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- PDB-8f8t: Cryo-EM structure of the Tropomodulin-capped pointed end of F-actin -

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Basic information

Entry
Database: PDB / ID: 8f8t
TitleCryo-EM structure of the Tropomodulin-capped pointed end of F-actin
Components
  • Actin, alpha skeletal muscle
  • Tropomodulin-1
KeywordsSTRUCTURAL PROTEIN / Actin cytoskeleton / filament / Tmod
Function / homology
Function and homology information


pointed-end actin filament capping / lens fiber cell development / myofibril assembly / Striated Muscle Contraction / cytoskeletal motor activator activity / tropomyosin binding / cortical cytoskeleton / myofibril / myosin heavy chain binding / mesenchyme migration ...pointed-end actin filament capping / lens fiber cell development / myofibril assembly / Striated Muscle Contraction / cytoskeletal motor activator activity / tropomyosin binding / cortical cytoskeleton / myofibril / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / sarcomere / adult locomotory behavior / filopodium / muscle contraction / actin filament organization / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / lamellipodium / cell body / actin binding / cytoskeleton / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / membrane / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Tropomodulin / Tropomodulin / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin ...Tropomodulin / Tropomodulin / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / Leucine-rich repeat domain superfamily / ATPase, nucleotide binding domain
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Tropomodulin-1 / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesHomo sapiens (human)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.26 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

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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: Actin, alpha skeletal muscle
T: Tropomodulin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)338,54922
Polymers335,3898
Non-polymers3,16114
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.: 7 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135
#2: Protein Tropomodulin-1 / / Erythrocyte tropomodulin / E-Tmod


Mass: 40619.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TMOD1, D9S57E, TMOD / Production host: Escherichia coli (E. coli) / References: UniProt: P28289
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / 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
1Tropomodulin-capped pointed end of F-actinCOMPLEX#1-#20MULTIPLE SOURCES
2Tropomodulin (Tmod)COMPLEX#21RECOMBINANT
3Actin filamentMicrofilamentCOMPLEX#11NATURAL
Molecular weightExperimental value: NO
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
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: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25004 / 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_d0.00522871
ELECTRON MICROSCOPYf_angle_d0.91631027
ELECTRON MICROSCOPYf_dihedral_angle_d14.0028558
ELECTRON MICROSCOPYf_chiral_restr0.0573449
ELECTRON MICROSCOPYf_plane_restr0.0083975

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