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- PDB-5ooc: Cryo-EM structure of jasplakinolide-stabilized F-actin in complex... -

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Basic information

Entry
Database: PDB / ID: 5ooc
TitleCryo-EM structure of jasplakinolide-stabilized F-actin in complex with ADP
ComponentsActin, alpha skeletal muscle
KeywordsSTRUCTURAL PROTEIN / Cytoskeleton / nucleotide states / filament stability / cell migration
Function / homology
Function and homology information


cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / actin filament bundle / troponin I binding / filamentous actin / mesenchyme migration / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament ...cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / actin filament bundle / troponin I binding / filamentous actin / mesenchyme migration / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament / skeletal muscle thin filament assembly / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / lamellipodium / cell body / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
Actin; Chain A, domain 2 / Actin; Chain A, domain 2 / ATPase, substrate binding domain, subdomain 4 / Actin; Chain A, domain 4 / ATPase, nucleotide binding domain / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. ...Actin; Chain A, domain 2 / Actin; Chain A, domain 2 / ATPase, substrate binding domain, subdomain 4 / Actin; Chain A, domain 4 / ATPase, nucleotide binding domain / 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 / Nucleotidyltransferase; domain 5 / Roll / Alpha-Beta Complex / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Chem-9ZK / ADENOSINE-5'-DIPHOSPHATE / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsMerino, F. / Pospich, S. / Funk, J. / Kuellmer, F. / Arndt, H.-D. / Bieling, P. / Raunser, S.
Funding support Germany, 4items
OrganizationGrant numberCountry
Max Planck Society Germany
State of Thuringia43- 5572-321-12040-12 Germany
European Union615984 Germany
Studienstiftung des deutschen Volkes Germany
CitationJournal: Nat Struct Mol Biol / Year: 2018
Title: Structural transitions of F-actin upon ATP hydrolysis at near-atomic resolution revealed by cryo-EM.
Authors: Felipe Merino / Sabrina Pospich / Johanna Funk / Thorsten Wagner / Florian Küllmer / Hans-Dieter Arndt / Peter Bieling / Stefan Raunser /
Abstract: The function of actin is coupled to the nucleotide bound to its active site. ATP hydrolysis is activated during polymerization; a delay between hydrolysis and inorganic phosphate (P) release results ...The function of actin is coupled to the nucleotide bound to its active site. ATP hydrolysis is activated during polymerization; a delay between hydrolysis and inorganic phosphate (P) release results in a gradient of ATP, ADP-P and ADP along actin filaments (F-actin). Actin-binding proteins can recognize F-actin's nucleotide state, using it as a local 'age' tag. The underlying mechanism is complex and poorly understood. Here we report six high-resolution cryo-EM structures of F-actin from rabbit skeletal muscle in different nucleotide states. The structures reveal that actin polymerization repositions the proposed catalytic base, His161, closer to the γ-phosphate. Nucleotide hydrolysis and P release modulate the conformational ensemble at the periphery of the filament, thus resulting in open and closed states, which can be sensed by coronin-1B. The drug-like toxin jasplakinolide locks F-actin in an open state. Our results demonstrate in detail how ATP hydrolysis links to F-actin's conformational dynamics and protein interaction.
History
DepositionAug 7, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 13, 2018Provider: repository / Type: Initial release
Revision 1.0Jun 13, 2018Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.1Oct 16, 2019Group: Data collection / Database references / Category: pdbx_database_related
Revision 1.2Apr 9, 2025Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update
Revision 1.1Apr 9, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / Category: database_2 / em_admin / Data content type: EM metadata / EM metadata / EM metadata
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)215,00520
Polymers209,3785
Non-polymers5,62715
Water00
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16260 Å2
ΔGint-137 kcal/mol
Surface area73410 Å2
MethodPISA

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Components

#1: Protein
Actin, alpha skeletal muscle / Alpha-actin-1


Mass: 41875.633 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / Tissue: skeletal muscle / References: UniProt: P68135
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-9ZK / (4~{R},7~{R},10~{S},13~{S},15~{E},19~{S})-10-(4-azanylbutyl)-4-(4-hydroxyphenyl)-7-(1~{H}-indol-3-ylmethyl)-8,13,15,19-tetramethyl-1-oxa-5,8,11-triazacyclononadec-15-ene-2,6,9,12-tetrone


Mass: 673.841 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C38H51N5O6
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction
Crystal symmetry∠γ: 90 ° / A: 1 Å / B: 1 Å / C: 1 Å / Space group name H-M: P1

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Sample preparation

ComponentName: Filamentous jasplakinolide-stabilized alpha actin in complex with ADP
Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: Skeletal Muscle
Buffer solutionpH: 7.5
Details: 5 mM Tris pH 7.5, 2 mM NaN3, 1 mM DTT, 100 mM KCl and 2 mM MgCl2, 0.12 %(v/v) DMSO
Buffer component
IDConc.NameFormulaBuffer-ID
15 mMTRISC4H11NO31
22 mMSodium azideNaN31
31 mMDTTC4H10O2S21
4100 mMPotassium chlorideKCl1
52 mMMagnesium chlorideMgCl21
60.12 % (v/v)DMSOC2H6OS1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Rise 27.56 A, Twist -166.70 degrees
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 298 K
Details: Manual backside blotting using Whatman filter paper No.5.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Cs-corrected microscope
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2.1 sec. / Electron dose: 71 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of real images: 1754
EM imaging opticsSpherical aberration corrector: Cs-corrected microscope
Image scansMovie frames/image: 40 / Used frames/image: 1-8

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Processing

EM software
IDNameVersionCategoryDetails
1SPARXparticle selectionsxhelixboxer.py
2EPUimage acquisition
4CTFFINDv4CTF correction
5RELION2CTF correction
8UCSF Chimeramodel fitting
10Rosettamodel refinement
11NAMDmodel refinement
12RELION2initial Euler assignment
13RELION2final Euler assignment
15RELION23D reconstruction
Crystal symmetry∠γ: 90 ° / A: 1 Å / B: 1 Å / C: 1 Å / Space group name H-M: P1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 385766
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 365209 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: Rosetta iterative refinement was combined with MDFF.

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