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- EMDB-10365: Cryo-EM structure of jasplakinolide-stabilized F-actin (aged) -

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Basic information

Entry
Database: EMDB / ID: EMD-10365
TitleCryo-EM structure of jasplakinolide-stabilized F-actin (aged)
Map dataMap of the full filament filtered to local resolution
Sample
  • Complex: Filamentous jasplakinolide-stabilized alpha actin in complex with ADP-Pi
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHATE IONPhosphate
  • Ligand: MAGNESIUM ION
  • Ligand: (4~{R},7~{R},10~{S},13~{S},15~{E},19~{S})-10-(4-azanylbutyl)-4-(4-hydroxyphenyl)-7-(1~{H}-indol-3-ylmethyl)-8,13,15,19-tetramethyl-1-oxa-5,8,11-triazacyclononadec-15-ene-2,6,9,12-tetrone
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
Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit) / Rabbit (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsPospich S / Merino F / Raunser S
Funding support Germany, 2 items
OrganizationGrant numberCountry
Max Planck Society Germany
European Union615984 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.
History
DepositionOct 7, 2019-
Header (metadata) releaseOct 23, 2019-
Map releaseMar 4, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.048
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.048
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6t23
  • Surface level: 0.048
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6t23
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10365.png

Downloads & links

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Map

FileDownload / File: emd_10365.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of the full filament filtered to local resolution
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.048 / Movie #1: 0.048
Minimum - Maximum-0.14992727 - 0.30757803
Average (Standard dev.)0.0009253964 (±0.009246892)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 268.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z268.800268.800268.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1500.3080.001

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Supplemental data

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Mask #1

Fileemd_10365_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_10365_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_10365_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Filamentous jasplakinolide-stabilized alpha actin in complex with...

EntireName: Filamentous jasplakinolide-stabilized alpha actin in complex with ADP-Pi
Components
  • Complex: Filamentous jasplakinolide-stabilized alpha actin in complex with ADP-Pi
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHATE IONPhosphate
  • Ligand: MAGNESIUM ION
  • Ligand: (4~{R},7~{R},10~{S},13~{S},15~{E},19~{S})-10-(4-azanylbutyl)-4-(4-hydroxyphenyl)-7-(1~{H}-indol-3-ylmethyl)-8,13,15,19-tetramethyl-1-oxa-5,8,11-triazacyclononadec-15-ene-2,6,9,12-tetrone

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Supramolecule #1: Filamentous jasplakinolide-stabilized alpha actin in complex with...

SupramoleculeName: Filamentous jasplakinolide-stabilized alpha actin in complex with ADP-Pi
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: skeletal muscle

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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) / Tissue: skeletal muscle
Molecular weightTheoretical: 41.875633 KDa
SequenceString: DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GII TNW DDMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSG DG VTHNVPIYEG ...String:
DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GII TNW DDMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSG DG VTHNVPIYEG YALPHAIMRL DLAGRDLTDY LMKILTERGY SFVTTAEREI VRDIKEKLCY VALDFENEMA TAASSSSL E KSYELPDGQV ITIGNERFRC PETLFQPSFI GMESAGIHET TYNSIMKCDI DIRKDLYANN VMSGGTTMYP GIADRMQKE ITALAPSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ITKQEYDEAG PSIVHRKCF

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Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / 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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Macromolecule #3: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 3 / Number of copies: 5 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION / Phosphate

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: (4~{R},7~{R},10~{S},13~{S},15~{E},19~{S})-10-(4-azanylbutyl)-4-(4...

MacromoleculeName: (4~{R},7~{R},10~{S},13~{S},15~{E},19~{S})-10-(4-azanylbutyl)-4-(4-hydroxyphenyl)-7-(1~{H}-indol-3-ylmethyl)-8,13,15,19-tetramethyl-1-oxa-5,8,11-triazacyclononadec-15-ene-2,6,9,12-tetrone
type: ligand / ID: 5 / Number of copies: 5 / Formula: 9ZK
Molecular weightTheoretical: 673.841 Da
Chemical component information

ChemComp-9ZK:
(4~{R},7~{R},10~{S},13~{S},15~{E},19~{S})-10-(4-azanylbutyl)-4-(4-hydroxyphenyl)-7-(1~{H}-indol-3-ylmethyl)-8,13,15,19-tetramethyl-1-oxa-5,8,11-triazacyclononadec-15-ene-2,6,9,12-tetrone

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationFormulaName
5.0 mMC4H11NO3TRIS
2.0 mMNaN3Sodium azide
1.0 mMC4H10O2S2DTT
100.0 mMKClPotassium chloride
2.0 mMMgCl2Magnesium chloride
0.2 % (v/v)C2H6OSDimethyl sulfoxide
0.02 % (v/w)C58H114O26Tween 20

Details: 5 mM Tris pH 7.5, 2 mM NaN3, 1 mM DTT, 100 mM KCl and 2 mM MgCl2, 0.2 %(v/v) DMSO, 0.02 %(v/w) Tween 20
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 286 K / Instrument: FEI VITROBOT MARK IV
DetailsRise 27.7 A, Twist -166.7 degrees

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 15 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 2856 / Average exposure time: 15.0 sec. / Average electron dose: 78.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 467060
CTF correctionSoftware: (Name: Gctf, RELION (ver. 3))
Startup modelType of model: INSILICO MODEL
In silico model: Initial model was generated from PDB 5JLF and filtered to 25 A using SPARX.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 336783
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6t1y

chain_id: C

5ood

chain_id: C, residue_range: 402-404
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-6t23:
Cryo-EM structure of jasplakinolide-stabilized F-actin (aged)

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