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- EMDB-4603: Volta phase plate in situ tomogram of a Synechocystis cell -

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Basic information

Entry
Database: EMDB / ID: EMD-4603
TitleVolta phase plate in situ tomogram of a Synechocystis cell
Map dataVolta phase plate in situ tomogram of a Synechocystis cell
Sample
  • Cell: Volta phase plate in situ tomogram of a Synechocystis cell
Biological speciesSynechocystis sp. PCC 6803 (bacteria)
Methodelectron tomography / cryo EM
AuthorsRast A / Wan W / Pfeffer S / Engel BD
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research FoundationFOR2092 Germany
CitationJournal: Nat Plants / Year: 2019
Title: Biogenic regions of cyanobacterial thylakoids form contact sites with the plasma membrane.
Authors: Anna Rast / Miroslava Schaffer / Sahradha Albert / William Wan / Stefan Pfeffer / Florian Beck / Jürgen M Plitzko / Jörg Nickelsen / Benjamin D Engel /
Abstract: Little is known about how the photosynthetic machinery is arranged in time and space during the biogenesis of thylakoid membranes. Using in situ cryo-electron tomography to image the three- ...Little is known about how the photosynthetic machinery is arranged in time and space during the biogenesis of thylakoid membranes. Using in situ cryo-electron tomography to image the three-dimensional architecture of the cyanobacterium Synechocystis, we observed that the tips of multiple thylakoids merge to form a substructure called the 'convergence membrane'. This high-curvature membrane comes into close contact with the plasma membrane at discrete sites. We generated subtomogram averages of 70S ribosomes and array-forming phycobilisomes, then mapped these structures onto the native membrane architecture as markers for protein synthesis and photosynthesis, respectively. This molecular localization identified two distinct biogenic regions in the thylakoid network: thylakoids facing the cytosolic interior of the cell that were associated with both marker complexes, and convergence membranes that were decorated by ribosomes but not phycobilisomes. We propose that the convergence membranes perform a specialized biogenic function, coupling the synthesis of thylakoid proteins with the integration of cofactors from the plasma membrane and the periplasmic space.
History
DepositionFeb 11, 2019-
Header (metadata) releaseFeb 20, 2019-
Map releaseFeb 20, 2019-
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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Supplemental images

emd_4603.png

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Map

FileDownload / File: emd_4603.map.gz / Format: CCP4 / Size: 762.2 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationVolta phase plate in situ tomogram of a Synechocystis cell
Voxel sizeX=Y=Z: 13.68 Å
Density
Minimum - Maximum-258 - 116
Average (Standard dev.)2.1207142 (±10.92974)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
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
start NX/NY/NZ212211153
NX/NY/NZ80102186
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS928928464
D min/max/mean-258.000116.0002.121

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Supplemental data

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Sample components

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Entire : Volta phase plate in situ tomogram of a Synechocystis cell

EntireName: Volta phase plate in situ tomogram of a Synechocystis cell
Components
  • Cell: Volta phase plate in situ tomogram of a Synechocystis cell

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Supramolecule #1: Volta phase plate in situ tomogram of a Synechocystis cell

SupramoleculeName: Volta phase plate in situ tomogram of a Synechocystis cell
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)

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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
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 90 % / Chamber temperature: 297 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time: 10 sec Blot force: 10.
DetailsVitrious Synechocystis cell milled with a Ga2+ focused ion beam.
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: 3600 sec. / Focused ion beam - Temperature: 93 K / Focused ion beam - Initial thickness: 2000 nm / Focused ion beam - Final thickness: 150 nm
Focused ion beam - Details: 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 0.0001 µm / Nominal defocus min: 0.0001 µm / Nominal magnification: 42000
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
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 / 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. Higher tilts had longer exposures.
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) / Number images used: 58

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