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- EMDB-20837: Equine spleen apoferritin prepared with the Shake-it-off device -

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Basic information

Entry
Database: EMDB / ID: EMD-20837
TitleEquine spleen apoferritin prepared with the Shake-it-off device
Map dataapoferritin prepared by the shake-it-off device
Sample
  • Complex: apoferritinFerritin
Function / homology
Function and homology information


intracellular ferritin complex / intracellular sequestering of iron ion / ferric iron binding / ferrous iron binding / iron ion transport / iron ion binding / cytoplasm
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
Ferritin light chain
Similarity search - Component
Biological speciesEquus caballus (horse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsRubinstein JL / Guo H / Ripstein ZA / Benlekbir S
Funding support Canada, 1 items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (Canada)Discovery Grant Canada
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2019
Title: Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device.
Authors: John L Rubinstein / Hui Guo / Zev A Ripstein / Ali Haydaroglu / Aaron Au / Christopher M Yip / Justin M Di Trani / Samir Benlekbir / Timothy Kwok /
Abstract: Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen-preparation methods have lagged behind. Most strategies ...Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen-preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM specimen-preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.
History
DepositionOct 16, 2019-
Header (metadata) releaseDec 11, 2019-
Map releaseDec 11, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.84
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.84
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20837.png

Downloads & links

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Map

FileDownload / File: emd_20837.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationapoferritin prepared by the shake-it-off device
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 1.84 / Movie #1: 1.84
Minimum - Maximum-4.141582 - 6.842949
Average (Standard dev.)0.014816575 (±0.53738534)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 190.79999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z190.800190.800190.800
α/β/γ90.00090.00090.000
start NX/NY/NZ79740
NX/NY/NZ93103213
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-4.1426.8430.015

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Supplemental data

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Mask #1

Fileemd_20837_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B - from cryoSPARC

Fileemd_20837_half_map_1.map
AnnotationHalf map B - from cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A - from cryoSPARC

Fileemd_20837_half_map_2.map
AnnotationHalf map A - from cryoSPARC
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

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Supramolecule #1: apoferritin

SupramoleculeName: apoferritin / type: complex / ID: 1 / Parent: 0
Details: purified from equine spleen, commercially available
Source (natural)Organism: Equus caballus (horse) / Organ: Spleen
Molecular weightTheoretical: 480 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

Concentration5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
50.0 mM(HOCH2)3CNH2tris base
GridSupport film - topology: HOLEY ARRAY / Support film - Film thickness: 35.0 nm / Pretreatment - Atmosphere: AIR
Details: 5 msec spray onto homemade self-wicking copper nanowire grids grown on Cu/Rh 400 mesh grid with 2 micrometer holes spaced 4 micrometer centre-to-centre in sputtered gold.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 40 % / Chamber temperature: 295 K / Details: Experimental Shake-it-off grid freezing device.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Sampling interval: 14.0 µm / Number grids imaged: 1 / Number real images: 324 / Average exposure time: 30.0 sec. / Average electron dose: 21.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 308752
CTF correctionSoftware - Name: cryoSPARC (ver. 2)
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 2) / Details: SGD in cryoSPARC v2
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 2)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: O (octahedral) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 172469

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