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- EMDB-4600: In situ subtomogram average of ribosome dimer from Synechocystis -

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Basic information

Entry
Database: EMDB / ID: EMD-4600
TitleIn situ subtomogram average of ribosome dimer from Synechocystis
Map dataIn situ subtomogram average of a ribosome dimer from Synechocystis sp. PCC 6803
Sample
  • Complex: Ribosome dimer within the native cellular environment of Synechocystis
Biological speciesSynechocystis sp. PCC 6803 (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 33.0 Å
AuthorsRast A / Wan W / Pfeffer S / Engel BD
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-
UpdateApr 17, 2019-
Current statusApr 17, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4600.png

Downloads & links

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Map

FileDownload / File: emd_4600.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn situ subtomogram average of a ribosome dimer from Synechocystis sp. PCC 6803
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.42 Å/pix.
x 256 pix.
= 875.52 Å		3.42 Å/pix.
x 256 pix.
= 875.52 Å		3.42 Å/pix.
x 256 pix.
= 875.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.42 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.34334403 - 0.36626112
Average (Standard dev.)-0.000019420098 (±0.01616791)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 875.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.423.423.42
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z875.520875.520875.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.3430.366-0.000

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Supplemental data

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Sample components

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Entire : Ribosome dimer within the native cellular environment of Synechocystis

EntireName: Ribosome dimer within the native cellular environment of Synechocystis
Components
  • Complex: Ribosome dimer within the native cellular environment of Synechocystis

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Supramolecule #1: Ribosome dimer within the native cellular environment of Synechocystis

SupramoleculeName: Ribosome dimer within the native cellular environment of Synechocystis
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria) / Location in cell: Cytosol
Molecular weightTheoretical: 5 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
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.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
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.
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: 0.006 µm / Nominal defocus min: 0.004 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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 33.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PyTom (ver. 0.97) / Number subtomograms used: 2391
ExtractionNumber tomograms: 20 / Number images used: 10884 / Software - Name: PyTom (ver. 0.97)
Details: Template matching in PyTom with a de novo reference.
CTF correctionSoftware - Name: IMOD (ver. 4.9)
Final 3D classificationNumber classes: 50 / Avg.num./class: 200 / Software - Name: PyTom (ver. 0.97)
Details: Dimers were identified during the classification step.
Final angle assignmentType: OTHER / Software - Name: PyTom (ver. 0.97) / Details: template matching

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