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- EMDB-10780: In situ cryo-electron tomogram of the Chlamydomonas chloroplast (... -

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Basic information

Entry
Database: EMDB / ID: EMD-10780
TitleIn situ cryo-electron tomogram of the Chlamydomonas chloroplast (Volta phase plate, bin4)
Map dataIn situ cryo-electron tomogram of the Chlamydomonas chloroplast (Volta phase plate, bin4)
Sample
  • Cell: Whole Chlamydomonas cells
Biological speciesChlamydomonas reinhardtii (plant)
Methodelectron tomography / cryo EM
AuthorsSchaffer M / Wietrzynski W / Tegunov D / Albert S / Plitzko J / Baumeister W / Engel BD
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)FOR2092 (EN 1194/1-1) Germany
CitationJournal: Elife / Year: 2020
Title: Charting the native architecture of thylakoid membranes with single-molecule precision.
Authors: Wojciech Wietrzynski / Miroslava Schaffer / Dimitry Tegunov / Sahradha Albert / Atsuko Kanazawa / Jürgen M Plitzko / Wolfgang Baumeister / Benjamin D Engel /
Abstract: Thylakoid membranes scaffold an assortment of large protein complexes that work together to harness the energy of light. It has been a longstanding challenge to visualize how the intricate thylakoid ...Thylakoid membranes scaffold an assortment of large protein complexes that work together to harness the energy of light. It has been a longstanding challenge to visualize how the intricate thylakoid network organizes these protein complexes to finely tune the photosynthetic reactions. Previously, we used in situ cryo-electron tomography to reveal the native architecture of thylakoid membranes (Engel et al., 2015). Here, we leverage technical advances to resolve the individual protein complexes within these membranes. Combined with a new method to visualize membrane surface topology, we map the molecular landscapes of thylakoid membranes inside green algae cells. Our tomograms provide insights into the molecular forces that drive thylakoid stacking and reveal that photosystems I and II are strictly segregated at the borders between appressed and non-appressed membrane domains. This new approach to charting thylakoid topology lays the foundation for dissecting photosynthetic regulation at the level of single protein complexes within the cell.
History
DepositionMar 21, 2020-
Header (metadata) releaseApr 1, 2020-
Map releaseApr 1, 2020-
UpdateSep 30, 2020-
Current statusSep 30, 2020Processing 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)
  • Imaged by UCSF Chimera
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Movie viewer
Supplemental images

emd_10780.png

Downloads & links

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Map

FileDownload / File: emd_10780.map.gz / Format: CCP4 / Size: 762.2 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationIn situ cryo-electron tomogram of the Chlamydomonas chloroplast (Volta phase plate, bin4)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
13.68 Å/pix.
x 464 pix.
= 6347.52 Å		13.68 Å/pix.
x 928 pix.
= 12695.04 Å		13.68 Å/pix.
x 928 pix.
= 12695.04 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: 13.68 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-162 - 131
Average (Standard dev.)5.400458 (±10.212014)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00232
Dimensions928928464
Spacing928928464
CellA: 12695.04 Å / B: 12695.04 Å / C: 6347.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z13.6813.6813.68
M x/y/z928928464
origin x/y/z0.0000.0000.000
length x/y/z12695.04012695.0406347.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS00232
NC/NR/NS928928464
D min/max/mean-162.000131.0005.400

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Supplemental data

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Sample components

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Entire : Whole Chlamydomonas cells

EntireName: Whole Chlamydomonas cells
Components
  • Cell: Whole Chlamydomonas cells

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Supramolecule #1: Whole Chlamydomonas cells

SupramoleculeName: Whole Chlamydomonas cells / type: cell / ID: 1 / Parent: 0
Details: Grown suspended in TAP media, with normal atmosphere aeration and constant light
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: mat3-4

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7 / Details: TAP media
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV / Details: blotted from back for 10 seconds before plunging.
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.03 nA / Focused ion beam - Duration: 2000 sec. / Focused ion beam - Temperature: 95 K / Focused ion beam - Initial thickness: 1000 nm / Focused ion beam - Final thickness: 75 nm
Focused ion beam - Details: See https://bio-protocol.org/e1575 for detailed procedure.. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Quanta FIB. This is not in a list of ...Focused ion beam - Details: See https://bio-protocol.org/e1575 for detailed procedure.. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Quanta FIB. This is not in a list of allowed values set(['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
DetailsBidirectional tilt scheme, separated at 0 degrees, Volta Phase Plate
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 61 / Average exposure time: 1.5 sec. / Average electron dose: 1.5 e/Å2
Details: Images were collected in movie-mode at 12 frames per second
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 42000
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 / Number images used: 61
CTF correctionSoftware - Name: IMOD

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