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- EMDB-20694: Actin phalloidin at BeFx state -

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Basic information

Entry
Database: EMDB / ID: EMD-20694
TitleActin phalloidin at BeFx state
Map dataActin phalloidin at BeFx state
Sample
  • Organelle or cellular component: Actin phalloidin, BeFx state
    • Protein or peptide: Actin, alpha skeletal muscle
  • Protein or peptide: PHALLOIDIN Derivative
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Function / homology
Function and homology information


cytoskeletal motor activator activity / tropomyosin binding / mesenchyme migration / troponin I binding / myosin heavy chain binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle assembly ...cytoskeletal motor activator activity / tropomyosin binding / mesenchyme migration / troponin I binding / myosin heavy chain binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle assembly / 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
Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit) / Rabbit (rabbit) / synthetic construct (others)
Methodhelical reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsDas S / Ge P / Durer ZAO / Grintsevich EE / Zhou ZH / Reisler E
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM071940 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM077190 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI094386 United States
National Science Foundation (NSF, United States)XSEDE MCB140140 United States
CitationJournal: Structure / Year: 2020
Title: D-loop Dynamics and Near-Atomic-Resolution Cryo-EM Structure of Phalloidin-Bound F-Actin.
Authors: Sanchaita Das / Peng Ge / Zeynep A Oztug Durer / Elena E Grintsevich / Z Hong Zhou / Emil Reisler /
Abstract: Detailed molecular information on G-actin assembly into filaments (F-actin), and their structure, dynamics, and interactions, is essential for understanding their cellular functions. Previous studies ...Detailed molecular information on G-actin assembly into filaments (F-actin), and their structure, dynamics, and interactions, is essential for understanding their cellular functions. Previous studies indicate that a flexible DNase I binding loop (D-loop, residues 40-50) plays a major role in actin's conformational dynamics. Phalloidin, a "gold standard" for actin filament staining, stabilizes them and affects the D-loop. Using disulfide crosslinking in yeast actin D-loop mutant Q41C/V45C, light-scattering measurements, and cryoelectron microscopy reconstructions, we probed the constraints of D-loop dynamics and its contribution to F-actin formation/stability. Our data support a model of residues 41-45 distances that facilitate G- to F-actin transition. We report also a 3.3-Å resolution structure of phalloidin-bound F-actin in the ADP-Pi-like (ADP-BeFx) state. This shows the phalloidin-binding site on F-actin and how the relative movement between its two protofilaments is restricted by it. Together, our results provide molecular details of F-actin structure and D-loop dynamics.
History
DepositionSep 6, 2019-
Header (metadata) releaseOct 2, 2019-
Map releaseMay 13, 2020-
UpdateMay 20, 2020-
Current statusMay 20, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
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  • Surface view with fitted model
  • Atomic models: PDB-6u96
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6u96
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20694.png

Downloads & links

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Map

FileDownload / File: emd_20694.map.gz / Format: CCP4 / Size: 22.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationActin phalloidin at BeFx state
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.029988598 - 0.088445336
Average (Standard dev.)0.0020726256 (±0.0068329675)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-180
Dimensions128128360
Spacing128128360
CellA: 136.96 Å / B: 136.96 Å / C: 385.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z128128360
origin x/y/z0.0000.0000.000
length x/y/z136.960136.960385.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ240240240
MAP C/R/S123
start NC/NR/NS-64-64-180
NC/NR/NS128128360
D min/max/mean-0.0300.0880.002

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Supplemental data

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Sample components

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Entire : Actin phalloidin, BeFx state

EntireName: Actin phalloidin, BeFx state
Components
  • Organelle or cellular component: Actin phalloidin, BeFx state
    • Protein or peptide: Actin, alpha skeletal muscle
  • Protein or peptide: PHALLOIDIN Derivative
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Actin phalloidin, BeFx state

SupramoleculeName: Actin phalloidin, BeFx state / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Rabbit (rabbit)
Molecular weightTheoretical: 42.109973 KDa
SequenceString: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIE(HIC)G IIT NWDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLD SG DGVTHNVPIY ...String:
MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIE(HIC)G IIT NWDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLD SG DGVTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSS S LEKSYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVMSGGTTM YPGIADRMQ KEITALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWITKQEYDE AGPSIVHRKC F

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Macromolecule #2: PHALLOIDIN Derivative

MacromoleculeName: PHALLOIDIN Derivative / type: protein_or_peptide / ID: 2 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 808.899 Da
SequenceString:
(EEP)A(ALO)C(HYP)AW

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationNameFormula
10.0 mMHEPES
130.0 mMSaltNaCl
0.2 mMCalcium ChlorideCaCl2

Details: pH7.4, 10mM Tris, 50mM KCl, 1 mM MgC2, 0.2mM CaCl2, 1mM ATP, 1mM DTT, 0.2mM EGTA, 0.2mM BeCl2, 5mM NaF
GridSupport film - Material: CARBON / Support film - topology: HOLEY ARRAY / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 80.0 K / Max: 81.0 K
Specialist opticsEnergy filter - Name: GIF Quantum LS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 3-12 / Number grids imaged: 1 / Number real images: 5874 / Average exposure time: 8.0 sec. / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 4.2 µm / Calibrated defocus min: 2.2 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 29.508839 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.967625 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 181626
CTF correctionSoftware - Name: RELION (ver. 3)
Segment selectionNumber selected: 214555 / Software - Name: EMAN (ver. 1.8)
Startup modelType of model: OTHER / Details: An old, unpublished, low resolution F-actin map
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6u96:
Actin phalloidin at BeFx state

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