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- EMDB-15901: Cryo-EM map of Zebrafish (Danio rerio) Cardiac Thin Filament -

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Basic information

Entry
Database: EMDB / ID: EMD-15901
TitleCryo-EM map of Zebrafish (Danio rerio) Cardiac Thin Filament
Map data
Sample
  • Complex: Cardiac Thin Filament
KeywordsCardiac Thin Filament / Thin Filament / Troponin / Tropomyosin / Actin / Actin binding protein / CONTRACTILE PROTEIN
Biological speciesDanio rerio (zebrafish)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.4 Å
AuthorsBradshaw M / Paul DM
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
British Heart FoundationS100402-101 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
DepositionOct 3, 2022-
Header (metadata) releaseAug 2, 2023-
Map releaseAug 2, 2023-
UpdateOct 11, 2023-
Current statusOct 11, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15901.png

Downloads & links

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Map

FileDownload / File: emd_15901.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.47 Å/pix.
x 96 pix.
= 525. Å		5.47 Å/pix.
x 96 pix.
= 525. Å		5.47 Å/pix.
x 96 pix.
= 525. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.46875 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.9
Minimum - Maximum-11.874521 - 15.385061
Average (Standard dev.)-0.0030731587 (±0.54979056)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 525.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_15901_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15901_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cardiac Thin Filament

EntireName: Cardiac Thin Filament
Components
  • Complex: Cardiac Thin Filament

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Supramolecule #1: Cardiac Thin Filament

SupramoleculeName: Cardiac Thin Filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Danio rerio (zebrafish) / Organ: Heart / Tissue: Ventrical / Location in cell: Sarcomere

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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

Concentration0.8 mg/mL
BufferpH: 6.8
Component:
ConcentrationFormulaName
0.1 MNaClSodium Chloride
5.0 mMMgAcMagnesium acetate
2.0 mMC14H24N2O10EGTA
2.5 mMC10H13N5O13P3Adenosine triphosphate
10.0 mMC4H10N3O5PCreatine Phosphate

Details: Phosphate Buffer Protease Inhibitor Cocktail (0.44 mg/ml)
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.01 kPa / Details: 15 mAh
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: LEICA EM GP
DetailsFreshly excised heart tissue from Zebrafish. Monodispersed filaments of varying lengths.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
SoftwareName: EPU
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Frames/image: 1-35 / Number grids imaged: 2 / Number real images: 5622 / Average electron dose: 38.85 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 1.0 µm / Calibrated defocus min: 0.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.9 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 18593 / Details: Manually Picked
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 15.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 9821
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cisTEM (ver. 1.0)
Final 3D classificationNumber classes: 350 / Avg.num./class: 53 / Software - Name: cisTEM (ver. 1.0)
FSC plot (resolution estimation)

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