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- EMDB-28933: Cryo-EM structure of the CapZ-capped barbed end of F-actin -

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Basic information

Entry
Database: EMDB / ID: EMD-28933
TitleCryo-EM structure of the CapZ-capped barbed end of F-actin
Map dataSharpened final map of the CapZ-bound actin filament barbed end.
Sample
  • Complex: CapZ-capped barbed end of F-actin
    • Complex: CapZ (alpha and beta subunits)
      • Protein or peptide: F-actin-capping protein subunit alpha-1
      • Protein or peptide: F-actin-capping protein subunit beta
    • Complex: Actin filamentMicrofilament
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsBarbed end capping protein / F-actin / CapZ / STRUCTURAL PROTEIN
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
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) / rabbit (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.79 Å
AuthorsCarman PJ / Barrie KR / Dominguez R
Funding support United States, 3 items
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, 2022-
Header (metadata) releaseJun 7, 2023-
Map releaseJun 7, 2023-
UpdateJul 5, 2023-
Current statusJul 5, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28933.png

Downloads & links

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Map

FileDownload / File: emd_28933.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened final map of the CapZ-bound actin filament barbed end.
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.02645325 - 1.8826389
Average (Standard dev.)0.0007141245 (±0.017851708)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 432.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map of the CapZ-bound actin filament barbed end.

Fileemd_28933_additional_1.map
AnnotationUnsharpened map of the CapZ-bound actin filament barbed end.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A of the CapZ-bound actin filament barbed end.

Fileemd_28933_half_map_1.map
AnnotationHalf-map A of the CapZ-bound actin filament barbed end.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B of the CapZ-bound actin filament barbed end.

Fileemd_28933_half_map_2.map
AnnotationHalf-map B of the CapZ-bound actin filament barbed end.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CapZ-capped barbed end of F-actin

EntireName: CapZ-capped barbed end of F-actin
Components
  • Complex: CapZ-capped barbed end of F-actin
    • Complex: CapZ (alpha and beta subunits)
      • Protein or peptide: F-actin-capping protein subunit alpha-1
      • Protein or peptide: F-actin-capping protein subunit beta
    • Complex: Actin filamentMicrofilament
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: CapZ-capped barbed end of F-actin

SupramoleculeName: CapZ-capped barbed end of F-actin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: CapZ (alpha and beta subunits)

SupramoleculeName: CapZ (alpha and beta subunits) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Actin filament

SupramoleculeName: Actin filament / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: rabbit (rabbit)
Molecular weightTheoretical: 42.109973 KDa
SequenceString: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIE(HIC)G IIT NWDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLD SG DGVTHNVPIY ...String:
MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIE(HIC)G IIT NWDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLD SG DGVTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSS S LEKSYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVMSGGTTM YPGIADRMQ KEITALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWITKQEYDE AGPSIVHRKC F

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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: Homo sapiens (human)
Molecular weightTheoretical: 32.964727 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MADFDDRVSD EEKVRIAAKF ITHAPPGEFN EVFNDVRLLL NNDNLLREGA AHAFAQYNMD QFTPVKIEGY EDQVLITEHG DLGNSRFLD PRNKISFKFD HLRKEASDPQ PEEADGGLKS WRESCDSALR AYVKDHYSNG FCTVYAKTID GQQTIIACIE S HQFQPKNF ...String:
MADFDDRVSD EEKVRIAAKF ITHAPPGEFN EVFNDVRLLL NNDNLLREGA AHAFAQYNMD QFTPVKIEGY EDQVLITEHG DLGNSRFLD PRNKISFKFD HLRKEASDPQ PEEADGGLKS WRESCDSALR AYVKDHYSNG FCTVYAKTID GQQTIIACIE S HQFQPKNF WNGRWRSEWK FTITPPTAQV VGVLKIQVHY YEDGNVQLVS HKDVQDSLTV SNEAQTAKEF IKIIENAENE YQ TAISENY QTMSDTTFKA LRRQLPVTRT KIDWNKILSY KIGKEMQNA

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Macromolecule #3: F-actin-capping protein subunit beta

MacromoleculeName: F-actin-capping protein subunit beta / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 31.258289 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDQQLDCALD LMRRLPPQQI EKNLSDLIDL VPSLCEDLLS SVDQPLKIAR DKVVGKDYLL CDYNRDGDSY RSPWSNKYDP PLEDGAMPS ARLRKLEVEA NNAFDQYRDL YFEGGVSSVY LWDLDHGFAG VILIKKAGDG SKKIKGCWDS IHVVEVQEKS S GRTAHYKL ...String:
SDQQLDCALD LMRRLPPQQI EKNLSDLIDL VPSLCEDLLS SVDQPLKIAR DKVVGKDYLL CDYNRDGDSY RSPWSNKYDP PLEDGAMPS ARLRKLEVEA NNAFDQYRDL YFEGGVSSVY LWDLDHGFAG VILIKKAGDG SKKIKGCWDS IHVVEVQEKS S GRTAHYKL TSTVMLWLQT NKSGSGTMNL GGSLTRQMEK DETVSDCSPH IANIGRLVED MENKIRSTLN EIYFGKTKDI VN GLRSIDA IPDNQKFKQL QRELSQVLTQ RQIYIQPDN

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / 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 #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration1.05 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force 0 Blot time 2.5 s.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 44.7 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1102793 / Details: Topaz particle picking with training
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.79 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.0) / Number images used: 155516
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient
Output model

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

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