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- EMDB-29557: Mouse apoferritin from data collected on Titan Krios with K3 came... -

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Basic information

Entry
Database: EMDB / ID: EMD-29557
TitleMouse apoferritin from data collected on Titan Krios with K3 camera in counting mode without energy filter, in 150-200 nm ice thickness
Map dataMouse apoferritin from data collected on Titan Krios with K3 camera in counting mode without energy filter, in 150-200 nm ice thickness
Sample
  • Complex: Mouse apoferritin
KeywordsMouse apoferritin / Titan Krios / K3 camera / counting mode / 150-200 nm ice thickness / METAL BINDING PROTEIN
Biological speciesmouse (mice)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsNeselu K / Wang B / Rice WJ / Potter CS / Carragher B / Chua EYD
Funding support United States, 2 items
OrganizationGrant numberCountry
Simons FoundationSF349247 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
CitationJournal: J Struct Biol X / Year: 2023
Title: Measuring the effects of ice thickness on resolution in single particle cryo-EM.
Authors: Kasahun Neselu / Bing Wang / William J Rice / Clinton S Potter / Bridget Carragher / Eugene Y D Chua /
Abstract: Ice thickness is a critical parameter in single particle cryo-EM - too thin ice can break during imaging or exclude the sample of interest, while ice that is too thick contributes to more inelastic ...Ice thickness is a critical parameter in single particle cryo-EM - too thin ice can break during imaging or exclude the sample of interest, while ice that is too thick contributes to more inelastic scattering that precludes obtaining high resolution reconstructions. Here we present the practical effects of ice thickness on resolution, and the influence of energy filters, accelerating voltage, or detector mode. We collected apoferritin data with a wide range of ice thicknesses on three microscopes with different instrumentation and settings. We show that on a 300 kV microscope, using a 20 eV energy filter slit has a greater effect on improving resolution in thicker ice; that operating at 300 kV instead of 200 kV accelerating voltage provides significant resolution improvements at an ice thickness above 150 nm; and that on a 200 kV microscope using a detector operating in super resolution mode enables good reconstructions for up to 200 nm ice thickness, while collecting in counting instead of linear mode leads to improvements in resolution for ice of 50-150 nm thickness. Our findings can serve as a guide for users seeking to optimize data collection or sample preparation routines for both single particle and in situ cryo-EM. We note that most in situ data collection is done on samples in a range of ice thickness above 150 nm so these results may be especially relevant to that community.
History
DepositionJan 25, 2023-
Header (metadata) releaseMar 8, 2023-
Map releaseMar 8, 2023-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29557.png

Downloads & links

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Map

FileDownload / File: emd_29557.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMouse apoferritin from data collected on Titan Krios with K3 camera in counting mode without energy filter, in 150-200 nm ice thickness
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 277.248 Å		1.08 Å/pix.
x 256 pix.
= 277.248 Å		1.08 Å/pix.
x 256 pix.
= 277.248 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.083 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-0.9898655 - 1.8102202
Average (Standard dev.)-0.00022062393 (±0.097726166)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 277.248 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_29557_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29557_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Mouse apoferritin

EntireName: Mouse apoferritin
Components
  • Complex: Mouse apoferritin

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Supramolecule #1: Mouse apoferritin

SupramoleculeName: Mouse apoferritin / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: mouse (mice)
Molecular weightTheoretical: 450 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration8 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
30.0 mMHEPES
150.0 mMSodium chlorideNaCl
1.0 mMDithiothreitol
GridModel: UltrAuFoil R1.2/1.3 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Average exposure time: 2.0 sec. / Average electron dose: 51.22 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.1) / Number images used: 14000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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