Structure of Halothiobacillus neapolitanus carboxysomes by cryo-electron tomography

Abstract

Carboxysomes are polyhedral bodies consisting of a proteinaceous shell filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). They are found in the cytoplasm of all cyanobacteria and some chemoautotrophic bacteria. Previous studies of Halothiobacillus neapolitanus and Nitrobacter agilis carboxysomes suggest that the structures are either icosahedral or dodecahedral. To determine the protein shell structure more definitively, purified H. neapolitanus carboxysomes were re-examined by cryo-electron tomography and scanning transmission electron microscopy (STEM). Due to the limited tilt angles in the electron microscope, the tomographic reconstructions are distorted. Corrections were made in the 3D orientation searching and averaging of the computationally extracted carboxysomes to minimize the missing data effects. It was found that H. neapolitanus carboxysomes vary widely in size and mass as shown by cryo-electron tomography and STEM mass measurements, respectively. We have aligned and averaged carboxysomes in several size classes from the 3D tomographic reconstruction by methods that are not model-biased. The averages reveal icosahedral symmetry of the shell, but not of the density inside it, for all the size classes.

Figure 1

An image from the tilt series near the 0° tilt angle showing isolated carboxysomes embedded in a layer of vitreous ice. The entire series is represented in a movie as Supplemental Movie 1. The box outlines the approximate area of the tomogram shown in Figure 2a and b, about 400 pixels, or 432 nm on a side.

Figure 2a

A slice through the center of the tomographic volume in the area indicated in Figure 1. Four sections of the tomogram are averaged in z. A long arrow points to the thin shell, about 4 nm thick. Shorter arrows indicate the five-fold vertices of several particles where the shell is slightly thicker. Arrowheads indicate RuBisCO molecules inside the carboxysome shell. Some shells are fully packed, others (on the right, for instance) are rather emptier. 2b-Stereo image of a part of the tomogram, averaged by a factor of 4 (4.32 nm/pixel) from the original image sampling and volume-rendered. A variety of sizes and orientations of carboxysomes are visible, and the amount of RuBisCO in the interior of these carboxysomes is also seen to vary (note the carboxysome near the lower right which has less mass inside). The majority of the free particles outside the carboxysomes in this preparation are likely to be RuBisCO molecules that have been liberated from the particles during the preparation. They preferentially congregate at the top and bottom of the vitreous ice. “Below” the particle near the center is some ice contamination on the “bottom” surface of the vitreous ice layer. The field of view spans 432 nm in width. The entire tomogram is shown as a movie in Supplemental Movie 2.

Figure 3

A histogram of the diameters of the carboxysomes in the data set. The diameters of the particles were determined from the 1D radial density profiles of each of the 92 3D extracted and centered volumes. Each bin is 1 nm wide, from 88 to 108 nm. A large group of particles cluster around a size of 94–95 and 97–100 nm, but groups with larger and smaller size particles are clearly present.

Figure 4

a–The average of 21 3D reconstructions of particles averaging about 100 nm in diameter and surface-rendered. The average has no symmetry imposed, but it clearly displays approximate 5, 3 and 2-fold symmetry in the correct relationships to produce icosahedral symmetry. The direction along which the resolution is affected by the missing wedge in Fourier space is approximately horizontal in this view b–The average in Figure 4a was icosahedrally averaged. This average is also displayed in Figure 5f as part of the gallery of particle averages from all the size classes.

Figure 5

a–g A gallery of views down the 3-fold axis of all seven size classes, ranging in diameter from 88 to 103 nm in diameter. The surface has been colored radially to emphasize the low relief of the triangular faces. White arrows point to the different arrangement of density at the center of the triangular faces of two groups (5a and 5b). Differences like this occur in all the groups. The variation in arrangement of the surface densities may reflect a different organization of the shell in different sized particles. The number of particle 3D volumes that went into each size class shown was: 3, 3, 13, 18, 17, 21, and 15 for Figure 5a–g respectively. This is a reflection of the distribution of particle sizes shown in Figure 3.

Figure 6

a–A radial density distribution plot for one of the 3D particles that went into the average shown in Figure 4a. The layered nature of the interior RuBisCO molecules is clear for each individual particle, though the exact azimuthal location turns out to be variable. b–A projection of the central 12 nm thickness of the icosahedrally averaged particles of average size 100 nm (Figure 4b and 5f). The shell is of higher density because the interior is less regularly arranged and averaging decreases its density. However, the interior layers are spaced similar to the radial plot of the individual particles. The layer under the shell has the highest density, indicating that its average occupancy is higher than the layers closer to the center. The density threshold for this figure had to be reduced to about half of what is used for Figure 5f in order to make the interior visible at all.

Figure 7

The mass distribution of purified H. neapolitanus carboxysomes determined by STEM mass measurements. The bins start at the labeled mass and are 15MDa wide. The Y axis is the number of carboxysomes having the mass range indicated. It is interesting to note that the midpoint of this histogram (220 MDa) corresponds closely to a mass of protein forming a 100nm diameter sphere with a density corresponding to a typical protein crystal.