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- EMDB-28147: Rabbit muscle aldolase determined using single-particle cryo-EM w... -

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Basic information

Entry
Database: EMDB / ID: EMD-28147
TitleRabbit muscle aldolase determined using single-particle cryo-EM with Apollo camera.
Map datasharpened and masked map
Sample
  • Complex: Aldolase from rabbit muscleFructose-bisphosphate aldolase
    • Protein or peptide: Fructose-bisphosphate aldolase A
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 / glycolytic process / protein homotetramerization / positive regulation of cell migration
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 (rabbit) / rabbit (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.24 Å
AuthorsPeng R / Fu X / Mendez JH / Randolph PH / Bammes B / Stagg SM
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM143805 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM139616 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM119032 United States
CitationJournal: J Struct Biol X / Year: 2023
Title: Characterizing the resolution and throughput of the Apollo direct electron detector.
Authors: Ruizhi Peng / Xiaofeng Fu / Joshua H Mendez / Peter S Randolph / Benjamin E Bammes / Scott M Stagg /
Abstract: Advances in electron detection have been essential to the success of high-resolution cryo-EM structure determination. A new generation of direct electron detector called the Apollo, has been ...Advances in electron detection have been essential to the success of high-resolution cryo-EM structure determination. A new generation of direct electron detector called the Apollo, has been developed by Direct Electron. The Apollo uses a novel event-based MAPS detector custom designed for ultra-fast electron counting. We have evaluated this new camera, finding that it delivers high detective quantum efficiency (DQE) and low coincidence loss, enabling high-quality electron counting data acquisition at up to nearly 80 input electrons per pixel per second. We further characterized the performance of Apollo for single particle cryo-EM on real biological samples. Using mouse apoferritin, Apollo yielded better than 1.9 Å resolution reconstructions at all three tested dose rates from a half-day data collection session each. With longer collection time and improved specimen preparation, mouse apoferritin was reconstructed to 1.66 Å resolution. Applied to a more challenging small protein aldolase, we obtained a 2.24 Å resolution reconstruction. The high quality of the map indicates that the Apollo has sufficiently high DQE to reconstruct smaller proteins and complexes with high-fidelity. Our results demonstrate that the Apollo camera performs well across a broad range of dose rates and is capable of capturing high quality data that produce high-resolution reconstructions for large and small single particle samples.
History
DepositionSep 14, 2022-
Header (metadata) releaseDec 21, 2022-
Map releaseDec 21, 2022-
UpdateJan 11, 2023-
Current statusJan 11, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28147.png

Downloads & links

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Map

FileDownload / File: emd_28147.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened and masked map
Voxel sizeX=Y=Z: 0.599 Å
Density
Contour LevelBy AUTHOR: 1.23
Minimum - Maximum-4.7797413 - 6.7428656
Average (Standard dev.)0.00071511226 (±0.11193744)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 306.688 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28147_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_28147_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_28147_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Aldolase from rabbit muscle

EntireName: Aldolase from rabbit muscleFructose-bisphosphate aldolase
Components
  • Complex: Aldolase from rabbit muscleFructose-bisphosphate aldolase
    • Protein or peptide: Fructose-bisphosphate aldolase A

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Supramolecule #1: Aldolase from rabbit muscle

SupramoleculeName: Aldolase from rabbit muscle / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Details: Plays a key role in glycolysis and gluconeogenesis. In addition, may also function as scaffolding protein.
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: muscle
Molecular weightTheoretical: 14921 MDa

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Macromolecule #1: Fructose-bisphosphate aldolase A

MacromoleculeName: Fructose-bisphosphate aldolase A / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: fructose-bisphosphate aldolase
Source (natural)Organism: rabbit (rabbit) / Tissue: muscle
Molecular weightTheoretical: 39.394875 KDa
SequenceString: MPHSHPALTP EQKKELSDIA HRIVAPGKGI LAADESTGSI AKRLQSIGTE NTEENRRFYR QLLLTADDRV NPCIGGVILF HETLYQKAD DGRPFPQVIK SKGGVVGIKV DKGVVPLAGT NGETTTQGLD GLSERCAQYK KDGADFAKWR CVLKIGEHTP S ALAIMENA ...String:
MPHSHPALTP EQKKELSDIA HRIVAPGKGI LAADESTGSI AKRLQSIGTE NTEENRRFYR QLLLTADDRV NPCIGGVILF HETLYQKAD DGRPFPQVIK SKGGVVGIKV DKGVVPLAGT NGETTTQGLD GLSERCAQYK KDGADFAKWR CVLKIGEHTP S ALAIMENA NVLARYASIC QQNGIVPIVE PEILPDGDHD LKRCQYVTEK VLAAVYKALS DHHIYLEGTL LKPNMVTPGH AC TQKYSHE EIAMATVTAL RRTVPPAVTG VTFLSGGQSE EEASINLNAI NKCPLLKPWA LTFSYGRALQ ASALKAWGGK KEN LKAAQE EYVKRALANS LACQGKYTPS GQAGAAASES LFISNHAY

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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

Concentration4.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
30.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mMNaClSodium chloridesodium chloride
1.0 mMDTTDL-Dithiothreitol

Details: DTT are added freshly before use.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 80.0 µm / Calibrated magnification: 72621 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: OTHER / Digitization - Dimensions - Width: 8192 pixel / Digitization - Dimensions - Height: 8192 pixel / Number grids imaged: 1 / Number real images: 8682 / Average exposure time: 1.347 sec. / Average electron dose: 60.0 e/Å2
Details: Images were collected using Direct Electron Apollo camera at a fixed movie frame rate of 60 frames/second.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3805094
Details: particles were picked using templates projected from EMD-21023.
Startup modelType of model: EMDB MAP
EMDB ID:

21023

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Software - details: heterogenous refinement in cryosparc is used firstly
Final 3D classificationNumber classes: 3 / Avg.num./class: 608100 / Software - Name: cryoSPARC (ver. 3.3.2)
Software - details: heterogenous refinement is used for 3D classification
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Software - details: Non-uniform refinement is used for high resolution
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.24 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2)
Software - details: auto sharpened map in cryosparc refinement is used
Number images used: 722778
DetailsDirect Electron Apollo
FSC plot (resolution estimation)

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