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- EMDB-10712: Apoferritin from horse spleen, prepared using trEM workflow. -

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Basic information

Entry
Database: EMDB / ID: EMD-10712
TitleApoferritin from horse spleen, prepared using trEM workflow.
Map dataApoferritin from equine spleen, prepared using trEM workflow
Sample
  • Complex: Apoferritin from horse spleen
Function / homology
Function and homology information


ferritin complex / autolysosome / : / ferric iron binding / autophagosome / ferrous iron binding / iron ion transport / cytoplasmic vesicle / 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: 2.77 Å
AuthorsMaeots ME / Lee B / Nans A / Jeong SG / Esfahani MNM / Smith D / Lee CS / Lee SS / Peter M / Enchev RI
Funding support Korea, Republic Of, Switzerland, United Kingdom, 7 items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)NRF-2015K1A1A2033054 Korea, Republic Of
European Research Council (ERC) Switzerland
Swiss National Science Foundation Switzerland
Cancer Research UK United Kingdom
The Francis Crick Institute United Kingdom
Wellcome Trust United Kingdom
Medical Research Council (MRC, United Kingdom) United Kingdom
CitationJournal: Nat Commun / Year: 2020
Title: Modular microfluidics enables kinetic insight from time-resolved cryo-EM.
Authors: Märt-Erik Mäeots / Byungjin Lee / Andrea Nans / Seung-Geun Jeong / Mohammad M N Esfahani / Shan Ding / Daniel J Smith / Chang-Soo Lee / Sung Sik Lee / Matthias Peter / Radoslav I Enchev /
Abstract: Mechanistic understanding of biochemical reactions requires structural and kinetic characterization of the underlying chemical processes. However, no single experimental technique can provide this ...Mechanistic understanding of biochemical reactions requires structural and kinetic characterization of the underlying chemical processes. However, no single experimental technique can provide this information in a broadly applicable manner and thus structural studies of static macromolecules are often complemented by biophysical analysis. Moreover, the common strategy of utilizing mutants or crosslinking probes to stabilize intermediates is prone to trapping off-pathway artefacts and precludes determining the order of molecular events. Here we report a time-resolved sample preparation method for cryo-electron microscopy (trEM) using a modular microfluidic device, featuring a 3D-mixing unit and variable delay lines that enables automated, fast, and blot-free sample vitrification. This approach not only preserves high-resolution structural detail but also substantially improves sample integrity and protein distribution across the vitreous ice. We validate the method by visualising reaction intermediates of early RecA filament growth across three orders of magnitude on sub-second timescales. The trEM method reported here is versatile, reproducible, and readily adaptable to a broad spectrum of fundamental questions in biology.
History
DepositionFeb 28, 2020-
Header (metadata) releaseJul 22, 2020-
Map releaseJul 22, 2020-
UpdateJul 22, 2020-
Current statusJul 22, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0576
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0576
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10712.png

Downloads & links

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Map

FileDownload / File: emd_10712.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationApoferritin from equine spleen, prepared using trEM workflow
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.85 Å/pix.
x 250 pix.
= 211.25 Å		0.85 Å/pix.
x 250 pix.
= 211.25 Å		0.85 Å/pix.
x 250 pix.
= 211.25 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.845 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0576 / Movie #1: 0.0576
Minimum - Maximum-0.13326071 - 0.20716992
Average (Standard dev.)0.00000000000 (±0.012729209)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 211.25 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8450.8450.845
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z211.250211.250211.250
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ250250250
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.1330.2070.000

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Supplemental data

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Sample components

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Entire : Apoferritin from horse spleen

EntireName: Apoferritin from horse spleen
Components
  • Complex: Apoferritin from horse spleen

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Supramolecule #1: Apoferritin from horse spleen

SupramoleculeName: Apoferritin from horse spleen / type: complex / ID: 1 / Parent: 0 / Details: commercial apoferritin from Sigma Aldrich
Source (natural)Organism: Equus caballus (horse) / Organ: spleen
Molecular weightTheoretical: 443 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

Concentration2 mg/mL
BufferpH: 7.4 / Component - Name: PBSA
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 50mA
VitrificationCryogen name: ETHANE / Chamber temperature: 294 K / Instrument: HOMEMADE PLUNGER / Details: Plunging speed 1.1m/s, ambient conditions..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 33.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: O (octahedral) / Resolution.type: BY AUTHOR / Resolution: 2.77 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 9804
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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