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- EMDB-43528: Aldolase at 100 keV on the Alpine detector with a side-entry cryo... -

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Basic information

Entry
Database: EMDB / ID: EMD-43528
TitleAldolase at 100 keV on the Alpine detector with a side-entry cryoholder
Map datarabbit muscle aldolase determined at 100 keV on a Talos F200C with a Gatan Alpine detector using a Gatan 626 side-entry cryoholder
Sample
  • Complex: Fructose-bisphosphate aldolase A
    • Protein or peptide: Fructose-bisphosphate aldolase A
Keywordsfructose-bisphosphate aldolase activity / HYDROLASE
Function / homology
Function and homology information


negative regulation of Arp2/3 complex-mediated actin nucleation / fructose-bisphosphate aldolase / fructose-bisphosphate aldolase activity / M band / I band / fructose 1,6-bisphosphate metabolic process / glycolytic process / protein homotetramerization / positive regulation of cell migration / cytosol
Similarity search - Function
Fructose-bisphosphate aldolase class-I active site / Fructose-bisphosphate aldolase class-I active site. / Fructose-bisphosphate aldolase, class-I / Fructose-bisphosphate aldolase class-I / Aldolase-type TIM barrel
Similarity search - Domain/homology
Fructose-bisphosphate aldolase A
Similarity search - Component
Biological speciesOryctolagus cuniculus cuniculus (mammal) / Oryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 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_43528.png

Downloads & links

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Map

FileDownload / File: emd_43528.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationrabbit muscle aldolase determined at 100 keV on a Talos F200C with a Gatan Alpine detector using a Gatan 626 side-entry cryoholder
Voxel sizeX=Y=Z: 0.852 Å
Density
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-1.7709377 - 2.5401206
Average (Standard dev.)-0.0009348414 (±0.07017865)
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_43528_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Additional map: unsharpened map

Fileemd_43528_additional_1.map
Annotationunsharpened map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_43528_half_map_1.map
AnnotationHalf map B
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_43528_half_map_2.map
AnnotationHalf map A
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Histogram (log scale)

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Sample components

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Entire : Fructose-bisphosphate aldolase A

EntireName: Fructose-bisphosphate aldolase A
Components
  • Complex: Fructose-bisphosphate aldolase A
    • Protein or peptide: Fructose-bisphosphate aldolase A

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Supramolecule #1: Fructose-bisphosphate aldolase A

SupramoleculeName: Fructose-bisphosphate aldolase A / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: purchased as a lyophilized powder (Sigma-Aldrich)
Source (natural)Organism: Oryctolagus cuniculus cuniculus (mammal) / Tissue: Muscle
Molecular weightTheoretical: 156 KDa

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Macromolecule #1: Fructose-bisphosphate aldolase A

MacromoleculeName: Fructose-bisphosphate aldolase A / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: fructose-bisphosphate aldolase
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: Muscle
SequenceString: PHSHPALTPE QKKELSDIA H RIVAPGKG IL AADESTG SIA KRLQSI GTEN TEENR RFYRQ LLLT ADDRVN PCI GGVILFH ET LYQKADDG R PFPQVIKSK GGVVGIKVDK GVVPLAGTN G ETTTQGLD GL SERCAQY KKD GADFAK WRCV LKIGE ...String:
PHSHPALTPE QKKELSDIA H RIVAPGKG IL AADESTG SIA KRLQSI GTEN TEENR RFYRQ LLLT ADDRVN PCI GGVILFH ET LYQKADDG R PFPQVIKSK GGVVGIKVDK GVVPLAGTN G ETTTQGLD GL SERCAQY KKD GADFAK WRCV LKIGE HTPSA LAIM ENANVL ARY ASICQQN GI VPIVEPEI L PDGDHDLKR CQYVTEKVLA AVYKALSDH H IYLEGTLL KP NMVTPGH ACT QKYSHE EIAM ATVTA LRRTV PPAV TGVTFL SGG QSEEEAS IN LNAINKCP L LKPWALTFS YGRALQASAL KAWGGKKEN L KAAQEEYV KR ALANSLA CQG KYTPSG QAGA AASES LFISN HAY

UniProtKB: Fructose-bisphosphate aldolase A

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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

Concentration1.6 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHEPES
50.0 mMSodium ChlorideNaCl
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, 15 Watts
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER
Details: 3 microliters of purified aldolase was applied to grids and manually blotted for four to five seconds using Whatman No. 1 filter paper. Blot time counting commenced once the blotted sample ...Details: 3 microliters of purified aldolase was applied to grids and manually blotted for four to five seconds using Whatman No. 1 filter paper. Blot time counting commenced once the blotted sample on the filter paper stopped spreading, monitored visually using a lamp positioned behind the sample. At the end of 4-5 s, the blotting paper was pulled back and immediately plunged into a well of liquid ethane cooled by liquid nitrogen..
DetailsLyophillized rabbit muscle aldolase was purchased (Sigma Aldrich) and solubilized in 20 mM HEPES (pH 7.5), 50 mM NaCl at ~3 mg/ml. Aldolase was loaded onto a using a Sepharose 6 10/300 (GE Healthcare) column equilibrated in the solubilization buffer, and fractions corresponding to apoferritin were pooled and concentrated to 1.6 mg/ml.

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Electron microscopy

MicroscopeTFS TALOS F200C
TemperatureMin: 64.0 K / Max: 70.0 K
Image recordingFilm or detector model: OTHER / Digitization - Dimensions - Width: 2304 pixel / Digitization - Dimensions - Height: 3240 pixel / Number grids imaged: 1 / Number real images: 2476 / Average exposure time: 10.0 sec. / Average electron dose: 71.0 e/Å2 / Details: Gatan Alpine
Electron beamAcceleration voltage: 100 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 15.0 µm / Calibrated defocus min: 15.0 µm / Calibrated magnification: 58685 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 5.0 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

DetailsAlpine detector
Particle selectionNumber selected: 1914721 / Details: template-based picking in cryoSPARC
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1)
Details: Per-particle defocus, beam tilt, and trefoil aberration estimation were performed in RELION, and 3D auto-refinement with D2 symmetry and CTF refinement including 4th-order (symmetrical) aberration correction
Number images used: 674124
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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