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- EMDB-4601: In situ defocus subtomogram average of phycobilisome array from S... -

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Basic information

Entry
Database: EMDB / ID: EMD-4601
TitleIn situ defocus subtomogram average of phycobilisome array from Synechocystis sp. PCC 6803
Map dataIn situ defocus subtomogram average of phycobilisome array from Synechocystis sp. PCC 6803
Sample
  • Complex: In situ defocus subtomogram average of phycobilisome array from Synechocystis
Biological speciesSynechocystis sp. PCC 6803 (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 23.6 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: 0.065
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4601.png

Downloads & links

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Map

FileDownload / File: emd_4601.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn situ defocus subtomogram average of phycobilisome array from Synechocystis sp. PCC 6803
Voxel sizeX=Y=Z: 3.42 Å
Density
Contour LevelBy AUTHOR: 0.055 / Movie #1: 0.065
Minimum - Maximum-0.40795508 - 0.38164684
Average (Standard dev.)-0.001255502 (±0.10330404)
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-0.4080.382-0.001

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Supplemental data

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Sample components

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Entire : In situ defocus subtomogram average of phycobilisome array from S...

EntireName: In situ defocus subtomogram average of phycobilisome array from Synechocystis
Components
  • Complex: In situ defocus subtomogram average of phycobilisome array from Synechocystis

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Supramolecule #1: In situ defocus subtomogram average of phycobilisome array from S...

SupramoleculeName: In situ defocus subtomogram average of phycobilisome array from Synechocystis
type: complex / ID: 1 / Parent: 0
Details: Each unit of the array has a molecular weight of ~6 MDa.
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria) / Location in cell: Cytosol
Molecular weightTheoretical: 30 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
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: 0.006 µm / Nominal defocus min: 0.004 µm / Nominal magnification: 42000
Specialist opticsEnergy 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

ExtractionNumber tomograms: 20 / Number images used: 3850 / Software - Name: PyTom (ver. 0.97)
Details: Template matching followed by oversampling of splines along arrays. Duplicate points were removed following the alignment process.
CTF correctionSoftware - Name: IMOD (ver. 4.9)
Final angle assignmentType: OTHER / Details: Template matching with PyTom
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 23.6 Å / Resolution method: FSC 0.143 CUT-OFF / Details: Averaging was performed with STOPGAP 0.3.1 / Number subtomograms used: 1710

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