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- EMDB-13343: Structure of capping protein bound to the barbed end of a cytopla... -

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Basic information

Entry
Database: EMDB / ID: EMD-13343
TitleStructure of capping protein bound to the barbed end of a cytoplasmic actin filament
Map dataLocally filtered map with global B-factor
Sample
  • Complex: Complex between capping protein and the barbed end of cytoplasmic actin filaments
    • Protein or peptide: Isoform 2 of F-actin-capping protein subunit beta
    • Protein or peptide: F-actin-capping protein subunit alpha-1
    • Protein or peptide: Actin, cytoplasmic 1
    • Protein or peptide: Phalloidin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHATE IONPhosphate
Function / homology
Function and homology information


cytoskeletal calyx / Adherens junctions interactions / Cell-extracellular matrix interactions / RHOF GTPase cycle / Advanced glycosylation endproduct receptor signaling / RHOBTB2 GTPase cycle / RHOD GTPase cycle / Gap junction degradation / Formation of annular gap junctions / MAP2K and MAPK activation ...cytoskeletal calyx / Adherens junctions interactions / Cell-extracellular matrix interactions / RHOF GTPase cycle / Advanced glycosylation endproduct receptor signaling / RHOBTB2 GTPase cycle / RHOD GTPase cycle / Gap junction degradation / Formation of annular gap junctions / MAP2K and MAPK activation / EPHB-mediated forward signaling / Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / COPI-independent Golgi-to-ER retrograde traffic / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / WASH complex / sperm connecting piece / F-actin capping protein complex / COPI-mediated anterograde transport / negative regulation of filopodium assembly / structural constituent of postsynaptic actin cytoskeleton / VEGFA-VEGFR2 Pathway / dense body / Factors involved in megakaryocyte development and platelet production / Clathrin-mediated endocytosis / cell projection organization / cell junction assembly / MHC class II antigen presentation / barbed-end actin filament capping / actin polymerization or depolymerization / regulation of cell morphogenesis / regulation of lamellipodium assembly / NuA4 histone acetyltransferase complex / lamellipodium assembly / cortical cytoskeleton / brush border / asymmetric synapse / cytoskeleton organization / hippocampal mossy fiber to CA3 synapse / axonogenesis / actin filament / cell motility / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Schaffer collateral - CA1 synapse / cell morphogenesis / Z disc / actin filament binding / cell-cell junction / actin cytoskeleton / lamellipodium / actin binding / actin cytoskeleton organization / dendritic spine / postsynaptic density / cytoskeleton / hydrolase activity / axon / focal adhesion / neuronal cell body / synapse / protein kinase binding / protein-containing complex / ATP binding / membrane / nucleus / plasma membrane / 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
F-actin-capping protein subunit alpha-1 / F-actin-capping protein subunit beta / Actin, cytoplasmic 1
Similarity search - Component
Biological speciesMus musculus (house mouse) / Bovine (cattle) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsFunk J / Merino F / Schacks M / Rottner K / Raunser S / Bieling P
Funding support Germany, 1 items
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, 2021-
Header (metadata) releaseSep 1, 2021-
Map releaseSep 1, 2021-
UpdateOct 6, 2021-
Current statusOct 6, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
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  • Surface view colored by cylindrical radius
  • Surface level: 0.045
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  • Surface view with fitted model
  • Atomic models: PDB-7pdz
  • Surface level: 0.045
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7pdz
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13343.png

Downloads & links

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Map

FileDownload / File: emd_13343.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally filtered map with global B-factor
Voxel sizeX=Y=Z: 1.21 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.08255227 - 0.21015494
Average (Standard dev.)-0.00014572637 (±0.0043614074)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 464.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.211.211.21
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z464.640464.640464.640
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ640640640
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0830.210-0.000

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Supplemental data

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Mask #1

Fileemd_13343_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Locally filtered map using deepEMhancer's high resolution model

Fileemd_13343_additional_1.map
AnnotationLocally filtered map using deepEMhancer's high resolution model
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: Map filtered globally to a resolution of 3.9 A

Fileemd_13343_additional_2.map
AnnotationMap filtered globally to a resolution of 3.9 A
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_13343_half_map_1.map
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Half map: #1

Fileemd_13343_half_map_2.map
Projections & Slices
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Sample components

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Entire : Complex between capping protein and the barbed end of cytoplasmic...

EntireName: Complex between capping protein and the barbed end of cytoplasmic actin filaments
Components
  • Complex: Complex between capping protein and the barbed end of cytoplasmic actin filaments
    • Protein or peptide: Isoform 2 of F-actin-capping protein subunit beta
    • Protein or peptide: F-actin-capping protein subunit alpha-1
    • Protein or peptide: Actin, cytoplasmic 1
    • Protein or peptide: Phalloidin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHATE IONPhosphate

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Supramolecule #1: Complex between capping protein and the barbed end of cytoplasmic...

