponentName: Free barbed end of F-actin / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Buffer solutionpH: 7.5
SpecimenConc.: 1.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K / Details: Blot force 0 Blot time 2.5 s

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 44.7 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: Topaz particle picking with training
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 24855 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00420972
ELECTRON MICROSCOPYf_angle_d0.91828463
ELECTRON MICROSCOPYf_dihedral_angle_d6.7732921
ELECTRON MICROSCOPYf_chiral_restr0.0543161
ELECTRON MICROSCOPYf_plane_restr0.013639

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