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- EMDB-11790: Cryo-EM structure of F-actin stabilized by trans-optoJASP-8 -

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Basic information

Entry
Database: EMDB / ID: EMD-11790
TitleCryo-EM structure of F-actin stabilized by trans-optoJASP-8
Map dataSharpened map of the full filament filtered to local resolution (Bfactor optimised for optoJASP)
Sample
  • Complex: Filamentous alpha actin stabilized by trans-optoJASP-8 in complex with ADP-Pi
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHATE IONPhosphate
  • Ligand: MAGNESIUM ION
  • Ligand: ~{N}-[4-[(4~{R},7~{R},10~{S},13~{S},15~{E},19~{S})-4-(4-hydroxyphenyl)-7-(1~{H}-indol-3-ylmethyl)-8,13,15,19-tetramethyl-2,6,9,12-tetrakis(oxidanylidene)-1-oxa-5,8,11-triazacyclononadec-15-en-10-yl]butyl]-~{N}'-[5-methoxy-2-[(~{Z})-(3,4,5-trimethoxyphenyl)diazenyl]phenyl]butanediamide
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: 3.6 Å
AuthorsPospich S / Raunser S
Funding support Germany, 2 items
OrganizationGrant numberCountry
Max Planck Society Germany
European Union (EU)615984 Germany
CitationJournal: Angew Chem Int Ed Engl / Year: 2021
Title: Cryo-EM Resolves Molecular Recognition Of An Optojasp Photoswitch Bound To Actin Filaments In Both Switch States.
Authors: Sabrina Pospich / Florian Küllmer / Veselin Nasufović / Johanna Funk / Alexander Belyy / Peter Bieling / Hans-Dieter Arndt / Stefan Raunser /
Abstract: Actin is essential for key processes in all eukaryotic cells. Cellpermeable optojasps provide spatiotemporal control of the actin cytoskeleton, confining toxicity and potentially rendering F-actin ...Actin is essential for key processes in all eukaryotic cells. Cellpermeable optojasps provide spatiotemporal control of the actin cytoskeleton, confining toxicity and potentially rendering F-actin druggable by photopharmacology. Here, we report cryo electron microscopy (cryo-EM) structures of both isomeric states of one optojasp bound to actin filaments. The high-resolution structures reveal for the first time the pronounced effects of photoswitching a functionalized azobenzene. By characterizing the optojasp binding site and identifying conformational changes within F-actin that depend on the optojasp isomeric state, we refine determinants for the design of functional F-actin photoswitches.
History
DepositionSep 25, 2020-
Header (metadata) releaseJan 27, 2021-
Map releaseJan 27, 2021-
UpdateApr 14, 2021-
Current statusApr 14, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ahq
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7ahq
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11790.png

Downloads & links

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Map

FileDownload / File: emd_11790.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of the full filament filtered to local resolution (Bfactor optimised for optoJASP)
Voxel sizeX=Y=Z: 0.68 Å
Density
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.022
Minimum - Maximum-0.01669943 - 0.07294451
Average (Standard dev.)0.0005770129 (±0.0033051919)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 261.12 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.680.680.68
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z261.120261.120261.120
α/β/γ90.00090.00090.000
start NX/NY/NZ1081340
NX/NY/NZ13584349
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0170.0730.001

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Supplemental data

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Mask #1

Fileemd_11790_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened map of the central 120A of the...

Fileemd_11790_additional_1.map
AnnotationSharpened map of the central 120A of the filament filtered to nominal resolution (Bfactor optimised for optoJASP)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened map of the central 120A of thel...

Fileemd_11790_additional_2.map
AnnotationSharpened map of the central 120A of thel filament filtered to nominal resolution (automatic Bfactor)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of the full filament

Fileemd_11790_half_map_1.map
AnnotationHalf map of the full filament
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of the full filament

Fileemd_11790_half_map_2.map
AnnotationHalf map of the full filament
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 alpha actin stabilized by trans-optoJASP-8 in complex...

