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- EMDB-15202: Whole cell cryo-electron tomogram of Apilactobacillus kunkeei and... -

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Basic information

Entry
Database: EMDB / ID: EMD-15202
TitleWhole cell cryo-electron tomogram of Apilactobacillus kunkeei and secreted nanoparticles (TS_07.2)
Map data
Sample
  • Cell: Extracellular nanoparticles (membrane vesicles, protein complexes) secreted from the cell surface of Apilactobacillus kunkeei.
KeywordsMembrane vesicles / extracellular protein complexes / UNKNOWN FUNCTION
Biological speciesApilactobacillus kunkeei (bacteria)
Methodelectron tomography / cryo EM
AuthorsSeeger C / Andersson SGE
Funding support Sweden, 1 items
OrganizationGrant numberCountry
Knut and Alice Wallenberg Foundation Sweden
CitationJournal: To Be Published
Title: Whole cell cryo-electron tomogram of Apilactobacillus kunkeei and secreted nanoparticles (TS_07.2)
Authors: Seeger C / Andersson SGE
History
DepositionJun 16, 2022-
Header (metadata) releaseJul 5, 2023-
Map releaseJul 5, 2023-
UpdateJul 5, 2023-
Current statusJul 5, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15202.png

Downloads & links

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Map

FileDownload / File: emd_15202.map.gz / Format: CCP4 / Size: 365.6 MB / Type: IMAGE STORED AS SIGNED BYTE
Voxel sizeX=Y=Z: 8.452 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)-21.014595 (±20.972629999999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-208208-150
Dimensions14401024260
Spacing10241440260
CellA: 8654.848 Å / B: 12170.88 Å / C: 2197.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Extracellular nanoparticles (membrane vesicles, protein complexes...

EntireName: Extracellular nanoparticles (membrane vesicles, protein complexes) secreted from the cell surface of Apilactobacillus kunkeei.
Components
  • Cell: Extracellular nanoparticles (membrane vesicles, protein complexes) secreted from the cell surface of Apilactobacillus kunkeei.

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Supramolecule #1: Extracellular nanoparticles (membrane vesicles, protein complexes...

SupramoleculeName: Extracellular nanoparticles (membrane vesicles, protein complexes) secreted from the cell surface of Apilactobacillus kunkeei.
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Apilactobacillus kunkeei (bacteria) / Strain: A1401

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4 / Component - Concentration: 1.0 1 / Component - Name: HyClone / Details: HyClone
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: manual back-side blotting of 5 seconds.
DetailsCells were cultivated until log-phase (OD approx. 0.3), gently pelleted (500 x g, 2 min, RT) and re-suspended in HyClone to a final concentration of OD600 approx. 1.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Aurion / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 61 / Average exposure time: 0.4 sec. / Average electron dose: 85.0 e/Å2
Details: Datasets were collected using a Titan Krios G3i microscope (FEI/Thermo Fisher Scientific) outfitted with a K3 detector and BioQuantum imaging filter (Gatan) operated at 300 kV in nanoprobe ...Details: Datasets were collected using a Titan Krios G3i microscope (FEI/Thermo Fisher Scientific) outfitted with a K3 detector and BioQuantum imaging filter (Gatan) operated at 300 kV in nanoprobe and EF-TEM mode, a C2 aperture size of 50 micron, an objective aperture size of 100 micron, and an energy filter slit width of 20 eV in Zero-Loss mode. SerialEM software was used to acquire each tilt-series using a dose-symmetric tilt scheme with a range of +/-60deg, 2deg angular increment and a target defocus of -3 to -6 micron. Each tilt-series of 61 10-frame movies was recorded in counting mode with a pixel size of 2.11angstrom at a dose rate of 15 e-/px/s for 0.4 s and a total dose per tilt-series of ~83e-/A2.
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 3.0 µm / Nominal defocus min: 6.0 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe initial raw movies were aligned and dose-weight filtered using alignframes from the IMOD package. Tilt-series were aligned using the gold fiducial markers, and tomograms were reconstructed by weighted back-projection using programs within within IMOD v4.11.6
Final reconstructionSoftware - Name: IMOD (ver. v4.11.6) / Number images used: 61

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