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- PDB-8ord: Cryo-EM map of zebrafish cardiac F-actin -

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Basic information

Entry
Database: PDB / ID: 8ord
TitleCryo-EM map of zebrafish cardiac F-actin
ComponentsActin, alpha 1b, skeletal muscle
KeywordsCONTRACTILE PROTEIN / F-actin / Actin / Cardiac Actin / Filamentous
Function / homology
Function and homology information


embryonic heart tube development / skeletal muscle fiber development
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 / Actin, alpha 1b, skeletal muscle
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsBradshaw, M. / Squire, J.M. / Morris, E. / Atkinson, G. / Richardson, B. / Lees, J. / Paul, D.M.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
British Heart FoundationFS/14/18/30711 United Kingdom
CitationJournal: J Muscle Res Cell Motil / Year: 2023
Title: Zebrafish as a model for cardiac disease; Cryo-EM structure of native cardiac thin filaments from Danio Rerio.
Authors: Marston Bradshaw / John M Squire / Edward Morris / Georgia Atkinson / Rebecca Richardson / Jon Lees / Massimo Caputo / Giulia M Bigotti / Danielle M Paul /
Abstract: Actin, tropomyosin and troponin, the proteins that comprise the contractile apparatus of the cardiac thin filament, are highly conserved across species. We have used cryo-EM to study the three- ...Actin, tropomyosin and troponin, the proteins that comprise the contractile apparatus of the cardiac thin filament, are highly conserved across species. We have used cryo-EM to study the three-dimensional structure of the zebrafish cardiac thin and actin filaments. With 70% of human genes having an obvious zebrafish orthologue, and conservation of 85% of disease-causing genes, zebrafish are a good animal model for the study of human disease. Our structure of the zebrafish thin filament reveals the molecular interactions between the constituent proteins, showing that the fundamental organisation of the complex is the same as that reported in the human reconstituted thin filament. A reconstruction of zebrafish cardiac F-actin demonstrates no deviations from human cardiac actin over an extended length of 14 actin subunits. Modelling zebrafish homology models into our maps enabled us to compare, in detail, the similarity with human models. The structural similarities of troponin-T in particular, a region known to contain a hypertrophic cardiomyopathy 'hotspot', confirm the suitability of zebrafish to study these disease-causing mutations.
History
DepositionApr 13, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 2, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 11, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Actin, alpha 1b, skeletal muscle
B: Actin, alpha 1b, skeletal muscle
C: Actin, alpha 1b, skeletal muscle
D: Actin, alpha 1b, skeletal muscle
E: Actin, alpha 1b, skeletal muscle
hetero molecules


Theoretical massNumber of molelcules
Total (without water)212,72015
Polymers210,1095
Non-polymers2,61110
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15870 Å2
ΔGint-159 kcal/mol
Surface area69730 Å2

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Components

#1: Protein
Actin, alpha 1b, skeletal muscle / / actin / alpha skeletal muscle


Mass: 42021.887 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Danio rerio (zebrafish) / Organ: Heart / Tissue: Ventrical / References: UniProt: Q4KMI7
#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-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: PO4
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Cardiac F-actin / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Danio rerio (zebrafish) / Cellular location: Sarcomere / Organ: Heart / Tissue: Ventrical
Buffer solutionpH: 6.8
Details: Phosphate Buffer Protease Inhibitor Cocktail (0.44 mg/ml)
Buffer component
IDConc.NameFormulaBuffer-ID
10.1 MSodium ChlorideNaClSodium chloride1
25 mMMagnesium acetateMgAc1
32 mMEGTAC14H24N2O101
42.5 mMAdenosine triphosphateC10H13N5O13P31
510 mMCreatine PhosphateC4H10N3O5P1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Freshly excised heart tissue from Zebrafish. Monodispersed filaments of varying lengths.
Specimen supportDetails: 15 mAh / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 900 nm / Nominal defocus min: 400 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 1000 nm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 38.85 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5622
EM imaging opticsPhase plate: VOLTA PHASE PLATE
Image scansWidth: 4096 / Height: 4096 / Movie frames/image: 35 / Used frames/image: 1-35

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Processing

EM software
IDNameVersionCategory
1RELION2.1particle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
10cisTEM1initial Euler assignment
11RELION2.1final Euler assignment
12cisTEM1classification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 507184
Details: Relion auto helical picking using manually picked references
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 420108 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT

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