Visualizing Individual RuBisCO and Its Assembly into Carboxysomes in Marine Cyanobacteria by Cryo-Electron Tomography - PubMed (original) (raw)

Visualizing Individual RuBisCO and Its Assembly into Carboxysomes in Marine Cyanobacteria by Cryo-Electron Tomography

Wei Dai et al. J Mol Biol. 2018.

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 (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.

Keywords: RuBisCO; carboxysome biogenesis; convolutional neural network-based annotation; molecular dynamics simulation; zernike phase-contrast cryo-electron tomography.

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Figures

Figure 1.. Zernike phase contrast cryoET enables direct visualization of subcellular structures in Synechococcus sp. WH8109 cells.

(a) A 54Å slab taken from a WH8109 cell tomogram. (b) Volume rendering of the entire cell tomogram shown in (a). Carboxysome shell is segmented and colored in orange. RuBisCO complexes are colored in magenta. (c) Zoom-in view of the region boxed in (a).

Figure 2.. Applying automated neural-network based annotation and subtomogram analysis to study RuBisCO distribution and packing in individual carboxysomes.

Left: slice views of a carboxysome and a cytosolic RuBisCO cluster; Middle: isosurface views of a carboxysome and of a RuBisCO cluster. The carboxysome is radially colored with blue representing the innermost layer of RuBisCO, green, representing the middle layer, and red to represent the outermost layer. Right: top and side views of RuBisCO subtomogram averages from carboxysomes (top) and from cytosolic clusters (bottom).

Figure 3.. Comparison of distance to the nearest neighbors of RuBisCO complexes inside WH8109 cells.

The insets are representative clusters or particles in each class.

Figure 4.. Carboxysome size distribution and the numbers of icosahedral and non-icosahedral particles in each size group.

(a) Central slabs of 54 Å of representative icosahedral and non-icosahedral particles. (b) Carboxysome size distribution histogram including both non-symmetric and icosahedral particles in each size group. Intracellular carboxysome subtomograms were classified into various size groups based on their diameters by radial density plotting.

Figure 5.. Partially assembled carboxysomes identified in Synechococcus WH8109 cell tomograms.

(a) Section view of a tomogram of a cell that has just completed cell division showing a partially assembled carboxysome labeled by “C”. (b) Slice and color isosurface views of partially assembled carboxysomes. The carboxysomes were arranged from particles with a small number of RuBisCO and shell units, probably representing particles in the early stage of carboxysome biogenesis, to particles that are close to completion of assembly. Magenta: shell; yellow: RuBisCO.

Figure 6.. Molecular dynamic simulation vs experimental subtomogram averages of carboxysomes.

(a) Radial density plots of individual carboxysome simulations with variable numbers of RuBisCO (N) enclosed were compared to those of experimental subtomogram averages (red line). The red dashed lines denote the locations of the density peaks in the radial density plots. The cross sectional views of best matching simulation maps were shown with corresponding subtomogram average maps in (b).

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Visualizing Individual RuBisCO and Its Assembly into Carboxysomes in Marine Cyanobacteria by Cryo-Electron Tomography - PubMed (original) (raw)