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- PDB-6fhl: Cryo-EM structure of F-actin in complex with ADP-Pi -

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Basic information

Entry
Database: PDB / ID: 6fhl
TitleCryo-EM structure of F-actin in complex with ADP-Pi
ComponentsActin, alpha skeletal muscle
KeywordsSTRUCTURAL PROTEIN / Cytoskeleton / nucleotide states / filament stability / cell migration
Function / homology
Function and homology information


cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / actin filament bundle assembly / striated muscle thin filament ...cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / actin filament bundle assembly / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / stress fiber / skeletal muscle fiber development / 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
ADENOSINE-5'-DIPHOSPHATE / PHOSPHATE ION / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsMerino, F. / Pospich, S. / Funk, J. / Wagner, T. / 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
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
DepositionJan 15, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 13, 2018Provider: repository / Type: Initial release
Revision 1.1Aug 29, 2018Group: Data collection / Database references / Category: pdbx_database_related / Item: _pdbx_database_related.db_id

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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)212,11120
Polymers209,3785
Non-polymers2,73215
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17210 Å2
ΔGint-217 kcal/mol
Surface area68120 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-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
#4: Chemical
ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: PO4

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Filamentous alpha actin in complex with ADP-ADPPi / 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 HEPES pH 7.5, 0.05 M KCl, 2 mM MgCl2, 2 mM NaN3, 0.5 mM TCEP, 0.2 mM ADP, 50 mM potassium phosphate.
Buffer component
IDConc.NameFormulaBuffer-ID
15 mMHEPESC8H18N2O4S1
20.05 MPotassium chlorideKCl1
32 mMMagnesium chlorideMgCl21
40.5 mMTCEPC9H15O6P1
52 mMSodium azideNaN31
60.2 mMADPC10H15N5O10P21
750 mMPhosphatePO41
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Rise 26.9 A, Twist -166.7 degrees
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 286 K / Details: 8s blotting, 1s drain time, -25 force

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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: 1.5 sec. / Electron dose: 93 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 2614
EM imaging opticsSpherical aberration corrector: Cs-corrected microscope
Image scansMovie frames/image: 25 / Used frames/image: 1-5

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4Gctf0.5CTF correction
5RELION2CTF correction
8UCSF Chimeramodel fitting
10RELION2initial Euler assignment
11RELION2final Euler assignment
13RELION23D reconstruction
14Rosettamodel refinement
15NAMDmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1850662
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 933087 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Details: Rosetta iterative refinement combined with MDFF.

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