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- EMDB-30179: Phalloidin bound F-actin complex -

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Basic information

Entry
Database: EMDB / ID: EMD-30179
TitlePhalloidin bound F-actin complex
Map dataphalloidin stabilised F-actin
Sample
  • Complex: Filamentous actin in ADP state
    • Protein or peptide: Actin, alpha skeletal muscle
    • Protein or peptide: Phalloidin
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Function / homology
Function and homology information


Striated Muscle Contraction / skeletal muscle thin filament assembly / striated muscle thin filament / skeletal muscle fiber development / stress fiber / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / toxin activity / hydrolase activity / ATP binding
Similarity search - Function
Amanitin/phalloidin toxin / 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
Phalloidin proprotein / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesGallus gallus (chicken) / Chicken (chicken) / Amanita phalloides (death cap)
Methodhelical reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsKumari A / Ragunath VK / Sirajuddin M
Funding support India, 2 items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)IA/14/2/501533 India
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/2014 India
CitationJournal: EMBO J / Year: 2020
Title: Structural insights into actin filament recognition by commonly used cellular actin markers.
Authors: Archana Kumari / Shubham Kesarwani / Manjunath G Javoor / Kutti R Vinothkumar / Minhajuddin Sirajuddin /
Abstract: Cellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides, and proteins that bind actin. While the choice of these markers has been largely ...Cellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides, and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F-actin markers for a particular requirement. Here, we describe the electron cryomicroscopy structures of phalloidin, lifeAct, and utrophin bound to F-actin, providing a comprehensive high-resolution structural comparison of widely used actin markers and their influence towards F-actin. Our results show that phalloidin binding does not induce specific conformational change and lifeAct specifically recognizes closed D-loop conformation, i.e., ADP-Pi or ADP states of F-actin. The structural models aided designing of minimal utrophin and a shorter lifeAct, which can be utilized as F-actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin-binding proteins and thus offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants.
History
DepositionApr 1, 2020-
Header (metadata) releaseMay 20, 2020-
Map releaseMay 20, 2020-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bti
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7bti
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30179.png

Downloads & links

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Map

FileDownload / File: emd_30179.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationphalloidin stabilised F-actin
Voxel sizeX=Y=Z: 1.38 Å
Density
Contour LevelBy AUTHOR: 0.095 / Movie #1: 0.12
Minimum - Maximum-0.20386076 - 0.53642565
Average (Standard dev.)0.00089536177 (±0.018650627)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 353.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.381.381.38
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z353.280353.280353.280
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.2040.5360.001

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Supplemental data

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Sample components

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Entire : Filamentous actin in ADP state

EntireName: Filamentous actin in ADP state
Components
  • Complex: Filamentous actin in ADP state
    • Protein or peptide: Actin, alpha skeletal muscle
    • Protein or peptide: Phalloidin
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Filamentous actin in ADP state

SupramoleculeName: Filamentous actin in ADP state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Gallus gallus (chicken)

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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: Chicken (chicken)
Molecular weightTheoretical: 42.096953 KDa
SequenceString: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY ...String:
MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSSSL EK SYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVMSGGTTM YPGIADRMQK EIT ALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWITKQEYDE AGPSIVHRKC F

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Macromolecule #2: Phalloidin

MacromoleculeName: Phalloidin / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Amanita phalloides (death cap)
Molecular weightTheoretical: 808.899 Da
SequenceString:
(HYP)AW(G5G)A(ALO)C

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.0002 mg/mL
BufferpH: 7.5
Details: 50mM KCl,1mM MgCl2,0.2mM EGTA, 10mM Imidazole buffer pH 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.025 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK I / Details: blot for 3.5 seconds.
Details10 mole excess of lifeact was mixed with F-actin and used for sample preparation.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 75000 / Illumination mode: OTHER / Imaging mode: OTHER / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 100.0 K / Max: 120.0 K
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 529 / Average exposure time: 2.0 sec. / Average electron dose: 49.2 e/Å2 / Details: 30
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 349839 / Software - Name: RELION (ver. 3.0)
CTF correctionSoftware - Name: Gctf (ver. 1.06) / Details: GCTF for CTF correction
Startup modelType of model: INSILICO MODEL / In silico model: cylinder
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 4
Applied symmetry - Helical parameters - Δz: 27.89 Å
Applied symmetry - Helical parameters - Δ&Phi: -167 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 91245
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5onv


Chain - Chain ID: A / Chain - Residue range: 6-373
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 179
Output model

PDB-7bti:
Phalloidin bound F-actin complex

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