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- EMDB-10708: Anabaena sp. PCC 7120 Septal Junction Average from Lamellae gener... -

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Basic information

Entry
Database: EMDB / ID: EMD-10708
TitleAnabaena sp. PCC 7120 Septal Junction Average from Lamellae generated by Automated Milling
Map dataSeptal junction average generated from 343 particles and 5-fold symmetrizing. Lamellae were generated using an automated milling approach.
Sample
  • Complex: Septal Junction
Biological speciesNostoc sp. PCC 7120 (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 30.0 Å
AuthorsZachs T / Pilhofer M
Funding support Switzerland, European Union, 2 items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_179255 Switzerland
European Research Council (ERC)679209European Union
CitationJournal: Elife / Year: 2020
Title: Fully automated, sequential focused ion beam milling for cryo-electron tomography.
Authors: Tobias Zachs / Andreas Schertel / João Medeiros / Gregor L Weiss / Jannik Hugener / Joao Matos / Martin Pilhofer /
Abstract: Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and ...Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here, we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.
History
DepositionFeb 27, 2020-
Header (metadata) releaseMar 18, 2020-
Map releaseMar 18, 2020-
UpdateMar 18, 2020-
Current statusMar 18, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.72
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.72
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10708.png

Downloads & links

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Map

FileDownload / File: emd_10708.map.gz / Format: CCP4 / Size: 333 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSeptal junction average generated from 343 particles and 5-fold symmetrizing. Lamellae were generated using an automated milling approach.
Voxel sizeX=Y=Z: 6.757 Å
Density
Contour LevelBy AUTHOR: 4.72 / Movie #1: 4.72
Minimum - Maximum-35.85216 - 49.241894
Average (Standard dev.)-1.0315125 (±7.2475963)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions444444
Spacing444444
CellA=B=C: 297.30798 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.7576.7576.757
M x/y/z444444
origin x/y/z0.0000.0000.000
length x/y/z297.308297.308297.308
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS444444
D min/max/mean-35.85249.242-1.032

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Supplemental data

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Sample components

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Entire : Septal Junction

EntireName: Septal Junction
Components
  • Complex: Septal Junction

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Supramolecule #1: Septal Junction

SupramoleculeName: Septal Junction / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Nostoc sp. PCC 7120 (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 2.3 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: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: FSC 0.5 CUT-OFF / Number subtomograms used: 343
ExtractionNumber tomograms: 9 / Number images used: 343
Final angle assignmentType: OTHER

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