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- PDB-7pdz: Structure of capping protein bound to the barbed end of a cytopla... -

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Basic information

Entry
Database: PDB / ID: 7pdz
TitleStructure of capping protein bound to the barbed end of a cytoplasmic actin filament
Components
  • Actin, cytoplasmic 1
  • F-actin-capping protein subunit alpha-1
  • Isoform 2 of F-actin-capping protein subunit beta
  • Phalloidin
KeywordsSTRUCTURAL PROTEIN / Cytoskeleton / cell-shape remodelling / barbed end
Function / homology
Function and homology information


cytoskeletal calyx / regulation of protein kinase C signaling / Advanced glycosylation endproduct receptor signaling / COPI-independent Golgi-to-ER retrograde traffic / WASH complex / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / sperm head / negative regulation of filopodium assembly / COPI-mediated anterograde transport / sperm connecting piece ...cytoskeletal calyx / regulation of protein kinase C signaling / Advanced glycosylation endproduct receptor signaling / COPI-independent Golgi-to-ER retrograde traffic / WASH complex / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / sperm head / negative regulation of filopodium assembly / COPI-mediated anterograde transport / sperm connecting piece / F-actin capping protein complex / muscle cell development => GO:0055001 / structural constituent of postsynaptic actin cytoskeleton / dense body / cell projection organization / Factors involved in megakaryocyte development and platelet production / MHC class II antigen presentation / barbed-end actin filament capping / actin polymerization or depolymerization / NuA4 histone acetyltransferase complex / regulation of cell morphogenesis / negative regulation of microtubule polymerization / regulation of lamellipodium assembly / cell junction assembly / beta-tubulin binding / lamellipodium assembly / cortical cytoskeleton / brush border / asymmetric synapse / synaptic vesicle endocytosis / intercalated disc / calyx of Held / cell motility / cytoskeleton organization / axonogenesis / hippocampal mossy fiber to CA3 synapse / Schaffer collateral - CA1 synapse / actin filament / cell morphogenesis / actin cytoskeleton organization / Z disc / negative regulation of protein binding / neuron projection development / actin cytoskeleton / cell-cell junction / lamellipodium / actin binding / dendritic spine / postsynaptic density / cytoskeleton / synapse / axon / focal adhesion / neuronal cell body / protein kinase binding / protein-containing complex / membrane / ATP binding / plasma membrane / nucleus / cytosol / cytoplasm
Similarity search - Function
F-actin capping protein, beta subunit / F-actin-capping protein subunit beta, N-terminal domain / F-actin capping protein, beta subunit, conserved site / F-actin capping protein beta subunit signature. / F-actin-capping protein subunit beta / F-actin capping protein, alpha subunit, conserved site / F-actin capping protein alpha subunit signature 2. / F-actin capping protein alpha subunit signature 1. / F-actin-capping protein subunit alpha / F-actin-capping protein subunit alpha/beta, domain 2 ...F-actin capping protein, beta subunit / F-actin-capping protein subunit beta, N-terminal domain / F-actin capping protein, beta subunit, conserved site / F-actin capping protein beta subunit signature. / F-actin-capping protein subunit beta / F-actin capping protein, alpha subunit, conserved site / F-actin capping protein alpha subunit signature 2. / F-actin capping protein alpha subunit signature 1. / F-actin-capping protein subunit alpha / F-actin-capping protein subunit alpha/beta, domain 2 / F-actin-capping protein subunit alpha/beta / F-actin capping protein, alpha subunit, domain 1 / F-actin capping protein alpha subunit / Actins signature 1. / Actins signature 2. / Actin, conserved site / Actins and actin-related proteins signature. / Actin/actin-like conserved site / Actin / Actin family / Actin / ATPase, nucleotide binding domain
Similarity search - Domain/homology
F-actin-capping protein subunit alpha-1 / F-actin-capping protein subunit beta / Phalloidin / ADENOSINE-5'-DIPHOSPHATE / PHOSPHATE ION / Actin, cytoplasmic 1
Similarity search - Component
Biological speciesMus musculus (house mouse)
Bos taurus (cattle)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsFunk, J. / Merino, F. / Schacks, M. / Rottner, K. / Raunser, S. / Bieling, P.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Nat Commun / Year: 2021
Title: A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks.
Authors: Johanna Funk / Felipe Merino / Matthias Schaks / Klemens Rottner / Stefan Raunser / Peter Bieling /
Abstract: Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside ...Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP's C-terminal "tentacle" extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the β tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes-capping and nucleation-in branched actin network assembly.
History
DepositionAug 9, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 1, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 6, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Assembly

Deposited unit
E: Isoform 2 of F-actin-capping protein subunit beta
F: F-actin-capping protein subunit alpha-1
I: Actin, cytoplasmic 1
J: Actin, cytoplasmic 1
K: Actin, cytoplasmic 1
L: Actin, cytoplasmic 1
N: Actin, cytoplasmic 1
O: Actin, cytoplasmic 1
Q: Phalloidin
R: Phalloidin
S: Phalloidin
T: Phalloidin
P: Phalloidin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)321,55829
Polymers318,46913
Non-polymers3,08916
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: assay for oligomerization
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area37550 Å2
ΔGint-341 kcal/mol
Surface area109870 Å2
MethodPISA

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Components

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Protein , 3 types, 8 molecules EFIJKLNO

#1: Protein Isoform 2 of F-actin-capping protein subunit beta / CapZ beta


Mass: 30669.768 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Capzb, Cappb1 / Production host: Escherichia coli (E. coli) / References: UniProt: P47757
#2: Protein F-actin-capping protein subunit alpha-1 / CapZ alpha-1


Mass: 32980.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Capza1, Cappa1 / Production host: Escherichia coli (E. coli) / References: UniProt: P47753
#3: Protein
Actin, cytoplasmic 1 / / Beta-actin


Mass: 41795.680 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: P60712

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Protein/peptide , 1 types, 5 molecules QRSTP

#4: Protein/peptide
Phalloidin / /


Type: Cyclic peptide / Class: Toxin / Mass: 808.899 Da / Num. of mol.: 5 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) / References: Phalloidin

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Non-polymers , 3 types, 16 molecules

#5: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


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


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical
ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION

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Details

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

ComponentName: Complex between capping protein and the barbed end of cytoplasmic actin filaments
Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Buffer solutionpH: 7 / Details: KMEI buffer
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMPotassium chlorideKCl1
210 mMImidazole1
31.5 mMMagnesium chlorideMgCl21
41 mMEGTA1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Mild discharging with 5 mA current / Grid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / 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
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 3 sec. / Electron dose: 60 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 4204

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1.13CTF correction
7UCSF Chimeramodel fitting
9SPHIRE1.3initial Euler assignment
10SPHIRE1.3final Euler assignment
11SPHIRE1.3classification
12SPHIRE1.33D reconstruction
13ISOLDEmodel refinement
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 570724
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 60206 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 40 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 5ADX

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