EntireName: Filamentous alpha actin stabilized by trans-optoJASP-8 in complex with ADP-Pi
Components
  • Complex: Filamentous alpha actin stabilized by trans-optoJASP-8 in complex with ADP-Pi
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHATE IONPhosphate
  • Ligand: MAGNESIUM ION
  • Ligand: ~{N}-[4-[(4~{R},7~{R},10~{S},13~{S},15~{E},19~{S})-4-(4-hydroxyphenyl)-7-(1~{H}-indol-3-ylmethyl)-8,13,15,19-tetramethyl-2,6,9,12-tetrakis(oxidanylidene)-1-oxa-5,8,11-triazacyclononadec-15-en-10-yl]butyl]-~{N}'-[5-methoxy-2-[(~{Z})-(3,4,5-trimethoxyphenyl)diazenyl]phenyl]butanediamide

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Supramolecule #1: Filamentous alpha actin stabilized by trans-optoJASP-8 in complex...

SupramoleculeName: Filamentous alpha actin stabilized by trans-optoJASP-8 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)
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: 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: ~{N}-[4-[(4~{R},7~{R},10~{S},13~{S},15~{E},19~{S})-4-(4-hydroxyph...

MacromoleculeName: ~{N}-[4-[(4~{R},7~{R},10~{S},13~{S},15~{E},19~{S})-4-(4-hydroxyphenyl)-7-(1~{H}-indol-3-ylmethyl)-8,13,15,19-tetramethyl-2,6,9,12-tetrakis(oxidanylidene)-1-oxa-5,8,11-triazacyclononadec-15-en- ...Name: ~{N}-[4-[(4~{R},7~{R},10~{S},13~{S},15~{E},19~{S})-4-(4-hydroxyphenyl)-7-(1~{H}-indol-3-ylmethyl)-8,13,15,19-tetramethyl-2,6,9,12-tetrakis(oxidanylidene)-1-oxa-5,8,11-triazacyclononadec-15-en-10-yl]butyl]-~{N}'-[5-methoxy-2-[(~{Z})-(3,4,5-trimethoxyphenyl)diazenyl]phenyl]butanediamide
type: ligand / ID: 5 / Number of copies: 5 / Formula: RLZ
Molecular weightTheoretical: 1.073239 KDa
Chemical component information

ChemComp-RLZ:
~{N}-[4-[(4~{R},7~{R},10~{S},13~{S},15~{E},19~{S})-4-(4-hydroxyphenyl)-7-(1~{H}-indol-3-ylmethyl)-8,13,15,19-tetramethyl-2,6,9,12-tetrakis(oxidanylidene)-1-oxa-5,8,11-triazacyclononadec-15-en-10-yl]butyl]-~{N}'-[5-methoxy-2-[(~{Z})-(3,4,5-trimethoxyphenyl)diazenyl]phenyl]butanediamide

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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
0.7 % (v/v)C2H6OSDMSODimethyl 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, 0.7 %(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 III
Details: 1.5 mul sample, automatic blotting for 7-7.5s, blot force -25, drain time 1s..
DetailsRise 27.5 A, Twist -166.6 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: 0.0 mm
Specialist opticsSpherical aberration corrector: Cs-corrected
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsCs-corrected
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 7178 / Average exposure time: 1.5 sec. / Average electron dose: 92.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1008519
CTF correctionSoftware: (Name: CTFFIND (ver. 4.1.8), RELION (ver. 2 beta))
Startup modelType of model: INSILICO MODEL
In silico model: Initial model was generated from PDB 5OOD and filtered to 25 A using SPARX/EMAN2.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2 beta)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2 beta)
Details: No helical refinement or symmetry was applied. See original publication for details.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2 beta) / Number images used: 667688
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5ood


Chain - Chain ID: C
DetailsAn initial model of trans-optoJASP-8 was generated using elBow within Phenix inputting the SMILES string.
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-7ahq:
Cryo-EM structure of F-actin stabilized by trans-optoJASP-8

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