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- EMDB-12901: Cryo-EM structure of pyrococcus furiosus apoferritin in nanofluid... -

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Basic information

Entry
Database: EMDB / ID: EMD-12901
TitleCryo-EM structure of pyrococcus furiosus apoferritin in nanofluidic channels
Map data
Sample
  • Complex: 24-mer of pyrococcus furiosus apoferritin
    • Protein or peptide: Ferritin
Function / homology
Function and homology information


ferric iron binding / iron ion transport / intracellular iron ion homeostasis
Similarity search - Function
Ferritin, prokaryotic-type / 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 speciesPyrococcus furiosus COM1 (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsHuber ST / Sarajlic E / Huijink R / Evers WH / Jakobi AJ
Funding supportEuropean Union, Netherlands, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)852880European Union
Netherlands Organisation for Scientific Research (NWO)NWO.STU.018-2.007 Netherlands
Citation
Journal: Elife / Year: 2022
Title: Nanofluidic chips for cryo-EM structure determination from picoliter sample volumes.
Authors: Stefan T Huber / Edin Sarajlic / Roeland Huijink / Felix Weis / Wiel H Evers / Arjen J Jakobi /
Abstract: Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness ...Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.
#1: Journal: Biorxiv / Year: 2021
Title: Nanofluidic chips for cryo-EM structure determination from picoliter sample volumes
Authors: Huber ST / Sarajlic E / Huijink R / Weis F / Evers WH / Jakobi AJ
History
DepositionMay 10, 2021-
Header (metadata) releaseAug 11, 2021-
Map releaseAug 11, 2021-
UpdateFeb 16, 2022-
Current statusFeb 16, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ohf
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7ohf
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12901.png

Downloads & links

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Map

FileDownload / File: emd_12901.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.8127 Å
Density
Contour LevelBy AUTHOR: 0.45 / Movie #1: 0.45
Minimum - Maximum-1.5604395 - 2.243183
Average (Standard dev.)-0.00037154698 (±0.10487074)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 260.064 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.81270.81270.8127
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z260.064260.064260.064
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-1.5602.243-0.000

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Supplemental data

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Mask #1

Fileemd_12901_msk_1.map
Projections & Slices
AxesZYX

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Additional map: #1

Fileemd_12901_additional_1.map
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Additional map: #2

Fileemd_12901_additional_2.map
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Half map: #1

Fileemd_12901_half_map_1.map
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Half map: #2

Fileemd_12901_half_map_2.map
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Sample components

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Entire : 24-mer of pyrococcus furiosus apoferritin

EntireName: 24-mer of pyrococcus furiosus apoferritin
Components
  • Complex: 24-mer of pyrococcus furiosus apoferritin
    • Protein or peptide: Ferritin

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Supramolecule #1: 24-mer of pyrococcus furiosus apoferritin

SupramoleculeName: 24-mer of pyrococcus furiosus apoferritin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pyrococcus furiosus COM1 (archaea)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 492 KDa

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Macromolecule #1: Ferritin

MacromoleculeName: Ferritin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus furiosus COM1 (archaea)
Molecular weightTheoretical: 20.535395 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MAMLSERMLK ALNDQLNREL YSAYLYFAMA AYFEDLGLEG FANWMKAQAE EEIGHALRFY NYIYDRNGRV ELDEIPKPPK EWESPLKAF EAAYEHEKFI SKSIYELAAL AEEEKDYSTR AFLEWFINEQ VEEEASVKKI LDKLKFAKDS PQILFMLDKE L SARAPKLP GLLMQGGE

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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

Concentration3.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMNaClSodium chloridesodium chloride
20.0 mMTristris(hydroxymethyl)aminomethane
GridModel: Homemade / Material: SILICON NITRIDE
VitrificationCryogen name: ETHANE / Instrument: LEICA PLUNGER
Details: The sample was filled into cryoChips through the cantilever and then transferred within ~10 seconds to the Leica plunger for freezing..
DetailsThe sample was filled into nanofluidic channels.

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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 defocus max: -2.0 µm / Nominal defocus min: -1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 3 / Number real images: 948 / Average exposure time: 9.0 sec. / Average electron dose: 63.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 3.1)
Startup modelType of model: OTHER / Details: Stochastic gradient decent in cryoSPARC 3.1
Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 1 / Software - Name: cryoSPARC (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 21238
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2x17

Output model

PDB-7ohf:
Cryo-EM structure of pyrococcus furiosus apoferritin in nanofluidic channels

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