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- PDB-6t20: Cryo-EM structure of phalloidin-stabilized F-actin (aged) -

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Basic information

Entry
Database: PDB / ID: 6t20
TitleCryo-EM structure of phalloidin-stabilized F-actin (aged)
Components
  • Actin, alpha skeletal muscle
  • phalloidin
KeywordsSTRUCTURAL PROTEIN / Cytoskeleton / phalloidin / stabilized-actin filament
Function / homology
Function and homology information


positive regulation of microfilament motor activity => GO:0120081 / mesenchyme migration / tropomyosin binding / myosin heavy chain binding / troponin I binding / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle / filamentous actin / actin filament bundle assembly ...positive regulation of microfilament motor activity => GO:0120081 / mesenchyme migration / tropomyosin binding / myosin heavy chain binding / troponin I binding / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle / filamentous actin / actin filament bundle assembly / actin monomer binding / skeletal muscle fiber development / skeletal muscle myofibril / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / calcium-dependent protein binding / cell body / lamellipodium / protein domain specific binding / positive regulation of gene expression / calcium ion binding / magnesium ion binding / ATP binding / identical protein binding / cytoplasm
Actin / ATPase, nucleotide binding domain / Actin/actin-like conserved site / Actin, conserved site / Actin family
Actin, alpha skeletal muscle
Biological speciesOryctolagus cuniculus (rabbit)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsPospich, S. / Merino, F. / Raunser, S.
Funding support Germany, 2items
OrganizationGrant numberCountry
Max Planck Society Germany
European Union (EU)615984 Germany
CitationJournal: Structure / Year: 2020
Title: Structural Effects and Functional Implications of Phalloidin and Jasplakinolide Binding to Actin Filaments.
Authors: Sabrina Pospich / Felipe Merino / Stefan Raunser /
Abstract: Actin undergoes structural transitions during polymerization, ATP hydrolysis, and subsequent release of inorganic phosphate. Several actin-binding proteins sense specific states during this ...Actin undergoes structural transitions during polymerization, ATP hydrolysis, and subsequent release of inorganic phosphate. Several actin-binding proteins sense specific states during this transition and can thus target different regions of the actin filament. Here, we show in atomic detail that phalloidin, a mushroom toxin that is routinely used to stabilize and label actin filaments, suspends the structural changes in actin, likely influencing its interaction with actin-binding proteins. Furthermore, high-resolution cryoelectron microscopy structures reveal structural rearrangements in F-actin upon inorganic phosphate release in phalloidin-stabilized filaments. We find that the effect of the sponge toxin jasplakinolide differs from the one of phalloidin, despite their overlapping binding site and similar interactions with the actin filament. Analysis of structural conformations of F-actin suggests that stabilizing agents trap states within the natural conformational space of actin.
Validation Report
SummaryFull reportAbout validation report
History
DepositionOct 7, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 4, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 15, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-10364
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  • Superimposition on EM map
  • EMDB-10364
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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: phalloidin
G: phalloidin
H: phalloidin
I: phalloidin
J: phalloidin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)215,68020
Polymers213,42310
Non-polymers2,25810
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area19810 Å2
ΔGint-172 kcal/mol
Surface area72720 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: Protein/peptide
phalloidin /


Mass: 808.899 Da / Num. of mol.: 5 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION / Magnesium


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

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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

Component
IDNameTypeEntity IDParent-IDSource
1Filamentous phalloidin-stabilized alpha actin in complex with ADPCOMPLEX#1-#20MULTIPLE SOURCES
2Filamentous alpha actin in complex with ADPCOMPLEX#11NATURAL
3PhalloidinCOMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDTissue
12Oryctolagus cuniculus (rabbit)9986skeletal muscle
23synthetic construct (others)32630
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 7.5
Details: 5 mM Tris pH 7.5, 2 mM NaN3, 1 mM DTT, 100 mM KCl and 2 mM MgCl2, 2.0 %(v/v) MeOH, 0.03 %(v/w) Tween 20
Buffer component
IDConc.NameFormulaBuffer-ID
15 mMTRISC4H11NO31
22 mMSodium azideNaN31
31 mMDTTC4H10O2S21
4100 mMPotassium chlorideKCl1
52 mMMagnesium chlorideMgCl21
62.0 % (v/v)MethanolCH3OH1
70.03 % (v/w)Tween 20C58H114O261
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Rise 27.6 A, Twist -166.9 degrees
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 286 K

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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 FIELDBright-field microscopy / Cs: 0 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 87 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5213
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
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1.5CTF correction
5RELION2-betaCTF correction
8UCSF Chimeramodel fitting
10Rosettamodel refinement
11RELION2-betainitial Euler assignment
12RELION2-betafinal Euler assignment
14RELION2-beta3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1209605
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 513783 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID3D fitting-IDPdb chain residue range
15ONVC1
26T1YC1378-378

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