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- EMDB-0733: Cryo electron tomogram of cryo-lamella of spinach leaf -

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Basic information

Entry
Database: EMDB / ID: EMD-0733
TitleCryo electron tomogram of cryo-lamella of spinach leaf
Map dataThe pixel size of original image is 2.65, this reconstructed tomogram has a binning factor of 4.
Sample
  • Tissue: cryo-lamella of spinach leaf
Biological speciesSpinacia oleracea (spinach)
Methodelectron tomography / cryo EM
AuthorsZhang J / Zhang D / Sun L / 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 7, 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
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
  • Download
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Movie viewer
Supplemental images

emd_0733.png

Downloads & links

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Map

FileDownload / File: emd_0733.map.gz / Format: CCP4 / Size: 883.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe pixel size of original image is 2.65, this reconstructed tomogram has a binning factor of 4.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
10.6 Å/pix.
x 260 pix.
= 2756. Å		10.6 Å/pix.
x 928 pix.
= 9836.801 Å		10.6 Å/pix.
x 960 pix.
= 10176. Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 10.6 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-3581.8308 - 3705.2969
Average (Standard dev.)81.13198 (±566.7743)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0060
Dimensions928960260
Spacing960928260
CellA: 10176.0 Å / B: 9836.801 Å / C: 2756.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z10.610.60000107758610.6
M x/y/z960928260
origin x/y/z0.0000.0000.000
length x/y/z10176.0009836.8012756.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS0060
NC/NR/NS960928260
D min/max/mean-3581.8313705.29781.132

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Supplemental data

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Sample components

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Entire : cryo-lamella of spinach leaf

EntireName: cryo-lamella of spinach leaf
Components
  • Tissue: cryo-lamella of spinach leaf

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Supramolecule #1: cryo-lamella of spinach leaf

SupramoleculeName: cryo-lamella of spinach leaf / type: tissue / ID: 1 / Parent: 0
Details: A puncher was used to cut a circular slice of leaf at about 2 mm diameter. The slice was then fitting into carrier for high pressure freezing.
Source (natural)Organism: Spinacia oleracea (spinach)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statetissue

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Sample preparation

BufferpH: 7 / Details: phosphate buffered saline (PBS)
VitrificationCryogen name: NITROGEN
DetailsLeaf slice was put in the recess of the carrier and cryoprotectant 1-hexadecene was added to fill the surrounding area. Then a sapphire disk was loaded on top of the carrier before the whole composed sandwich was frozen.
High pressure freezingInstrument: OTHER
Details: Leaf slice was put in the recess of the carrier and cryoprotectant 1-hexadecene was added to fill the surrounding area. Then a sapphire disk was loaded on top of the carrier before the whole ...Details: Leaf slice was put in the recess of the carrier and cryoprotectant 1-hexadecene was added to fill the surrounding area. Then a sapphire disk was loaded on top of the carrier before the whole composed sandwich was frozen.. The value given for _emd_high_pressure_freezing.instrument is HPF COMPACT 01. This is not in a list of allowed values {'BAL-TEC HPM 010', 'LEICA EM HPM100', 'OTHER', 'LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID IMMERSION FREEZER'} so OTHER is written into the XML file.
Cryo protectant1-hexadecene
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.08 nA / Focused ion beam - Duration: 3600 sec. / Focused ion beam - Temperature: 93 K / Focused ion beam - Initial thickness: 200 nm / Focused ion beam - Final thickness: 17 nm
Focused ion beam - Details: Then the carrier was transfer with the cryo-transfer shuttle into the SEM chamber by using Quorum PP3000T cryotransfer system under -180 degree. To improve sample ...Focused ion beam - Details: Then the carrier was transfer with the cryo-transfer shuttle into the SEM chamber by using Quorum PP3000T cryotransfer system under -180 degree. To improve sample conductivity and reduce curtaining artifacts, the samples were deposited with organometallic platinum using the in situ gas injection system (GIS) operated at 5 seconds gas injection time before milling. During the cryo-FIB milling process, the milling angle is nearly in parallel with the carrier, and the milling was performed parallel from both sides of the sample platform to produce lamella. Rough milling is produced with the accelerating voltage of the ion beam at 30 kV, and current at 0.79 nA-0.43 nA. The initial milling width is about 20 um and depth is about 20 um. To facilitate tomography data collection, ice at the notch above lamella was removed to get a trapezoid-shaped milling pattern. After rough milling, one side of the lamella is jagged from the main platform. When the thickness of lamella reaches about 1 um the ion current is reduced to 0.23 nA or 80 pA until thickness finally reaching 150 to 250 nm.. The value given for _emd_sectioning_focused_ion_beam.instrument is Helios NanoLab 600i. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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: 1-21 / Average exposure time: 1.0 sec. / Average electron dose: 3.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 5.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.9.2) / Number images used: 42

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