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- EMDB-24799: Rabbit muscle aldolase determined in the presence of 20% v/v glycerol. -

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Basic information

Entry
Database: EMDB / ID: EMD-24799
TitleRabbit muscle aldolase determined in the presence of 20% v/v glycerol.
Map dataRabbit muscle aldolase determined in the presence of 20% v/v glycerol.
Sample
  • Complex: Aldolase
    • Protein or peptide: Aldolase
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 (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsBasanta B / Hirschi M / Lander GC
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS095892 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R21GM142196 United States
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2022
Title: A case for glycerol as an acceptable additive for single-particle cryoEM samples.
Authors: Benjamin Basanta / Marscha M Hirschi / Danielle A Grotjahn / Gabriel C Lander /
Abstract: Buffer-composition and sample-preparation guidelines for cryo-electron microscopy are geared towards maximizing imaging contrast and reducing electron-beam-induced motion. These pursuits often ...Buffer-composition and sample-preparation guidelines for cryo-electron microscopy are geared towards maximizing imaging contrast and reducing electron-beam-induced motion. These pursuits often involve the minimization or the complete removal of additives that are commonly used to facilitate proper protein folding and minimize aggregation. Among these admonished additives is glycerol, a widely used osmolyte that aids protein stability. In this work, it is shown that the inclusion of glycerol does not preclude high-resolution structure determination by cryoEM, as demonstrated by an ∼2.3 Å resolution reconstruction of mouse apoferritin (∼500 kDa) and an ∼3.3 Å resolution reconstruction of rabbit muscle aldolase (∼160 kDa) in the presence of 20%(v/v) glycerol. While it was found that generating thin ice that is amenable to high-resolution imaging requires long blot times, the addition of glycerol did not result in increased beam-induced motion or an inability to pick particles. Overall, these findings indicate that glycerol should not be discounted as a cryoEM sample-buffer additive, particularly for large, fragile complexes that are prone to disassembly or aggregation upon its removal.
History
DepositionSep 1, 2021-
Header (metadata) releaseOct 6, 2021-
Map releaseOct 6, 2021-
UpdateApr 20, 2022-
Current statusApr 20, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00595
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.00595
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24799.png

Downloads & links

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Map

FileDownload / File: emd_24799.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRabbit muscle aldolase determined in the presence of 20% v/v glycerol.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 256 pix.
= 267.52 Å		1.05 Å/pix.
x 256 pix.
= 267.52 Å		1.05 Å/pix.
x 256 pix.
= 267.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.045 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00595 / Movie #1: 0.00595
Minimum - Maximum-0.022284174 - 0.02859089
Average (Standard dev.)2.1287444e-05 (±0.00074590405)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 267.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0451.0451.045
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z267.520267.520267.520
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ448448448
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0220.0290.000

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Supplemental data

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Mask #1

Fileemd_24799_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Rabbit muscle aldolase determined in the presence of...

Fileemd_24799_half_map_1.map
AnnotationRabbit muscle aldolase determined in the presence of 20% v/v glycerol. Half map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Rabbit muscle aldolase determined in the presence of...

Fileemd_24799_half_map_2.map
AnnotationRabbit muscle aldolase determined in the presence of 20% v/v glycerol. Half 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

EntireName: Aldolase
Components
  • Complex: Aldolase
    • Protein or peptide: Aldolase

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Supramolecule #1: Aldolase

SupramoleculeName: Aldolase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 157 KDa

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Macromolecule #1: Aldolase

MacromoleculeName: Aldolase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
SequenceString: PHSHPALTPE QKKELSDIAH RIVAPGKGIL AADESTGSIA KRLQSIGTEN TEENRRFYRQ LLLTADDRVN PCIGGVILFH ETLYQKADDG RPFPQVIKSK GGVVGIKVDK GVVPLAGTNG ETTTQGLDGL SERCAQYKKD GADFAKWRCV LKIGEHTPSA LAIMENANVL ...String:
PHSHPALTPE QKKELSDIAH RIVAPGKGIL AADESTGSIA KRLQSIGTEN TEENRRFYRQ LLLTADDRVN PCIGGVILFH ETLYQKADDG RPFPQVIKSK GGVVGIKVDK GVVPLAGTNG ETTTQGLDGL SERCAQYKKD GADFAKWRCV LKIGEHTPSA LAIMENANVL ARYASICQQN GIVPIVEPEI LPDGDHDLKR CQYVTEKVLA AVYKALSDHH IYLEGTLLKP NMVTPGHACT QKYSHEEIAM ATVTALRRTV PPAVTGVTFL SGGQSEEEAS INLNAINKCP LLKPWALTFS YGRALQASAL KAWGGKKENL KAAQEEYVKR ALANSLACQG KYTPSGQAGA AASESLFISN HAY

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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:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
20.0 % v/vC3H8O3glycerol
50.0 mMNaClSodium Chloride
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 9.33257 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K / Max: 77.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 2591 / Average exposure time: 10.0 sec. / Average electron dose: 52.5 e/Å2
Details: Images were collected using stage position navigation to the center of 4 holes and then beam shift was used to target exposures.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 1.7 µm / Calibrated defocus min: 1.3 µm / Calibrated magnification: 47846 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.7 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1255598
CTF correctionSoftware - Name: RELION (ver. 3.1)
Startup modelType of model: EMDB MAP
EMDB ID:

8743


Details: Low-pass filtered to 15 Angstroms
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 21155
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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