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- EMDB-0732: Cryo electron tomogram of cryo-lamella of rat skeleton muscle -

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Basic information

Entry
Database: EMDB / ID: EMD-0732
TitleCryo electron tomogram of cryo-lamella of rat skeleton muscle
Map data
Sample
  • Tissue: cryo-lamella of rat skeleton muscle
Biological speciesMus musculus (house mouse)
Methodelectron tomography
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 6, 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
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Supplemental images

emd_0732.png

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Map

FileDownload / File: emd_0732.map.gz / Format: CCP4 / Size: 700 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 26.1 Å
Density
Minimum - Maximum-3.3304439 - 2.5546393
Average (Standard dev.)-0.054471496 (±0.4088539)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin512512-250
Dimensions10241024175
Spacing10241024175
CellA: 26726.4 Å / B: 26726.4 Å / C: 4567.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z26.126.126.1
M x/y/z10241024175
origin x/y/z0.0000.0000.000
length x/y/z26726.40026726.4004567.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS512512-250
NC/NR/NS10241024175
D min/max/mean-3.3302.555-0.054

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Supplemental data

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Sample components

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Entire : cryo-lamella of rat skeleton muscle

EntireName: cryo-lamella of rat skeleton muscle
Components
  • Tissue: cryo-lamella of rat skeleton muscle

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Supramolecule #1: cryo-lamella of rat skeleton muscle

SupramoleculeName: cryo-lamella of rat skeleton muscle / type: tissue / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse) / Tissue: skeleton muscle

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Experimental details

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Structure determination

Processingelectron tomography
Aggregation statetissue

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Sample preparation

BufferpH: 7 / Details: phosphate buffered saline (PBS)
DetailsMice were sacrificed and muscle was removed and cut into small bulks with a razor blade. Then a bulk of muscle was fixed on the specimen holder of VT1200S vibrating blade microtome for slicing. The thickness of each slice was set to 80-100 um. The generated muscle slice was cut into appropriate size for high pressure freezing.
High pressure freezingInstrument: OTHER
Details: Muscle tissue 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 ...Details: Muscle tissue 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: 20 nm
Focused ion beam - Details: Then the carrier was transfer with the cryo-transfer shuttle17 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 shuttle17 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

MicroscopeTFS TALOS F200C
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 10.0 µm / Calibrated defocus min: 8.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 13500
Sample stageSpecimen holder model: GATAN 910 MULTI-SPECIMEN SINGLE TILT CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 14.0 µm / Number grids imaged: 1 / Number real images: 82 / Average exposure time: 1.0 sec. / Average electron dose: 3.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: FOURIER SPACE / Software - Name: ICON (ver. 1.2.9)
Details: ICON version 1.2.9 was used for the final reconstruction and the reconstruction algorithm is iterative compressed-sensing optimized NUFFT.
Number images used: 43

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