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- EMDB-43685: Structure of murine heavy chain apoferritin collected at 200 keV ... -

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Basic information

Entry
Database: EMDB / ID: EMD-43685
TitleStructure of murine heavy chain apoferritin collected at 200 keV with the Gatan Alpine direct detector
Map dataApoferritin from Alpine at 200 keV, full map
Sample
  • Complex: mouse heavy chain apoferritin
Keywordsapoferritin / complex / METAL TRANSPORT
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.76 Å
AuthorsMaker A / Bulkley DP / Verba KA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)fill in later United States
CitationJournal: J Struct Biol / Year: 2024
Title: High-resolution single-particle imaging at 100-200 keV with the Gatan Alpine direct electron detector.
Authors: Lieza M Chan / Brandon J Courteau / Allison Maker / Mengyu Wu / Benjamin Basanta / Hev Mehmood / David Bulkley / David Joyce / Brian C Lee / Stephen Mick / Cory Czarnik / Sahil Gulati / ...Authors: Lieza M Chan / Brandon J Courteau / Allison Maker / Mengyu Wu / Benjamin Basanta / Hev Mehmood / David Bulkley / David Joyce / Brian C Lee / Stephen Mick / Cory Czarnik / Sahil Gulati / Gabriel C Lander / Kliment A Verba /
Abstract: Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments ...Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments indicate advantages to imaging at 100 keV, energies for which the current detectors have not been optimized. In this study, we evaluated the Gatan Alpine detector, designed for operation at 100 and 200 keV. Compared to the Gatan K3, Alpine demonstrated a significant DQE improvement at these energies, specifically a ∼ 4-fold improvement at Nyquist at 100 keV. In single-particle cryo-EM experiments, Alpine datasets yielded better than 2 Å resolution reconstructions of apoferritin at 120 and 200 keV on a ThermoFisher Scientific (TFS) Glacios microscope fitted with a non-standard SP-Twin lens. We also achieved a ∼ 3.2 Å resolution reconstruction of a 115 kDa asymmetric protein complex, proving Alpine's effectiveness with complex biological samples. In-depth analysis revealed that Alpine reconstructions are comparable to K3 reconstructions at 200 keV, and remarkably, reconstruction from Alpine at 120 keV on a TFS Glacios surpassed all but the 300 keV data from a TFS Titan Krios with GIF/K3. Additionally, we show Alpine's capability for high-resolution data acquisition and screening on lower-end systems by obtaining ∼ 3 Å resolution reconstructions of apoferritin and aldolase at 100 keV and detailed 2D averages of a 55 kDa sample using a side-entry cryo holder. Overall, we show that Gatan Alpine performs well with the standard 200 keV imaging systems and may potentially capture the benefits of lower accelerating voltages, bringing smaller sized particles within the scope of cryo-EM.
History
DepositionFeb 12, 2024-
Header (metadata) releaseDec 11, 2024-
Map releaseDec 11, 2024-
UpdateDec 11, 2024-
Current statusDec 11, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43685.png

Downloads & links

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Map

FileDownload / File: emd_43685.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationApoferritin from Alpine at 200 keV, full map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.46 Å/pix.
x 512 pix.
= 236.032 Å		0.46 Å/pix.
x 512 pix.
= 236.032 Å		0.46 Å/pix.
x 512 pix.
= 236.032 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.461 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.175
Minimum - Maximum-0.0886183 - 0.42336205
Average (Standard dev.)0.0021308684 (±0.027830273)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 236.032 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43685_msk_1.map
Projections & Slices
AxesZYX

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Additional map: Apoferritin from Alpine at 200 keV, sharpened map

Fileemd_43685_additional_1.map
AnnotationApoferritin from Alpine at 200 keV, sharpened map
Projections & Slices
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Half map: Apoferritin from Alpine at 200 keV, half map B

Fileemd_43685_half_map_1.map
AnnotationApoferritin from Alpine at 200 keV, half map B
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Apoferritin from Alpine at 200 keV, half map A

Fileemd_43685_half_map_2.map
AnnotationApoferritin from Alpine at 200 keV, half map A
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : mouse heavy chain apoferritin

EntireName: mouse heavy chain apoferritin
Components
  • Complex: mouse heavy chain apoferritin

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Supramolecule #1: mouse heavy chain apoferritin

SupramoleculeName: mouse heavy chain apoferritin / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse)

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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

Concentration10 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHEPES
300.0 mMsodium chlorideNaCl
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: OTHER / Average electron dose: 45.5 e/Å2 / Details: Gatan Alpine detector
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm

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Image processing

DetailsGatan Alpine
Startup modelType of model: NONE / Details: Ab initio
Final reconstructionApplied symmetry - Point group: O (octahedral) / Resolution.type: BY AUTHOR / Resolution: 1.76 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2) / Number images used: 204387
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2)
FSC plot (resolution estimation)

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