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- EMDB-21951: Cryo-EM Structure of Human Apoferritin Light Chain Vitrified Usin... -

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Basic information

Entry
Database: EMDB / ID: EMD-21951
TitleCryo-EM Structure of Human Apoferritin Light Chain Vitrified Using Back-it-up
Map dataSharpened map
Sample
  • Organelle or cellular component: Human apoferritin light chain
    • Protein or peptide: Ferritin light chain
  • Ligand: CALCIUM ION
  • Ligand: water
KeywordsHuman / apoferritin / ferritin / light chain / back-it-up / through-grid wicking / METAL BINDING PROTEIN
Function / homologyPeroxisomal testis-specific protein 1 / Peroxisomal testis-specific protein 1 / peroxisome / positive regulation of apoptotic process / nucleus / Peroxisomal testis-specific protein 1
Function and homology information
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.0 Å
AuthorsTan YZ / Rubinstein JL
Funding support Canada, 3 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
Natural Sciences and Engineering Research Council (NSERC, Canada) Canada
Canada Excellence Research Chair Award Canada
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2020
Title: Through-grid wicking enables high-speed cryoEM specimen preparation.
Authors: Yong Zi Tan / John L Rubinstein /
Abstract: Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air-water interface. Here, ...Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air-water interface. Here, it is shown that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass-fiber filter held against the opposite side, often called the `back', of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. Ultrasonic specimen application and through-grid wicking were combined in a high-speed specimen-preparation device that was named `Back-it-up' or BIU. The high liquid-absorption capacity of the glass fiber compared with self-wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The speed of the device increases the number of views for a specimen that suffers from preferred orientations.
History
DepositionMay 9, 2020-
Header (metadata) releaseMay 20, 2020-
Map releaseMay 20, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wx6
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21951.png

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Map

FileDownload / File: emd_21951.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 288 pix.
= 235.008 Å		0.82 Å/pix.
x 288 pix.
= 235.008 Å		0.82 Å/pix.
x 288 pix.
= 235.008 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.816 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-3.960802 - 8.172722
Average (Standard dev.)-0.005302647 (±0.43088546)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 235.008 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8160.8160.816
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z235.008235.008235.008
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-3.9618.173-0.005

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Supplemental data

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Mask #1

Fileemd_21951_msk_1.map
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Additional map: Raw map

Fileemd_21951_additional_1.map
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Additional map: Local resolution map

Fileemd_21951_additional_2.map
AnnotationLocal resolution map
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Additional map: 3DFSC - Thresholded and Binarized

Fileemd_21951_additional_3.map
Annotation3DFSC - Thresholded and Binarized
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Additional map: 3DFSC - Thresholded

Fileemd_21951_additional_4.map
Annotation3DFSC - Thresholded
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Additional map: 3DFSC - Raw

Fileemd_21951_additional_5.map
Annotation3DFSC - Raw
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Half map: Half map 1

Fileemd_21951_half_map_1.map
AnnotationHalf map 1
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Half map: Half map 2

Fileemd_21951_half_map_2.map
AnnotationHalf map 2
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Sample components

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Entire : Human apoferritin light chain

EntireName: Human apoferritin light chain
Components
  • Organelle or cellular component: Human apoferritin light chain
    • Protein or peptide: Ferritin light chain
  • Ligand: CALCIUM ION
  • Ligand: water

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Supramolecule #1: Human apoferritin light chain

SupramoleculeName: Human apoferritin light chain / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 580 KDa

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Macromolecule #1: Ferritin light chain

MacromoleculeName: Ferritin light chain / type: protein_or_peptide / ID: 1 / Number of copies: 24 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.275898 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: TGWSHPQFEK LKGGSSRGGG GGSGGSGGSG GSMSSQIRQN YSTDVEAAVN SLVNLYLQAS YTYLSLGFYF DRDDVALEGV SHFFRELAE EKREGYERLL KMQNQRGGRA LFQDIKKPAE DEWGKTPDAM KAAMALEKKL NQALLDLHAL GSARTDPHLC D FLETHFLD ...String:
TGWSHPQFEK LKGGSSRGGG GGSGGSGGSG GSMSSQIRQN YSTDVEAAVN SLVNLYLQAS YTYLSLGFYF DRDDVALEGV SHFFRELAE EKREGYERLL KMQNQRGGRA LFQDIKKPAE DEWGKTPDAM KAAMALEKKL NQALLDLHAL GSARTDPHLC D FLETHFLD EEVKLIKKMG DHLTNLHRLG GPEAGLGEYL FERLTLRHDG GSGGSGGSGG SGGGASGGS

UniProtKB: Peroxisomal testis-specific protein 1

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 8 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 2420 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

BufferpH: 8
GridModel: Homemade / Material: COPPER/RHODIUM / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR
Details: Both sides of the grid were glow discharged for 120 seconds.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 50 % / Chamber temperature: 298 K / Instrument: HOMEMADE PLUNGER
Details: Back-it-up (ultrasonic specimen application and through-grid wicking in a high-speed specimen preparation device) was used.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 2 / Number real images: 3168 / Average exposure time: 9.0 sec. / Average electron dose: 27.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
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: 673794
Startup modelType of model: INSILICO MODEL
In silico model: Made using cryoSPARC 2 ab initio refinement
Final reconstructionApplied symmetry - Point group: O (octahedral) / Resolution.type: BY AUTHOR / Resolution: 2.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 594259
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2ffx


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 14.91
Output model

PDB-6wx6:
Cryo-EM Structure of Human Apoferritin Light Chain Vitrified Using Back-it-up

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