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- EMDB-0734: Spinach 80S ribosome -

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Basic information

Entry
Database: EMDB / ID: EMD-0734
TitleSpinach 80S ribosome
Map data
Sample
  • Complex: Spinach 80S ribosome
Biological speciesSpinacia olerac (spinach)
Methodsubtomogram averaging / cryo EM / Resolution: 34.0 Å
AuthorsZhang J / Zhang D / Sun L / Ji G / Huang X / Niu T / Sun F
Funding support China, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China31830020 China
Ministry of Science and Technology (China)2017YFA0504702 China
Citation
Journal: J Struct Biol / Year: 2021
Title: VHUT-cryo-FIB, a method to fabricate frozen hydrated lamellae from tissue specimens for in situ cryo-electron tomography.
Authors: Jianguo Zhang / Danyang Zhang / Lei Sun / Gang Ji / Xiaojun Huang / Tongxin Niu / Jiashu Xu / Chengying Ma / Yun Zhu / Ning Gao / Wei Xu / Fei Sun /
Abstract: Cryo-electron tomography (cryo-ET) provides a promising approach to study intact structures of macromolecules in situ, but the efficient preparation of high-quality cryosections represents a ...Cryo-electron tomography (cryo-ET) provides a promising approach to study intact structures of macromolecules in situ, but the efficient preparation of high-quality cryosections represents a bottleneck. Although cryo-focused ion beam (cryo-FIB) milling has emerged for large and flat cryo-lamella preparation, its application to tissue specimens remains challenging. Here, we report an integrated workflow, VHUT-cryo-FIB, for efficiently preparing frozen hydrated tissue lamella that can be readily used in subsequent cryo-ET studies. The workflow includes vibratome slicing, high-pressure freezing, ultramicrotome cryo-trimming and cryo-FIB milling. Two strategies were developed for loading cryo-lamella via a side-entry cryo-holder or an FEI AutoGrid. The workflow was validated by using various tissue specimens, including rat skeletal muscle, rat liver and spinach leaf specimens, and in situ structures of ribosomes were obtained at nanometer resolution from the spinach and liver samples.
#1: Journal: Biorxiv / Year: 2019
Title: VHUT-cryo-FIB, a method to fabricate frozen-hydrated lamella 1of tissue specimen for in situcryo-electron tomography
Authors: Zhang J / Zhang D / Sun L / Ji G / Huang X / Niu T / Sun F
History
DepositionAug 9, 2019-
Header (metadata) releaseAug 12, 2020-
Map releaseAug 12, 2020-
UpdateJul 21, 2021-
Current statusJul 21, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.52
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.52
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0734.png

Downloads & links

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Map

FileDownload / File: emd_0734.map.gz / Format: CCP4 / Size: 2.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 5.3 Å
Density
Contour LevelBy AUTHOR: 0.52 / Movie #1: 0.52
Minimum - Maximum-0.2550366 - 0.97346705
Average (Standard dev.)0.0414296 (±0.15774563)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions848484
Spacing848484
CellA=B=C: 445.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.35.35.3
M x/y/z848484
origin x/y/z0.0000.0000.000
length x/y/z445.200445.200445.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS848484
D min/max/mean-0.2550.9730.041

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Supplemental data

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Sample components

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Entire : Spinach 80S ribosome

EntireName: Spinach 80S ribosome
Components
  • Complex: Spinach 80S ribosome

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Supramolecule #1: Spinach 80S ribosome

SupramoleculeName: Spinach 80S ribosome / type: complex / ID: 1 / Parent: 0
Details: Ribosomes were found in cryo-lamella of spinach leaf tissue.
Source (natural)Organism: Spinacia olerac (spinach)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7
VitrificationCryogen name: NITROGEN
DetailsA puncher was used to cut a slice at about 2 mm diameter from the leaf of spinach. The leaf was then put in the recess of the carrier and frozen by HPF. A lamella was milled from the frozen sample through FIB. Tilt series data was collected on the lamella.

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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
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-20 / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 3.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 3 / Number images used: 1341 / Software: (Name: IMOD (ver. 4.9.2), RELION (ver. 1.4))
Details: Particles were manually picked from reconstructed tomogram.
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 1.4)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 34.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number subtomograms used: 923
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6ek0
PDB Unreleased entry

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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