SupramoleculeName: Complex between capping protein and the barbed end of cytoplasmic actin filaments
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4

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Macromolecule #1: Isoform 2 of F-actin-capping protein subunit beta

MacromoleculeName: Isoform 2 of F-actin-capping protein subunit beta / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 30.669768 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSDQQLDCAL DLMRRLPPQQ IEKNLSDLID LVPSLCEDLL SSVDQPLKIA RDKVVGKDYL LCDYNRDGDS YRSPWSNKYD PPLEDGAMP SARLRKLEVE ANNAFDQYRD LYFEGGVSSV YLWDLDHGFA GVILIKKAGD GSKKIKGCWD SIHVVEVQEK S SGRTAHYK ...String:
MSDQQLDCAL DLMRRLPPQQ IEKNLSDLID LVPSLCEDLL SSVDQPLKIA RDKVVGKDYL LCDYNRDGDS YRSPWSNKYD PPLEDGAMP SARLRKLEVE ANNAFDQYRD LYFEGGVSSV YLWDLDHGFA GVILIKKAGD GSKKIKGCWD SIHVVEVQEK S SGRTAHYK LTSTVMLWLQ TNKSGSGTMN LGGSLTRQME KDETVSDCSP HIANIGRLVE DMENKIRSTL NEIYFGKTKD IV NGLRSVQ TFADKSKQEA LKNDLVEALK RKQQC

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Macromolecule #2: F-actin-capping protein subunit alpha-1

MacromoleculeName: F-actin-capping protein subunit alpha-1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 32.980703 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MADFEDRVSD EEKVRIAAKF ITHAPPGEFN EVFNDVRLLL NNDNLLREGA AHAFAQYNMD QFTPVKIEGY DDQVLITEHG DLGNSRFLD PRNQISFKFD HLRKEASDPQ PEDVDGGLKS WRESCDSALR AYVKDHYSNG FCTVYAKTID GQQTIIACIE S HQFQPKNF ...String:
MADFEDRVSD EEKVRIAAKF ITHAPPGEFN EVFNDVRLLL NNDNLLREGA AHAFAQYNMD QFTPVKIEGY DDQVLITEHG DLGNSRFLD PRNQISFKFD HLRKEASDPQ PEDVDGGLKS WRESCDSALR AYVKDHYSNG FCTVYAKTID GQQTIIACIE S HQFQPKNF WNGRWRSEWK FTITPPSAQV VGVLKIQVHY YEDGNVQLVS HKDVQDSVTV SNEVQTTKEF IKIIESAENE YQ TAISENY QTMSDTTFKA LRRQLPVTRT KIDWNKILSY KIGKEMQNA

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Macromolecule #3: Actin, cytoplasmic 1

MacromoleculeName: Actin, cytoplasmic 1 / type: protein_or_peptide / ID: 3 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Bovine (cattle)
Molecular weightTheoretical: 41.79568 KDa
SequenceString: MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GIV TNW DDMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSG DG VTHTVPIYEG ...String:
MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GIV TNW DDMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSG DG VTHTVPIYEG YALPHAILRL DLAGRDLTDY LMKILTERGY SFTTTAEREI VRDIKEKLCY VALDFEQEMA TAASSSSL E KSYELPDGQV ITIGNERFRC PEALFQPSFL GMESCGIHET TFNSIMKCDV DIRKDLYANT VLSGGTTMYP GIADRMQKE ITALAPSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ISKQEYDESG PSIVHRKCF

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Macromolecule #4: Phalloidin

MacromoleculeName: Phalloidin / type: protein_or_peptide / ID: 4 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 808.899 Da
SequenceString:
(HYP)AW(EEP)A(DTH)C

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Macromolecule #5: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 6 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 7 / Number of copies: 4 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION / Phosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
Component:
ConcentrationFormulaName
50.0 mMKClPotassium chloride
10.0 mMImidazole
1.5 mMMgCl2Magnesium chloride
1.0 mMEGTA

Details: KMEI buffer
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: Mild discharging with 5 mA current
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 286 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 2 / Number real images: 4204 / Average exposure time: 3.0 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 570724
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.13)
Startup modelType of model: OTHER
Details: Artificially built actin filament. Generated from 6FHL
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE (ver. 1.3)
Final 3D classificationNumber classes: 3 / Software - Name: SPHIRE (ver. 1.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE (ver. 1.3)
Final reconstructionNumber classes used: 1 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE (ver. 1.3) / Number images used: 60206
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5adx

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 40
Output model

PDB-7pdz:
Structure of capping protein bound to the barbed end of a cytoplasmic actin filament

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