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- PDB-3g37: Cryo-EM structure of actin filament in the presence of phosphate -

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Basic information

Entry
Database: PDB / ID: 3g37
TitleCryo-EM structure of actin filament in the presence of phosphate
ComponentsActin, alpha skeletal muscle
KeywordsCONTRACTILE PROTEIN / actin / cytoskeleton / cell adhesion / cellular signaling / cytokinesis / muscle / cryo-EM / ATP-binding / Methylation / Muscle protein / Nucleotide-binding / Phosphoprotein
Function / homology
Function and homology information


cytoskeletal motor activator activity / tropomyosin binding / 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 ...cytoskeletal motor activator activity / tropomyosin binding / 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 / 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
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
ADENOSINE-5'-DIPHOSPHATE / PHOSPHATE ION / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6 Å
AuthorsWakabayshi, T. / Murakami, K. / Yasunaga, T. / Noguchi, T.Q. / Uyeda, T.Q.
CitationJournal: Cell / Year: 2010
Title: Structural basis for actin assembly, activation of ATP hydrolysis, and delayed phosphate release.
Authors: Kenji Murakami / Takuo Yasunaga / Taro Q P Noguchi / Yuki Gomibuchi / Kien X Ngo / Taro Q P Uyeda / Takeyuki Wakabayashi /
Abstract: Assembled actin filaments support cellular signaling, intracellular trafficking, and cytokinesis. ATP hydrolysis triggered by actin assembly provides the structural cues for filament turnover in vivo. ...Assembled actin filaments support cellular signaling, intracellular trafficking, and cytokinesis. ATP hydrolysis triggered by actin assembly provides the structural cues for filament turnover in vivo. Here, we present the cryo-electron microscopic (cryo-EM) structure of filamentous actin (F-actin) in the presence of phosphate, with the visualization of some α-helical backbones and large side chains. A complete atomic model based on the EM map identified intermolecular interactions mediated by bound magnesium and phosphate ions. Comparison of the F-actin model with G-actin monomer crystal structures reveals a critical role for bending of the conserved proline-rich loop in triggering phosphate release following ATP hydrolysis. Crystal structures of G-actin show that mutations in this loop trap the catalytic site in two intermediate states of the ATPase cycle. The combined structural information allows us to propose a detailed molecular mechanism for the biochemical events, including actin polymerization and ATPase activation, critical for actin filament dynamics.
History
DepositionFeb 2, 2009Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Nov 3, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Dec 4, 2019Group: Author supporting evidence / Data collection ...Author supporting evidence / Data collection / Database references / Derived calculations
Category: database_2 / em_image_scans ...database_2 / em_image_scans / em_imaging / em_imaging_optics / em_single_particle_entity / struct_conn / struct_ref_seq_dif
Item: _em_imaging.energy_filter / _em_imaging_optics.energyfilter_name ..._em_imaging.energy_filter / _em_imaging_optics.energyfilter_name / _struct_conn.pdbx_leaving_atom_flag / _struct_ref_seq_dif.details
Revision 1.3Dec 18, 2019Group: Data collection / Category: em_software / Item: _em_software.image_processing_id

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Assembly

Deposited unit
O: Actin, alpha skeletal muscle
P: Actin, alpha skeletal muscle
Q: Actin, alpha skeletal muscle
R: Actin, alpha skeletal muscle
S: Actin, alpha skeletal muscle
T: Actin, alpha skeletal muscle
U: Actin, alpha skeletal muscle
V: Actin, alpha skeletal muscle
W: Actin, alpha skeletal muscle
X: Actin, alpha skeletal muscle
Y: Actin, alpha skeletal muscle
Z: Actin, alpha skeletal muscle
hetero molecules


Theoretical massNumber of molelcules
Total (without water)513,115132
Polymers502,82012
Non-polymers10,295120
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 41901.668 Da / Num. of mol.: 12 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / Tissue: muscleSkeletal muscle / References: UniProt: P68135
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#3: Chemical...
ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 36 / Source method: obtained synthetically / Formula: PO4
#4: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 72 / 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: actin filament in the presence of phosphateMicrofilament
Type: COMPLEX
Details: 50 mM NaCl, 5 mM MgCl2, 0.025 mM ATP, 10 mM sodium phosphate (pH 7.4), 0.05 %NaN3, 0.7 mM DTT
Buffer solutionName: phosphate buffer / pH: 7.4 / Details: phosphate buffer
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Temp: 77 K / Humidity: 90 %

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Electron microscopy imaging

MicroscopyModel: HITACHI EF2000
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 100000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 1000 nm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN
Specimen holder type: Side entry liquid nitrogen-cooled cryo specimen holder
Temperature: 77 K / Temperature (max): 77 K / Temperature (min): 77 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 15 e/Å2 / Film or detector model: GENERIC CCD
Details: CCD camera with an epitaxially-grown CsI scintillator
EM imaging opticsEnergyfilter name: In-column Omega Filter

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Processing

EM software
IDNameCategoryDetails
1NAMDmodel fitting
2Omodel fitting
3EOS3D reconstructionEOS (Yasunaga & Wakabayshi 1996)
CTF correctionDetails: FSC at 0.143 cut-off
3D reconstructionMethod: single particleSingle particle analysis / Resolution: 6 Å / Num. of particles: 8000 / Nominal pixel size: 2.275 Å / Actual pixel size: 2.275 Å / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: pdbRhofit (Yasunaga & Wakabayashi, 1996)
Details: METHOD--local refinement REFINEMENT PROTOCOL--real-space refinement
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms35232 0 576 0 35808

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