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- EMDB-24797: Mouse heavy chain apoferritin determined in presence of 20% glyce... -

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Basic information

Entry
Database: EMDB / ID: EMD-24797
TitleMouse heavy chain apoferritin determined in presence of 20% glycerol (300keV)
Map dataMouse heavy chain apoferritin in buffer with 20% glycerol.
Sample
  • Complex: ApoferritinFerritin
    • Protein or peptide: ApoferritinFerritin
Function / homology
Function and homology information


ferric iron binding / iron ion transport / intracellular iron ion homeostasis
Similarity search - Function
Ferritin iron-binding regions signature 1. / Ferritin iron-binding regions signature 2. / Ferritin, conserved site / Ferritin / Ferritin-like diiron domain / Ferritin-like diiron domain profile. / Ferritin/DPS protein domain / Ferritin-like domain / Ferritin-like / Ferritin-like superfamily
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.1 Å
AuthorsHirschi M / Basanta B / 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: 0.153
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.153
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24797.png

Downloads & links

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Map

FileDownload / File: emd_24797.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMouse heavy chain apoferritin in buffer with 20% glycerol.
Voxel sizeX=Y=Z: 1.03 Å
Density
Contour LevelBy AUTHOR: 0.153 / Movie #1: 0.153
Minimum - Maximum-0.284906 - 0.5272622
Average (Standard dev.)0.0004721306 (±0.033544477)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 197.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z197.760197.760197.760
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ448448448
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.2850.5270.000

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Supplemental data

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Mask #1

Fileemd_24797_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Mouse heavy chain apoferritin in buffer with 20%...

Fileemd_24797_half_map_1.map
AnnotationMouse heavy chain apoferritin in buffer with 20% glycerol. Half map 2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Mouse heavy chain apoferritin in buffer with 20%...

Fileemd_24797_half_map_2.map
AnnotationMouse heavy chain apoferritin in buffer with 20% glycerol. Half map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Apoferritin

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

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Supramolecule #1: Apoferritin

SupramoleculeName: Apoferritin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 487.32 KDa

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Macromolecule #1: Apoferritin

MacromoleculeName: Apoferritin / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SPSQVRQNYH QDAEAAINRQ INLELYASYV YLSMSCYFDR DDVALKNFAK YFLHQSHEER EHAEKLMKLQ NQRGGRIFLQ DIKKPDRDDW ESGLNAMECA LHLEKSVNQS LLELHKLATD KNDPHLCDFI ETYYLSEQVK SIKELGDHVT NLRKMGAPEA GMAEYLFDKH TLGH

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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

Concentration5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
30.0 mMC8H18N2O4SHEPES
20.0 % v/vC3H8O3glycerol
150.0 mMNaClSodium chlorideSodium Chloride
1.0 mMC4H10O2S2Dithiothreitol
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 1.5 µm / Calibrated defocus min: 0.5 µm / Calibrated magnification: 48732 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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 - Frames/image: 1-65 / Number grids imaged: 1 / Number real images: 1859 / Average exposure time: 6.5 sec. / Average electron dose: 55.25 e/Å2
Details: Images were collected using stage position navigation to the center of 16 holes and then beam shift was used to target exposures.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1757706
CTF correctionSoftware - Name: RELION (ver. 3.1)
Startup modelType of model: EMDB MAP
EMDB ID:

21024


Details: Low-pass filtered to 15 Angstroms
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: O (octahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 778762
FSC plot (resolution estimation)

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