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- EMDB-43700: Cryo-EM map of LKB1-STRADalpha-MO25alpha from TFS Glacios with Ga... -

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Entry
Database: EMDB / ID: EMD-43700
TitleCryo-EM map of LKB1-STRADalpha-MO25alpha from TFS Glacios with Gatan Alpine detector at 120 keV
Map dataCryo-EM map of LKB1-STRADalpha-MO25alpha from TFS Glacios with Gatan Alpine detector at 120 keV
Sample
  • Complex: Heterotrimeric complex of serine/threonine kinase LKB1 with psuedokinase STRADa and scaffolding MO25a
Keywordsserine/threonine kinase / pseudokinase / complex / TRANSFERASE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsChan LM / Courteau BJ / Verba KA
Funding support1 items
OrganizationGrant numberCountry
Not funded
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) releaseJul 24, 2024-
Map releaseJul 24, 2024-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43700.png

Downloads & links

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Map

FileDownload / File: emd_43700.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of LKB1-STRADalpha-MO25alpha from TFS Glacios with Gatan Alpine detector at 120 keV
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 256 pix.
= 217.6 Å		0.85 Å/pix.
x 256 pix.
= 217.6 Å		0.85 Å/pix.
x 256 pix.
= 217.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.523
Minimum - Maximum-2.001316 - 2.905169
Average (Standard dev.)-0.00076490775 (±0.083313726)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 217.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Additional Map

Fileemd_43700_additional_1.map
AnnotationAdditional Map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_43700_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_43700_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Heterotrimeric complex of serine/threonine kinase LKB1 with psued...

EntireName: Heterotrimeric complex of serine/threonine kinase LKB1 with psuedokinase STRADa and scaffolding MO25a
Components
  • Complex: Heterotrimeric complex of serine/threonine kinase LKB1 with psuedokinase STRADa and scaffolding MO25a

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Supramolecule #1: Heterotrimeric complex of serine/threonine kinase LKB1 with psued...

SupramoleculeName: Heterotrimeric complex of serine/threonine kinase LKB1 with psuedokinase STRADa and scaffolding MO25a
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 140.5 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

BufferpH: 7.6
Component:
ConcentrationNameFormula
25.0 mMTris HCl
350.0 mMsodium chlorideNaCl
5.0 %glycerol
0.5 mMDTT
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN ALPINE (2.3k x 3.2k) / Number grids imaged: 1 / Number real images: 3635 / Average electron dose: 58.9 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.7 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 43000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER

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Image processing

Particle selectionNumber selected: 897749
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

2wtk

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 120684
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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