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- EMDB-15516: Cryo-EM Snapshots of Nanodisc-Embedded Native Eukaryotic Membrane... -

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Basic information

Entry
Database: EMDB / ID: EMD-15516
TitleCryo-EM Snapshots of Nanodisc-Embedded Native Eukaryotic Membrane Proteins
Map data
Sample
  • Complex: SB-DIBMA solubilized Chaetomium thermophilum membranes mMW fraction
Biological speciesThermochaetoides thermophila (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 18.59 Å
AuthorsJanson K / Kyrilis FL / Tueting C / Alfes M / Das M / Traeger TK / Schmidt C / Hamdi F / Keller S / Meister A / Kastritis PL
Funding support Germany, European Union, France, Austria, 8 items
OrganizationGrant numberCountry
German Federal Ministry for Education and Research03Z22HN22 Germany
German Federal Ministry for Education and Research03Z22HN23 Germany
German Federal Ministry for Education and Research03Z22HI2 Germany
European Regional Development FundZS/2016/04/78115European Union
European Regional Development FundEuropean Union
Agence Nationale de la Recherche (ANR)ME 4165/2-1 France
Agence Nationale de la Recherche (ANR)KE 1478/7-1 France
Austrian Science FundI 5359-N Austria
CitationJournal: Biomacromolecules / Year: 2022
Title: Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs.
Authors: Kevin Janson / Fotis L Kyrilis / Christian Tüting / Marie Alfes / Manabendra Das / Toni K Träger / Carla Schmidt / Farzad Hamdi / Carolyn Vargas / Sandro Keller / Annette Meister / Panagiotis L Kastritis /
Abstract: New technologies for purifying membrane-bound protein complexes in combination with cryo-electron microscopy (EM) have recently allowed the exploration of such complexes under near-native conditions. ...New technologies for purifying membrane-bound protein complexes in combination with cryo-electron microscopy (EM) have recently allowed the exploration of such complexes under near-native conditions. In particular, polymer-encapsulated nanodiscs enable the study of membrane proteins at high resolution while retaining protein-protein and protein-lipid interactions within a lipid bilayer. However, this powerful technology has not been exploited to address the important question of how endogenous─as opposed to overexpressed─membrane proteins are organized within a lipid environment. In this work, we demonstrate that biochemical enrichment protocols for native membrane-protein complexes from in combination with polymer-based lipid-bilayer nanodiscs provide a substantial improvement in the quality of recovered endogenous membrane-protein complexes. Mass spectrometry results revealed ∼1123 proteins, while multiple 2D class averages and two 3D reconstructions from cryo-EM data furnished prominent structural signatures. This integrated methodological approach to enriching endogenous membrane-protein complexes provides unprecedented opportunities for a deeper understanding of eukaryotic membrane proteomes.
History
DepositionAug 1, 2022-
Header (metadata) releaseFeb 8, 2023-
Map releaseFeb 8, 2023-
UpdateFeb 8, 2023-
Current statusFeb 8, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15516.png

Downloads & links

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Map

FileDownload / File: emd_15516.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.59 Å/pix.
x 350 pix.
= 207.13 Å		0.59 Å/pix.
x 350 pix.
= 207.13 Å		0.59 Å/pix.
x 350 pix.
= 207.13 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.5918 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0868
Minimum - Maximum-0.29168278 - 0.39386806
Average (Standard dev.)0.005636673 (±0.02391745)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 207.12999 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_15516_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15516_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : SB-DIBMA solubilized Chaetomium thermophilum membranes mMW fraction

EntireName: SB-DIBMA solubilized Chaetomium thermophilum membranes mMW fraction
Components
  • Complex: SB-DIBMA solubilized Chaetomium thermophilum membranes mMW fraction

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Supramolecule #1: SB-DIBMA solubilized Chaetomium thermophilum membranes mMW fraction

SupramoleculeName: SB-DIBMA solubilized Chaetomium thermophilum membranes mMW fraction
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Details: Sample was created by solubilizing native chaetomium termophilum membranes with the aid of the copolymer SB-DIBMA
Source (natural)Organism: Thermochaetoides thermophila (fungus) / Tissue: mycelia

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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

Concentration0.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
200.0 mMNaClsodium chloride
50.0 mMC4H11NO3Tris

Details: Solutions were freshly prepared, sterile filtrated, and sonicated before usage
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time of 12 s Blotforce of 0.
DetailsThis sample was purified by size exclusion chromatography. Subsequently, multiple fractions in the medium molecular weight region were pooled together to obtain a higher protein concentration.

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Electron microscopy

MicroscopeTFS GLACIOS
TemperatureMin: 77.15 K / Max: 103.15 K
Alignment procedureComa free - Residual tilt: 14.7 mrad
DetailsGrid screening was performed manually until criteria for good acquisition areas was narrowed down.
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4048 pixel / Digitization - Dimensions - Height: 4048 pixel / Number grids imaged: 1 / Number real images: 5912 / Average electron dose: 64.72 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 236566 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 240000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 1720004
Details: particles were picked automatically with the blob picker function of cryosparc
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 18.59 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 7253
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 80 / Avg.num./class: 900 / Software - Name: cryoSPARC (ver. 3.1)
FSC plot (resolution estimation)

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