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- PDB-7ahn: Cryo-EM structure of F-actin stabilized by cis-optoJASP-8 -

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Basic information

Entry
Database: PDB / ID: 7ahn
TitleCryo-EM structure of F-actin stabilized by cis-optoJASP-8
ComponentsActin, alpha skeletal muscle
KeywordsSTRUCTURAL PROTEIN / Cytoskeleton / jasplakinolide / azobenzene photoswitch / stabilized-actin filament
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
ADENOSINE-5'-DIPHOSPHATE / PHOSPHATE ION / Chem-RLZ / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsPospich, S. / Raunser, S.
Funding support Germany, 2items
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 24, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 27, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 14, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title

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Assembly

Deposited unit
C: Actin, alpha skeletal muscle
A: Actin, alpha skeletal muscle
E: Actin, alpha skeletal muscle
D: Actin, alpha skeletal muscle
B: Actin, alpha skeletal muscle
hetero molecules


Theoretical massNumber of molelcules
Total (without water)218,64825
Polymers210,5505
Non-polymers8,09920
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18640 Å2
ΔGint-196 kcal/mol
Surface area68720 Å2
MethodPISA

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Components

#1: Protein
Actin, alpha skeletal muscle / / Alpha-actin-1


Mass: 42109.973 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
#4: Chemical
ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: PO4
#5: Chemical
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 / cis-optoJASP-8;trans-optoJASP-8


Mass: 1073.239 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C58H72N8O12 / Feature type: SUBJECT OF INVESTIGATION
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: helical reconstruction

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Sample preparation

ComponentName: Filamentous alpha actin stabilized by cis-optoJASP-8 in complex with ADP-Pi
Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: skeletal muscle
Buffer solutionpH: 7.5
Details: 5 mM Tris pH 7.5, 2 mM NaN3, 1 mM DTT, 100 mM KCl and 2 mM MgCl2, 0.4 %(v/v) DMSO, 0.02 %(v/w) Tween 20
Buffer component
IDConc.NameFormulaBuffer-ID
15 mMTRISC4H11NO31
22 mMSodium azideNaN31
31 mMDTTC4H10O2S21
4100 mMPotassium chlorideKCl1
52 mMMagnesium chlorideMgCl21
60.4 % (v/v)DMSODimethyl sulfoxideC2H6OS1
70.02 % (v/w)Tween 20C58H114O261
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Rise 27.5 A, Twist -166.8 degrees
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 286 K
Details: 1.5 mul sample, automatic blotting for 7-7.5s, blot force -25, drain time 1s.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15 sec. / Electron dose: 86 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4771
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4CTFFIND4.1.5CTF correction
5SPHIRE1.2CTF correction
8UCSF Chimeramodel fitting
10SPHIRE1.2initial Euler assignmentsp_meridien_alpha.py
11SPHIRE1.2final Euler assignmentsp_meridien_alpha.py
13SPHIRE1.23D reconstructionsp_meridien_alpha.py
14PHENIX1.10 and 1.17model refinementreal space refinement and elBow
15ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 166.8 ° / Axial rise/subunit: 27.5 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 806158
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 676237 / Symmetry type: HELICAL
Atomic model buildingProtocol: OTHER / Space: REAL
Details: An initial model of cis-opto-ASP-8 was generated using elBow within Phenix inputting the SMILES string.
Atomic model buildingPDB-ID: 6FHL

6fhl


Pdb chain-ID: C
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01215275
ELECTRON MICROSCOPYf_angle_d1.11520735
ELECTRON MICROSCOPYf_dihedral_angle_d20.3675585
ELECTRON MICROSCOPYf_chiral_restr0.0662265
ELECTRON MICROSCOPYf_plane_restr0.0292625

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