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- EMDB-12727: Tomogram of the Drosophila central nervous system vitrified by pl... -

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Basic information

Entry
Database: EMDB / ID: EMD-12727
TitleTomogram of the Drosophila central nervous system vitrified by plunge freezing upon incubation in 10% glycerol
Map dataTomogram of the Drosophila central nervous system vitrified by plunge freezing upon incubation in 10% glycerol
Sample
  • Tissue: Tomogram of the Drosophila central nervous system vitrified by plunge freezing upon incubation in 10% glycerol
Biological speciesDrosophila melanogaster (fruit fly)
Methodelectron tomography / cryo EM
AuthorsBauerlein FJB / Pastor-Pareja JC / Fernandez-Busnadiego R
Funding support Germany, China, 3 items
OrganizationGrant numberCountry
German Research Foundation (DFG)EXC 2067/1- 390729940 Germany
National Natural Science Foundation of China (NSFC)91854207 China
National Natural Science Foundation of China (NSFC)91854207 China
CitationJournal: To Be Published
Title: Cryo-electron tomography of native Drosophila tissues vitrified by plunge freezing
Authors: Bauerlein FJB / Pastor-Pareja JC / Fernandez-Busnadiego R
History
DepositionApr 6, 2021-
Header (metadata) releaseJun 9, 2021-
Map releaseJun 9, 2021-
UpdateJun 9, 2021-
Current statusJun 9, 2021Processing site: PDBe / 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)
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Supplemental images

emd_12727.png

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Map

FileDownload / File: emd_12727.map.gz / Format: CCP4 / Size: 328.5 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationTomogram of the Drosophila central nervous system vitrified by plunge freezing upon incubation in 10% glycerol
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
17.56 Å/pix.
x 200 pix.
= 3512. Å		17.56 Å/pix.
x 928 pix.
= 16295.68 Å		17.56 Å/pix.
x 928 pix.
= 16295.68 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: 17.56 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-5104.0 - 4885.0
Average (Standard dev.)65.76221 (±410.39392)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-111
Dimensions928928200
Spacing928928200
CellA: 16295.68 Å / B: 16295.68 Å / C: 3512.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z17.5617.5617.56
M x/y/z928928200
origin x/y/z0.0000.0000.000
length x/y/z16295.68016295.6803512.000
α/β/γ90.00090.00090.000
start NX/NY/NZ727265
NX/NY/NZ157157169
MAP C/R/S123
start NC/NR/NS00-111
NC/NR/NS928928200
D min/max/mean-5104.0004885.00065.762

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Supplemental data

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Sample components

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Entire : Tomogram of the Drosophila central nervous system vitrified by pl...

EntireName: Tomogram of the Drosophila central nervous system vitrified by plunge freezing upon incubation in 10% glycerol
Components
  • Tissue: Tomogram of the Drosophila central nervous system vitrified by plunge freezing upon incubation in 10% glycerol

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Supramolecule #1: Tomogram of the Drosophila central nervous system vitrified by pl...

SupramoleculeName: Tomogram of the Drosophila central nervous system vitrified by plunge freezing upon incubation in 10% glycerol
type: tissue / ID: 1 / Parent: 0
Source (natural)Organism: Drosophila melanogaster (fruit fly) / Strain: wild type Canton-S strain / Organ: Ventral nerve cord / Tissue: Central nervous tissue

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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: 7.4 / Component - Name: PBS
GridModel: Quantifoil R2/1 / Material: MOLYBDENUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE-PROPANE / Instrument: HOMEMADE PLUNGER
Details: The grids were mounted on a manual plunger, blotted from the back side using Whatman paper #1 (Sigma-Aldrich) and plunged into a 2:1 ethane:propane mixture cooled down by liquid nitrogen by ...Details: The grids were mounted on a manual plunger, blotted from the back side using Whatman paper #1 (Sigma-Aldrich) and plunged into a 2:1 ethane:propane mixture cooled down by liquid nitrogen by the Martinsried-Plunger..
DetailsA dissected intact Drosophila nervous system was deposited on an EM grid.
Cryo protectant10% glycerol
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.05 nA / Focused ion beam - Duration: 3600 sec. / Focused ion beam - Temperature: 90 K / Focused ion beam - Initial thickness: 80000 nm / Focused ion beam - Final thickness: 140 nm
Focused ion beam - Details: To prepare thin electron transparent lamellae into the tissue, plunge-frozen grids were first mounted into Autogrid frames (FEI). The grids were then mounted into a dual- ...Focused ion beam - Details: To prepare thin electron transparent lamellae into the tissue, plunge-frozen grids were first mounted into Autogrid frames (FEI). The grids were then mounted into a dual-beam Quanta 3D FIB/SEM (FEI) using a custom-built transfer shuttle and a cryo-transfer system (PP3000T, Quorum). The samples were kept at -180 C throughout FIB milling by the cryo-stage. To improve SEM imaging, a thin layer of pure metallic Pt was sputtered onto the sample under cryo conditions in the PP3000T transfer system to increase its electrical conductivity. The following parameters were used: 10 mA sputtering current, 500 V between stage and sputtering target and 30 s of exposure at 4.5x10-2 mbar. To interpret and annotate the topographical anatomy of the tissue, overview maps of the EM grid were acquired by SEM at 10 kV at 100-250x magnification (object pixel size 1.1-0.4 um) and by secondary electrons induced by the Ga+ focused ion beam at 30 kV at 338x magnification (object pixel size 0.7 um). To protect the milling front of the lamellae, gaseous organic platinum was frozen on top of the grid using a gas injection system. To prevent bending of the lamella during the preparation, micro-expansion joints were milled left and right of the intended lamella preparation site. 10-20 um wide lamellae were prepared into the tissue with the ion beam at 30 kV at shallow angles (8-14 deg) in four consecutive steps: for the thicker tissue regions (e.g. VNC or skeletal muscle), the areas above and below the intended lamella were first removed with an ion beam current of 5 nA and 10 um spacing. This step was not necessary for thinner tissues such as the peripheral nerves. Further rectangular patterns were defined above and below the intended lamella with 2 um spacing for the rough milling step (ion beam current of 500-1000 pA), followed by fine milling with 800 nm spacing (100 pA) and a final polishing step down to the final lamella thickness of 100-200 nm (50 pA). To reach a uniform thickness, the lamella was tilted by +-0.5 deg and milled on each side separately with 50 pA current. The thickness of the lamella during the polishing step was assessed by SEM at 3-5 kV, 4.1 pA: the loss of charging effects in the lamella, visualized as the vanishing of bright areas, indicates a thickness <300 nm at 5 kV or <200 nm at 3 kV. Biological structures inside the lamella at the surface were imaged at each step by SEM at 2.5 kV, 4.1 pA in integration mode (64x). To reduce lamella charging during phase plate cryo-ET data acquisition, a thin layer of pure metallic Pt was sputtered onto the lamella under cryo conditions in the PP3000T transfer system with the following parameters: 5 mA sputtering current, 500 V between stage and sputtering target and 10 s of exposure at 4.5x10-2 mbar.. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Quanta 3D FIB/SEM. 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
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
DetailsAdditional Volta phase plate alignment.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3868 pixel / Digitization - Dimensions - Height: 3868 pixel / Average electron dose: 3.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus min: 0.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 33000
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. 4.9.0) / Number images used: 38

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