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- PDB-5ooe: Cryo-EM structure of F-actin in complex with AppNHp (AMPPNP) -

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Entry
Database: PDB / ID: 5ooe
TitleCryo-EM structure of F-actin in complex with AppNHp (AMPPNP)
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 / 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
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 ...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 / Alpha-Beta Complex / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / 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 Thuring43-5572-321-12040-12 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

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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)212,03115
Polymers209,3785
Non-polymers2,65310
Water00
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13830 Å2
ΔGint-99 kcal/mol
Surface area71210 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-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


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

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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 AppNHp (AMPPNP)
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, 0.1 M KCl, 2 mM MgCl2, 2 mM, 2 mM NaN3, 0.5 mM TCEP and 0.4 mM AppNHp.
Buffer component
IDConc.NameFormulaBuffer-ID
15 mMHEPESC8H18N2O4S1
20.1 MPotassium chlorideKCl1
32 mMMagnesium chlorideMgCl21
40.5 mMTCEPC9H15O6P1
52 mMSodium azideNaN31
60.4 mMAppNHpC10H17N6O12P31
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Rise 27.36 A, Twist -166.58 degrees
Specimen supportGrid material: COPPER / 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: 1.5 sec. / Electron dose: 110 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of real images: 2416
EM imaging opticsSpherical aberration corrector: Cs-corrected microscope
Image scansMovie frames/image: 25 / Used frames/image: 1-4

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Processing

EM software
IDNameVersionCategoryDetails
1SPARXparticle selectionsxhelixboxer.py
2EPUimage acquisition
4CTFFINDv4CTF correction
5RELION2CTF correction
8UCSF Chimeramodel fitting
10RELION2initial Euler assignment
11RELION2final Euler assignment
13RELION23D reconstruction
14Rosettamodel refinement
15NAMDmodel refinement
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 1 Å / B: 1 Å / C: 1 Å / Space group name: P1 / Space group num: 1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 317928
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 284554 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: Rosetta iterative refinement was combined with MDFF.

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