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- PDB-8f8q: Cryo-EM structure of the CapZ-capped barbed end of F-actin -

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Basic information

Entry
Database: PDB / ID: 8f8q
TitleCryo-EM structure of the CapZ-capped barbed end of F-actin
Components
  • Actin, alpha skeletal muscle
  • F-actin-capping protein subunit alpha-1
  • F-actin-capping protein subunit beta
KeywordsSTRUCTURAL PROTEIN / Barbed end capping protein / F-actin / CapZ
Function / homology
Function and homology information


WASH complex / sperm connecting piece / F-actin capping protein complex / negative regulation of filopodium assembly / COPI-independent Golgi-to-ER retrograde traffic / cell junction assembly / barbed-end actin filament capping / actin polymerization or depolymerization / RHOF GTPase cycle / RHOD GTPase cycle ...WASH complex / sperm connecting piece / F-actin capping protein complex / negative regulation of filopodium assembly / COPI-independent Golgi-to-ER retrograde traffic / cell junction assembly / barbed-end actin filament capping / actin polymerization or depolymerization / RHOF GTPase cycle / RHOD GTPase cycle / regulation of cell morphogenesis / regulation of lamellipodium assembly / Sensory processing of sound by inner hair cells of the cochlea / lamellipodium assembly / cytoskeletal motor activator activity / tropomyosin binding / cortical cytoskeleton / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / brush border / striated muscle thin filament / Advanced glycosylation endproduct receptor signaling / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / COPI-mediated anterograde transport / stress fiber / cytoskeleton organization / titin binding / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / hippocampal mossy fiber to CA3 synapse / MHC class II antigen presentation / actin filament polymerization / Gene and protein expression by JAK-STAT signaling after Interleukin-12 stimulation / sarcomere / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Schaffer collateral - CA1 synapse / cell morphogenesis / calcium-dependent protein binding / actin filament binding / actin cytoskeleton / lamellipodium / cell body / actin binding / Factors involved in megakaryocyte development and platelet production / actin cytoskeleton organization / protein-containing complex assembly / postsynaptic density / cytoskeleton / hydrolase activity / cadherin binding / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / extracellular exosome / extracellular region / ATP binding / membrane / identical protein binding / cytosol / cytoplasm
Similarity search - Function
F-actin-capping protein subunit beta / F-actin capping protein, beta subunit, conserved site / F-actin-capping protein subunit beta, N-terminal domain / F-actin capping protein, beta subunit / F-actin capping protein beta subunit signature. / F-actin capping protein, alpha subunit, conserved site / F-actin capping protein alpha subunit signature 1. / F-actin capping protein alpha subunit signature 2. / F-actin-capping protein subunit alpha / F-actin-capping protein subunit alpha/beta ...F-actin-capping protein subunit beta / F-actin capping protein, beta subunit, conserved site / F-actin-capping protein subunit beta, N-terminal domain / F-actin capping protein, beta subunit / F-actin capping protein beta subunit signature. / F-actin capping protein, alpha subunit, conserved site / F-actin capping protein alpha subunit signature 1. / F-actin capping protein alpha subunit signature 2. / F-actin-capping protein subunit alpha / F-actin-capping protein subunit alpha/beta / F-actin-capping protein subunit alpha/beta, domain 2 / F-actin capping protein, alpha subunit, domain 1 / F-actin capping protein alpha subunit / 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 / F-actin-capping protein subunit beta / F-actin-capping protein subunit alpha-1 / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesHomo sapiens (human)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.79 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
F: Actin, alpha skeletal muscle
G: F-actin-capping protein subunit alpha-1
H: F-actin-capping protein subunit beta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)319,59220
Polymers316,8838
Non-polymers2,70912
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.: 6 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135
#2: Protein F-actin-capping protein subunit alpha-1 / CapZ alpha-1


Mass: 32964.727 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CAPZA1 / Production host: Escherichia coli (E. coli) / References: UniProt: P52907
#3: Protein F-actin-capping protein subunit beta / CapZ beta


Mass: 31258.289 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CAPZB / Production host: Escherichia coli (E. coli) / References: UniProt: P47756
#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / 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

Component
IDNameTypeEntity IDParent-IDSource
1CapZ-capped barbed end of F-actinCOMPLEX#1-#30MULTIPLE SOURCES
2CapZ (alpha and beta subunits)COMPLEX#2-#31RECOMBINANT
3Actin filamentMicrofilamentCOMPLEX#11NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Oryctolagus cuniculus (rabbit)9986
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
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 software
IDNameVersionCategory
1Topaz0.2.4particle selection
4cryoSPARC3.3.2CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARC3.3.2initial Euler assignment
11cryoSPARC3.3.2final Euler assignment
13cryoSPARC4.0.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1102793 / Details: Topaz particle picking with training
3D reconstructionResolution: 2.79 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 155516 / 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_d0.00422476
ELECTRON MICROSCOPYf_angle_d0.66830478
ELECTRON MICROSCOPYf_dihedral_angle_d14.0948391
ELECTRON MICROSCOPYf_chiral_restr0.0493367
ELECTRON MICROSCOPYf_plane_restr0.0063918

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