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- EMDB-29592: Mouse apoferritin from data collected on Talos Arctica microscope... -

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Entry
Database: EMDB / ID: EMD-29592
TitleMouse apoferritin from data collected on Talos Arctica microscope equipped with a K3 camera operating in super resolution mode, in 100-150 nm ice thicknessFerritin
Map dataMouse apoferritin from data collected on Talos Arctica microscope equipped with a K3 camera operating in super resolution mode, in 100-150 nm ice thicknessFerritin
Sample
  • Complex: apoferritinFerritin
KeywordsMouse apoferritin / Talos Arctica / K3 camera / super resolution mode / 100-150 nm ice thickness / METAL BINDING PROTEIN
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.04 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 26, 2023-
Header (metadata) releaseMar 22, 2023-
Map releaseMar 22, 2023-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29592.png

Downloads & links

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Map

FileDownload / File: emd_29592.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMouse apoferritin from data collected on Talos Arctica microscope equipped with a K3 camera operating in super resolution mode, in 100-150 nm ice thickness
Voxel sizeX=Y=Z: 1.096 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-1.4901032 - 2.4333258
Average (Standard dev.)-0.003165254 (±0.12943888)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 280.576 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_29592_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29592_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 : apoferritin

EntireName: apoferritinFerritin
Components
  • Complex: apoferritinFerritin

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Supramolecule #1: apoferritin

SupramoleculeName: apoferritin / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse)
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 chlorideNaClSodium chloride
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.1) / Number images used: 14000
FSC plot (resolution estimation)

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