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- EMDB-43527: Apoferritin at 100 keV on Alpine detector with a side-entry cryoholder -

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Basic information

Entry
Database: EMDB / ID: EMD-43527
TitleApoferritin at 100 keV on Alpine detector with a side-entry cryoholder
Map datamouse apoferritin determined at 100 keV on a Talos 200C with a Gatan Alpine camera using a Gatan 626 side-entry cryoholder
Sample
  • Complex: Heavy chain apoferritin from mouse
    • Protein or peptide: Heavy chain apoferritin from mouse
KeywordsProtein complex / TRANSPORT PROTEIN
Function / homology
Function and homology information


Iron uptake and transport / Golgi Associated Vesicle Biogenesis / negative regulation of ferroptosis / ferroxidase / autolysosome / ferroxidase activity / Neutrophil degranulation / endocytic vesicle lumen / ferric iron binding / autophagosome ...Iron uptake and transport / Golgi Associated Vesicle Biogenesis / negative regulation of ferroptosis / ferroxidase / autolysosome / ferroxidase activity / Neutrophil degranulation / endocytic vesicle lumen / ferric iron binding / autophagosome / iron ion transport / intracellular iron ion homeostasis / immune response / iron ion binding / negative regulation of cell population proliferation / mitochondrion / extracellular region / identical protein binding / membrane / cytosol
Similarity search - Function
Ferritin iron-binding regions signature 1. / Ferritin iron-binding regions signature 2. / Ferritin, conserved site / Ferritin / Ferritin-like diiron domain / Ferritin-like diiron domain profile. / Ferritin/DPS protein domain / Ferritin-like domain / Ferritin-like / Ferritin-like superfamily
Similarity search - Domain/homology
Ferritin heavy chain
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsWu M / Lander GC
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM14305 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
DepositionJan 29, 2024-
Header (metadata) releaseJul 3, 2024-
Map releaseJul 3, 2024-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43527.png

Downloads & links

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Map

FileDownload / File: emd_43527.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmouse apoferritin determined at 100 keV on a Talos 200C with a Gatan Alpine camera using a Gatan 626 side-entry cryoholder
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 256 pix.
= 218.112 Å		0.85 Å/pix.
x 256 pix.
= 218.112 Å		0.85 Å/pix.
x 256 pix.
= 218.112 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.852 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.37
Minimum - Maximum-0.41709954 - 1.1301157
Average (Standard dev.)-0.0007328378 (±0.07865653)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 218.112 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43527_msk_1.map
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Additional map: unsharpened map

Fileemd_43527_additional_1.map
Annotationunsharpened map
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Half map: Half map A

Fileemd_43527_half_map_1.map
AnnotationHalf map A
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Half map: Half map B

Fileemd_43527_half_map_2.map
AnnotationHalf map B
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Sample components

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Entire : Heavy chain apoferritin from mouse

EntireName: Heavy chain apoferritin from mouse
Components
  • Complex: Heavy chain apoferritin from mouse
    • Protein or peptide: Heavy chain apoferritin from mouse

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Supramolecule #1: Heavy chain apoferritin from mouse

SupramoleculeName: Heavy chain apoferritin from mouse / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 487 KDa

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Macromolecule #1: Heavy chain apoferritin from mouse

MacromoleculeName: Heavy chain apoferritin from mouse / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: ferroxidase
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
SPSQVRQNYH QDAEAAINR Q INLELYAS YV YLSMSCY FDR DDVALK NFAK YFLHQ SHEER EHAE KLMKLQ NQR GGRIFLQ DI KKPDRDDW E SGLNAMECA LHLEKSVNQS LLELHKLAT D KNDPHLCD FI ETYYLSE QVK SIKELG DHVT NLRKM GAPEA GMAE YLFDKH TLG H

UniProtKB: Ferritin heavy chain

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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

Concentration5 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
30.0 mMHEPES
150.0 mMSodium ChlorideNaCl
1.0 mMDTT
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 6 sec. / Pretreatment - Atmosphere: OTHER / Details: Solarus plasma cleaner at 15 Watts
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER
Details: 3 uL of sample/grid was manually blotted for 4 seconds prior to immediate plunge-freezing in liquid nitrogen-cooled ethane.

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Electron microscopy

MicroscopeTFS TALOS F200C
TemperatureMin: 64.0 K / Max: 70.0 K
DetailsGrids loaded into Talos F200C with a Gatan 626 side-entry cryoholder
Image recordingFilm or detector model: OTHER / Digitization - Dimensions - Width: 2304 pixel / Digitization - Dimensions - Height: 3240 pixel / Number grids imaged: 1 / Number real images: 318 / Average exposure time: 20.0 sec. / Average electron dose: 195.0 e/Å2
Details: 318 movies were acquired in super resolution CDS mode with an exposure rate of 7.1 EPS, for a total of 20 sec (100 ms per frame, 200 frames)
Electron beamAcceleration voltage: 100 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 9.0 µm / Calibrated defocus min: 3.0 µm / Calibrated magnification: 58685 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 9.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 45000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsGatan Alpine Detector
Particle selectionNumber selected: 82977
Details: Particles were picked in cryoSPARC using scaled templates of apoferritin from a previously acquired dataset
Startup modelType of model: NONE / Details: cryoSPARC ab initio
Final reconstructionApplied symmetry - Point group: O (octahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1)
Details: Stack was imported into RELION 3.1 and particles were sorted into optics groups according to beam-image shift parameters stored in the Leginon database. Per-particle defocus, beam tilt, and ...Details: Stack was imported into RELION 3.1 and particles were sorted into optics groups according to beam-image shift parameters stored in the Leginon database. Per-particle defocus, beam tilt, and trefoil aberration estimation were subsequently performed.
Number images used: 51253
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.2.0)
FSC plot (resolution estimation)

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