(a) The operon structure for β-carboxysome genes in the cyanobacterium Synechocystis PCC 6803. The facets of the carboxysome shell (as shown in c) are believed to be constructed from hexagonal assemblies of the BMC shell protein paralogs, CcmK1, CcmK2, CcmK3, CcmK4, and CcmO. The pentamer vertices are encoded by the gene ccmL. The genes rbcL and rbcS encode the large and small subunits, respectively, of the enzyme RuBisCO, which is localized to the carboxysome interior. Carbonic anhydrase, encoded by the ccaA gene, is also located in the carboxysome. rbcX encodes a RuBisCO chaperone. ccmM and ccmN encode additional, non-BMC-type proteins believed to help organize other carboxysome proteins. (b) Surface rendering of the X-ray crystal structures of CcmK1, CcmK2, and CcmK4 hexamers (respective PDB ID codes 3bn4, 3cim, 2a18). Hexamers are the native oligomeric form for all single BMC domain proteins that have been investigated to date. (c) A schematic, idealized depiction of an icosahedral shell of the Synechocystis carboxysome. The shell is constructed from tightly packed BMC hexamers forming the facets and CcmL pentamers at the vertices. The diameters of carboxysomes range from 800 to 1400 Å and typically deviate from the perfectly icosahedral symmetry depicted here. (d) Image of intact Halothiobacillus neapolitanus carboxysomes recorded by low-dose electron cryomicroscopy. Bar = 100 nm.

(a) Projection maps from 2D crystals of the three CcmK proteins shown projected onto the molecular packings visualized in 3D crystals. In the case of CcmK4, hexamers in 3D crystals packed into uniformly ordered strips (illustrated), but not uniformly oriented layers. (b) 3D density map (tilted by 15° to enhance depth) showing the hexamer packing of CcmK1 in 2D lipid monolayer crystals (gray surface), which recapitulates the packing in the ab lattice plane in 3D crystals (blue ribbon) (pdb id. 3dn9). Center-to-center hexamer spacing is ∼68 Å for the EM map and ∼70 Å for the crystal structure. For comparison, the X-ray structure was manually docked into the EM density obtained from 3D reconstruction. The green line outlines the 2D unit cell with standard symbols at the axes of rotational symmetry.

(a) Schematic depiction of 2D lipid monolayer crystallization. Carboxysome BMC shell protein hexamers were localized at the lipid-aqueous interface of a mixed lipid layer with 4:1 (wt:wt) l-α-phosphatidylcholine (light spheres) plus DOGS-NTA-Ni²⁺ (dark spheres) by means of polyhistidine tag chelation via Ni²⁺ bound within the polar lipid layer. Resulting 2D crystals were transferred to an EM grid and stained for image diffraction analysis. (b) Negatively stained TEM image of CcmK1 2D crystals. Scale bar = 100 nm. (c) The underlying order is observed when the image is Fourier filtered, and corrections are made for lattice distortions. (d) The Fourier transform of the negatively stained image displays hexagonal symmetry. The arrow identifies the (2,2) reflection at 17 Å resolution. Images were contrast stretched to emphasize salient features.