The initial source of Earth’s organic carbon is atmospheric or dissolved CO2, which is fixed by enzymes found in green plants, algae and autotrophic bacteria. The carbon is then spread through the food chain to all other living organisms. The most important enzyme responsible for fixing carbon is ribulose bisphosphate carboxylase/oxygenase (RuBisCO), which catalyzes the first step in carbon fixation via the Calvin-Benson-Bassham cycle-the covalent attachment of CO2 to ribulose-1,5-bisphosphate and its subsequent cleavage into two molecules of 3-phosphoglycerate. Based on the immense biomass of photosynthetic and chemoautotrophic organisms on Earth, RuBisCO is estimated to be the most abundant protein known.
All cyanobacteria and some chemoautotrophic bacteria enhance their CO2
fixation by sequestering RuBisCO into polyhedral bodies called carboxysomes 1;2;3;4
. Halothiobacillus neapolitanus
carboxysomes are delimited by a proteinaceous shell and are filled with RuBisCO 5; 6; 7
. In previous electron microscopic studies, carboxysomes appeared hexagonal with a granular interior, a diameter of approximately 120 nm and a shell thickness of between 3 to 4 nm 8; 9
. Although the enhancement of carbon dioxide fixation by the carboxysome has been firmly established, the exact mechanism has not yet been elucidated.
carboxysomes consist of ten polypeptides 6; 7
. Of these, two polypeptides represent the small and large subunits of RuBisCO and six are known to make up the shell. One of these shell peptides has been identified as a unique carbonic anhydrase 10
. It is postulated that this enzyme may function to enhance carbon dioxide fixation by the carboxysome. The functions and locations of the two remaining polypeptides are not yet known. Carboxysomes are rapidly formed via de novo m-RNA and protein synthesis under low CO2
conditions 11; 12
. Their genes are operon-linked12
, so it is likely the proportions of their constituents are regulated.
Although much has been published on the occurrence, physiology, biochemistry and genetics of these microcompartments/organelles 5; 6; 7; 10
, only two major reports have analyzed the structure or symmetry of purified carboxysomes 9; 13
. Peters concluded that the carboxysomes of Nitrobacter agilis
obey icosahedral symmetry, based on negative stain projection images and heavy-metal shadowing of critical-point-dried carboxysomes 13
. Holthuijzen and co-workers, on the other hand, reported the shape of carboxysomes of H. neapolitanus
to be dodecahedral 9
There are reports that the RuBisCO inside the shell is paracrystalline and fills the interior 8
. Others have reported that the RuBisCO molecules are arranged in one or a few layers under the surface of the shell 9
. However, these conclusions were drawn from observations of either whole bacteria or isolated carboxysome preparations that are plastic-embedded, thin-sectioned, and/or negative stained. Using Hilbert Differential Contrast electron microscopy, paracrystalline arrays were observed in intact frozen cells 14
, but it is not possible in these 2D projections to tell whether the carboxysomes are filled, or have only one or a few layers of RuBisCO under the shell. Orus et al. 11
reported that the state of packaging of RuBisCO inside carboxysomes can vary with environmental conditions.
It is these aspects of the carboxysome structure that we intend to clarify using cryo-electron tomography of suspensions of carboxysomes isolated from the chemoautotrophic proteobacterium H. neapolitanus
and subsequent 3D alignment and averaging of particles computationally isolated from the tomogram. Averaging particles is necessary because of the low signal-to-noise ratio in any single instance of a particle from the tomogram, and because of the distortion introduced by the “missing wedge” of data in Fourier space. However, the search for the relative orientation of the 3D particle volumes is not a trivial step unless a 3D model of the particle with isotropic resolution already exists, as has previously been done15; 16
. In our study, an orientation search method that does not depend on such a prior model is used for aligning single particles from a tomogram with missing wedge data.