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- EMDB-16307: Subtomogram average of an intracellular microtubule filament -

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Basic information

Entry
Database: EMDB / ID: EMD-16307
TitleSubtomogram average of an intracellular microtubule filament
Map dataSubtomogram average of an intracellular microtubule filament
Sample
  • Organelle or cellular component: Microtubule
KeywordsMicrotubule / Cytoskeleton / STRUCTURAL PROTEIN
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 23.0 Å
AuthorsSharp TH / Last MGF
Funding supportEuropean Union, Netherlands, 3 items
OrganizationGrant numberCountry
European Research Council (ERC)759517European Union
Netherlands Organisation for Scientific Research (NWO)OCENW.KLEIN.291 Netherlands
Netherlands Organisation for Scientific Research (NWO)VI.Vidi.193.014 Netherlands
Citation
Journal: Sci Rep / Year: 2023
Title: Selecting optimal support grids for super-resolution cryogenic correlated light and electron microscopy.
Authors: Mart G F Last / Maarten W Tuijtel / Lenard M Voortman / Thomas H Sharp /
Abstract: Cryogenic transmission electron microscopy (cryo-TEM) and super-resolution fluorescence microscopy are two popular and ever improving methods for high-resolution imaging of biological samples. In ...Cryogenic transmission electron microscopy (cryo-TEM) and super-resolution fluorescence microscopy are two popular and ever improving methods for high-resolution imaging of biological samples. In recent years, the combination of these two techniques into one correlated workflow has gained attention as a promising route towards contextualizing and enriching cryo-TEM imagery. A problem that is often encountered in the combination of these methods is that of light-induced damage to the sample during fluorescence imaging that renders the sample structure unsuitable for TEM imaging. In this paper, we describe how absorption of light by TEM sample support grids leads to sample damage, and we systematically explore the importance of parameters of grid design. We explain how, by changing the grid geometry and materials, one can increase the maximum illumination power density in fluorescence microscopy by up to an order of magnitude. Finally, we demonstrate the significant improvements in super-resolution image quality that are enabled by the selection of support grids that are optimally suited for correlated cryo-microscopy.
#1: Journal: Selecting optimal support grids for super-resolution cryogenic correlated light and electron microscopy
Year: 2022
Title: Selecting optimal support grids for super-resolution cryogenic correlated light and electron microscopy
Authors: Sharp TH / Last MGF / Voortman LM / Tuijtel MW
History
DepositionDec 7, 2022-
Header (metadata) releaseDec 21, 2022-
Map releaseDec 21, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16307.png

Downloads & links

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Map

FileDownload / File: emd_16307.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of an intracellular microtubule filament
Voxel sizeX=Y=Z: 2.75 Å
Density
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-4.960396 - 5.9285374
Average (Standard dev.)-0.0021971029 (±1.0000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-90-90-90
Dimensions180180180
Spacing180180180
CellA=B=C: 495.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16307_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of a subtomogram average of an...

Fileemd_16307_half_map_1.map
AnnotationHalf map of a subtomogram average of an intracellular microtubule filament
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of a subtomogram average of an...

Fileemd_16307_half_map_2.map
AnnotationHalf map of a subtomogram average of an intracellular microtubule 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 : Microtubule

EntireName: Microtubule
Components
  • Organelle or cellular component: Microtubule

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Supramolecule #1: Microtubule

SupramoleculeName: Microtubule / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: Intracellular cytoskeletal filmanent composed of alpha and beta tubulin
Source (natural)Organism: Homo sapiens (human) / Strain: U2OS

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 50
VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 310 K / Instrument: LEICA EM GP
DetailsFilaments were picked from inside vitrified cells

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 3 / Average exposure time: 0.35 sec. / Average electron dose: 1.62 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 6.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 31000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 9.0 Å
Applied symmetry - Helical parameters - Δ&Phi: 27.6923 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 / Number subtomograms used: 1120
ExtractionNumber tomograms: 7 / Number images used: 1599 / Software - Name: EMAN2
Details: 24 MT filaments were picked manually. Particles were picked along the filaments automatically.
Final 3D classificationNumber classes: 1 / Software - Name: EMAN2
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: EMAN2
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT

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