- EMDB-43527: Apoferritin at 100 keV on Alpine detector with a side-entry cryoholder -
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Open data
ID or keywords:
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Basic information
Entry
Database: EMDB / ID: EMD-43527
Title
Apoferritin at 100 keV on Alpine detector with a side-entry cryoholder
Map data
mouse 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
Keywords
Protein complex / TRANSPORT PROTEIN
Function / homology
Function and homology information
Iron uptake and transport / Golgi Associated Vesicle Biogenesis / iron ion sequestering activity / autolysosome / ferroxidase / intracellular sequestering of iron ion / ferroxidase activity / negative regulation of fibroblast proliferation / endocytic vesicle lumen / Neutrophil degranulation ...Iron uptake and transport / Golgi Associated Vesicle Biogenesis / iron ion sequestering activity / autolysosome / ferroxidase / intracellular sequestering of iron ion / ferroxidase activity / negative regulation of fibroblast proliferation / endocytic vesicle lumen / Neutrophil degranulation / ferric iron binding / ferrous iron binding / iron ion transport / iron ion binding / immune response / negative regulation of cell population proliferation / mitochondrion / extracellular region / identical protein binding / membrane / cytoplasm / cytosol Similarity search - Function
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM14305
United States
Citation
Journal: 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.
Protein or peptide: Heavy chain apoferritin from mouse
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Supramolecule #1: Heavy chain apoferritin from mouse
Supramolecule
Name: Heavy chain apoferritin from mouse / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)
Organism: Mus musculus (house mouse)
Molecular weight
Theoretical: 487 KDa
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Macromolecule #1: Heavy chain apoferritin from mouse
Macromolecule
Name: Heavy chain apoferritin from mouse / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: ferroxidase
Source (natural)
Organism: Mus musculus (house mouse)
Recombinant expression
Organism: Escherichia coli BL21(DE3) (bacteria)
Sequence
String:
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
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
5 mg/mL
Buffer
pH: 7.5 Component:
Concentration
Name
Formula
30.0 mM
HEPES
150.0 mM
Sodium Chloride
NaCl
1.0 mM
DTT
Grid
Model: 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
Vitrification
Cryogen 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
Microscope
TFS TALOS F200C
Temperature
Min: 64.0 K / Max: 70.0 K
Details
Grids loaded into Talos F200C with a Gatan 626 side-entry cryoholder
Image recording
Film 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 beam
Acceleration voltage: 100 kV / Electron source: FIELD EMISSION GUN
Specimen 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
Details
Gatan Alpine Detector
Particle selection
Number selected: 82977 Details: Particles were picked in cryoSPARC using scaled templates of apoferritin from a previously acquired dataset
Startup model
Type of model: NONE / Details: cryoSPARC ab initio
Final reconstruction
Applied 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
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