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- EMDB-7855: Carboxysome subtomogram averaging of size group 106 nm -

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Basic information

Entry
Database: EMDB / ID: EMD-7855
TitleCarboxysome subtomogram averaging of size group 106 nm
Map dataSubtomogram averaging of carboxysome size group 106 nm
Sample
  • Organelle or cellular component: carboxysome
Biological speciesSynechococcus sp. WH 8109 (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 40.0 Å
AuthorsDai W / Chen M / Chiu W
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesP41GM103832 United States
National Institutes of Health/National Institute of General Medical SciencesR01GM080139 United States
National Institutes of Health/National Institute of General Medical SciencesR01GM037657 United States
Welch FoundationH-0037 United States
Welch FoundationQ1242 United States
CitationJournal: J Mol Biol / Year: 2018
Title: Visualizing Individual RuBisCO and Its Assembly into Carboxysomes in Marine Cyanobacteria by Cryo-Electron Tomography.
Authors: Wei Dai / Muyuan Chen / Christopher Myers / Steven J Ludtke / B Montgomery Pettitt / Jonathan A King / Michael F Schmid / Wah Chiu /
Abstract: Cyanobacteria are photosynthetic organisms responsible for ~25% of the organic carbon fixation on earth. A key step in carbon fixation is catalyzed by ribulose bisphosphate carboxylase/oxygenase ...Cyanobacteria are photosynthetic organisms responsible for ~25% of the organic carbon fixation on earth. A key step in carbon fixation is catalyzed by ribulose bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme in the biosphere. Applying Zernike phase-contrast electron cryo-tomography and automated annotation, we identified individual RuBisCO molecules and their assembly intermediates leading to the formation of carboxysomes inside Syn5 cyanophage infected cyanobacteria Synechococcus sp. WH8109 cells. Surprisingly, more RuBisCO molecules were found to be present as cytosolic free-standing complexes or clusters than as packaged assemblies inside carboxysomes. Cytosolic RuBisCO clusters and partially assembled carboxysomes identified in the cell tomograms support a concurrent assembly model involving both the protein shell and the enclosed RuBisCO. In mature carboxysomes, RuBisCO is neither randomly nor strictly icosahedrally packed within protein shells of variable sizes. A time-averaged molecular dynamics simulation showed a semi-liquid probability distribution of the RuBisCO in carboxysomes and correlated well with carboxysome subtomogram averages. Our structural observations reveal the various stages of RuBisCO assemblies, which could be important for understanding cellular function.
History
DepositionMay 4, 2018-
Header (metadata) releaseJul 25, 2018-
Map releaseSep 5, 2018-
UpdateAug 12, 2020-
Current statusAug 12, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7855.png

Downloads & links

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Map

FileDownload / File: emd_7855.map.gz / Format: CCP4 / Size: 38.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram averaging of carboxysome size group 106 nm
Voxel sizeX=Y=Z: 9.04 Å
Density
Contour LevelBy AUTHOR: 0.8 / Movie #1: 0.8
Minimum - Maximum-2.8100207 - 3.043319
Average (Standard dev.)0.037864655 (±0.32179418)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 1952.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z9.049.049.04
M x/y/z216216216
origin x/y/z0.0000.0000.000
length x/y/z1952.6401952.6401952.640
α/β/γ90.00090.00090.000
start NX/NY/NZ-163-114-126
NX/NY/NZ210124170
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS216216216
D min/max/mean-2.8103.0430.038

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Supplemental data

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Sample components

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Entire : carboxysome

EntireName: carboxysome
Components
  • Organelle or cellular component: carboxysome

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Supramolecule #1: carboxysome

SupramoleculeName: carboxysome / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: carboxysome from cyanobacteria Synechococcus sp. WH8109
Source (natural)Organism: Synechococcus sp. WH 8109 (bacteria) / Organelle: carboxysome / Location in cell: cytoplasm

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 8 / Component - Name: Artificial sea water
Details: Solutions were made fresh and autoclaved before use.
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 / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 2 seconds before plunging.
DetailsCarboxysome in Synechococcus sp. WH8109 cells

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Electron microscopy

MicroscopeJEOL 2200FS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 4.1 mm / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm / Nominal magnification: 25000
Specialist opticsPhase plate: ZERNIKE PHASE PLATE / Energy filter - Name: In-column Omega Filter / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average exposure time: 2.0 sec. / Average electron dose: 0.5 e/Å2

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Image processing

ExtractionNumber tomograms: 56 / Number images used: 198 / Reference model: none / Method: carboxysome subvolumes selected manually / Software - Name: EMAN2 (ver. 2.21a)
Final 3D classificationNumber classes: 2 / Details: Icosahedral/non-Icosahedral carboxysomes
Final angle assignmentType: OTHER / Software - Name: EMAN2 (ver. 2.21) / Details: Subtomogram alignment and averaging in EMAN2
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: I (icosahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 40.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2 (ver. 2.21) / Number subtomograms used: 28

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