National Institutes of Health/National Institute of General Medical Sciences
P41GM103832
United States
National Institutes of Health/National Institute of General Medical Sciences
R01GM080139
United States
National Institutes of Health/National Institute of General Medical Sciences
R01GM037657
United States
Welch Foundation
H-0037
United States
Welch Foundation
Q1242
United States
Citation
Journal: 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
Deposition
May 4, 2018
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Header (metadata) release
Jul 25, 2018
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Map release
Sep 5, 2018
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Update
Aug 12, 2020
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Current status
Aug 12, 2020
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
pH: 8 / Component - Name: Artificial sea water Details: Solutions were made fresh and autoclaved before use.
Grid
Model: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Vitrification
Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 2 seconds before plunging.
Details
Carboxysome in Synechococcus sp. WH8109 cells
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Electron microscopy
Microscope
JEOL 2200FS
Temperature
Min: 70.0 K
Specialist optics
Phase 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
Image recording
Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average exposure time: 2.0 sec. / Average electron dose: 0.5 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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