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- PDB-8f8p: Cryo-EM structure of F-actin in the ADP state -

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Basic information

Entry
Database: PDB / ID: 8f8p
TitleCryo-EM structure of F-actin in the ADP state
ComponentsActin, alpha skeletal muscle
KeywordsSTRUCTURAL PROTEIN / Actin cytoskeleton / filament / F-actin
Function / homology
Function and homology information


cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / actin filament bundle / troponin I binding / filamentous actin / mesenchyme migration / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament ...cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / actin filament bundle / troponin I binding / filamentous actin / mesenchyme migration / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament / skeletal muscle thin filament assembly / 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 / helical reconstruction / cryo EM / Resolution: 2.26 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

Downloads & links

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)212,80715
Polymers210,5505
Non-polymers2,25810
Water00
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.: 5 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135
#2: 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
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / 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: helical reconstruction

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Sample preparation

ComponentName: F-actin in the ADP state / 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 FIELD / 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) / Num. of grids imaged: 4 / Num. of real images: 18611

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
4cryoSPARC4CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
13cryoSPARC4.0.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.7 ° / Axial rise/subunit: 27.8 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 8187062 / Details: CryoSPARC filament tracer
3D reconstructionResolution: 2.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4773977 / Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00414960
ELECTRON MICROSCOPYf_angle_d0.79820305
ELECTRON MICROSCOPYf_dihedral_angle_d6.3522085
ELECTRON MICROSCOPYf_chiral_restr0.0492255
ELECTRON MICROSCOPYf_plane_restr0.0062595

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