3.1. Protein composition of the three-dimensional crystals
The phycobiliproteins of S. elongatus consist of a and b subunits. For C-PC, the amino acid sequences have been derived from gene sequencing (Swiss-Prot database primary accession numbers P50032 and P50033) and shown to have molecular masses of 17443 Da and 18186 Da respectively. In this study, the SDS-PAGE profile of the sample used for the crystallization trials indicated both a/b C-PC and A-PC to be present in the ~17 to 23 kDa apparent molecular weight range. This protein band profile is shown in Fig. 1A, where lane 1 is similar to that described previously (Ducret, Muller et al., 1998). Several trials containing 3D crystals, such as those shown in Fig. 1B, were pooled and the crystals separated from the supernatant by a brief centrifugation step. SDS-PAGE revealed that the crystals were of C-phycocyanin (Fig. 1A, lane 2) and the supernatant contained non-crystalline allophycocyanin (Fig. 1A, lane 3). No evidence for any linker proteins was found.

3.2. Crystallization
The optimal crystallization conditions were found by a novel method of separating the nucleation and growth phases of crystallization (based on Saridakis and Chayen, 2000). Screening around conditions that initially gave small, poor-quality crystals was performed in order to establish a working phase diagram for the crystallization of the protein. Dilutions across the super-solubility curve into the metastable zone of the phase diagram were carried out by transferring the cover-slips holding the hanging drops, previously incubated with conditions normally giving many small crystals, over reservoirs with concentrations that normally yielded clear drops. High quality crystals measured at least 250 x 250 x 150 µm (Fig. 1B) and were carried forward for X-ray diffraction studies.

3.3. Crystallographic results
Diffraction patterns from C-PC crystals (e.g. Fig. 2A) identified the space group as R32 (No. 155) with unit-cell dimensions of a = b = 187.99 Å and c = 60.54 Å. This c-cell edge was very close to that of F. diplosiphon and C. caldarium, suggesting similar packing. Assuming a molecular weight of 35.6 kDa per / C-PC heterodimer, and one heterodimer per asymmetric unit, then Vm = 2.89 Å3/Da, implying a solvent content occupying ~ 42% of the total volume. Vm decreased to 2.81 Å3/Da on inclusion of the 3 covalently linked chromophores. Diffraction was observed beyond the 1.4 Å limit (Fig. 2A), with the data being reliable up to 1.45Å. Relevant statistics are given in Table I. Crystallographic data have been deposited with the RCSB structural database (http://www.rcsb.org) under codes 1JBO for the co-ordinates and R1JBOSF for the structure factors.

3.4. Quality of structure
The model was adjusted to match the published sequence (Swiss-Prot database primary accession numbers P50032 and P50033). All the changes were confirmed with electron density in the map calculated at 1.45Å, including the post-translationally modified side chain of -Asn72, which was linked to the chromophore via an extra methylene group. The sequence numbering adopted for F. diplosiphon (1CPC) was retained for ease of comparison, i.e. with two gaps in the -chain (between positions 140 & 143, and 150 & 161) and one in the -chain (72 & 75). Difference density maps were used to locate the ordered solvent molecules, which were included in the refinement. No density could be seen in a continuous volume to suggest the presence of any linker protein. The contents of the asymmetric unit are detailed in Fig. 2B. The Ramachandran plot (not shown) indicates that residue Thr77 of chain B falls outside the favored region of the chart as discussed previously (Schirmer et al., 1987). An inspection of the model and electron density shows good match and high quality (see Fig.3). There is a close contact, 3.06Å, between the main-chain amide N and the 3-fold symmetry generated -84 chromophore. This appears to be a common feature in all C-phycocyanins, involved in locking a trimer rigidly, and possibly playing a part in the fine-tuning of the absorption and charge distribution properties of the protein.

3.5. Chromophores
The electron density around the three chromophores is shown in Fig. 3. All are covalently attached to the protein via cysteines -84, -84 and -155, denoted as CYC1, CYC2 and CYC3 respectively (see also Fig. 2B). In the case of CYC2 an extra methylene allows a linkage with -Asn72. The positions of -84 and -84 are reminiscent of the heme binding sites of myoglobin, with the thio-ether bonds having the R stereo-isomer conformation. The -155 binding site is located in a short insertion loop not found in the -subunit. Its thio-ether bond has the S stereo-isomer conformation. All 3 tetrapyrroles display Z configuration on the D ring. These features are consistent with those found for the 1.66 Å C-phycocyanin of F. diplosiphon (1CPC) and the 1.65 Å C-phycocyanin of C. caldarium structure (PDB code 1PHN). However, a least squares comparison between the chromophores reveals differences within the C-phycocyanin structures (see Fig. 3). The CYC1 chromophore is essentially identical in all 3 organisms (see Fig. 3A/D/G), but this is not true for CYC2 or CYC3. Although ring D of the CYC2 chromophore has a similar position in S. elongatus and F. diplosiphon, it differs in C. caldarium. In the latter, ring D is displaced more into the cavity of the protein as compared with S. elongatus and F. diplosiphon. The displacement is in the region of 1.9 Å and does not involve a change in its plane (see Fig. 3B/E/H). This seems to result from a partial unwinding of the -helix from the 105-115 region in C. caldarium (Stec et al., 1999). In contrast, ring D of CYC3 of S. elongatus is the same as that of C. caldarium and differs considerably from F. diplosiphon (see Fig. 3C/F/I). The difference involves a rotation such that the two dihedral angles between the C and D rings change by ~75 and 20 degrees. This structural difference is likely to reflect the different packing arrangement at the interface between the trimers, due to a lack of conservation of amino acid sequences (Adir et al., 2001).

3.6. Oligomerization and distances between chromophores
In the / heterodimer, the separation and geometry of the three chromophores allows only weak excitonic coupling. However, in the phycobilisome and also in the crystal, the / subunits oligomerize to form a hexamer consisting of two ab3 trimers (see Fig.4). When 3 monomers are assembled into an ab3 trimer the local environments around CYC1 and CYC2 chromophores change considerably. In the monomer, the distance between these two chromophores is ~50 Å based on the centers of gravity of the conjugated portions of the chromophores. In the case of the trimer, the distance between the adjacent CYC1/CYC2 reduces to 21.0 Å, facilitating a stronger excitonic interaction between them (see Table 2). For inter-chromophore distances within the hexamer, energy transfer is also favored between these chromophores to such an extent that we have calculated one of the highest transfer rates observed for such a protein, 236.4 ns-1. Furthermore, in the hexamer the CYC3 chromophores are close enough to permit efficient coupling, although this is less favorable compared to those within each trimer.