Newly determined crystal structures of the photosynthetic RC (reaction centre) from two substrains of the non-sulfur purple bacterium Blastochloris viridis strain DSM 133, together with analysis of their gene sequences, has revealed intraspecies evolutionary changes over a period of 14 years. Over 100 point mutations were identified between these two substrains in the four genes encoding the protein subunits of the RC, of which approximately one-fifth resulted in a total of 16 amino acid changes. The most interesting difference was in the M subunit where the change from a leucine residue to glycine in the carotenoid-binding pocket allowed NS5 (1,2-dihydroneurosporene) to adopt a more sterically favoured conformation, similar to the carotenoid conformation found in other related RCs. The results of the present study, together with a high rate of mutations in laboratory bacterial cultures described recently, suggest that bacteria evolve faster than has been generally recognized. The possibility that amino acid changes occur within protein sequences, without exhibiting any immediately observable phenotype, should be taken into account in studies that involve long-term continuous growth of pure bacterial cultures. The Blc. viridis RC is often studied with sophisticated biophysical techniques and changes such as those described here may well affect their outcome. In other words, there is a danger that laboratory-to-laboratory variation could well be due to different groups not realising that they are actually working with slightly different proteins. A way around this problem is suggested.

INTRODUCTION

Photosynthetic RCs (reaction centres) are integral membrane pigment–protein complexes that catalyse the primary electron-transfer reactions. The RC from the purple non-sulfur photosynthetic bacterium Blastochloris viridis (previously classified as Rhodopseudomonas viridis [1]) was the first integral membrane protein complex to have its structure determined by X-ray crystallography [26]. The gene and amino acid sequence of each polypeptide subunit was determined shortly after publication of the first crystal structure [79]. At the time of writing, the highest-resolution Blc. viridis RC structures [at 1.86 and 1.95 Å (1 Å=0.1 nm)] have been obtained from crystals grown by the LSP (lipidic sponge phase) technique (PDB codes 2WJN and 2WJM respectively) [10]. The Blc. viridis RC is composed of four polypeptide subunits, L, M, H and C (a tightly bound tetra-haem cytochrome c), and 14 cofactors as shown in Figure 1. Details of the electron-transfer reactions performed by the cofactors in purple bacterial RCs can be found in many excellent publications, for example see [1113].

Arrangement of cofactors in the Blc. viridis RC

Figure 1
Arrangement of cofactors in the Blc. viridis RC

The ‘active branch’, designated by subscript A, and the ‘inactive branch’, designated by subscript B that are predominantly associated with the L and M polypeptides respectively. The two bacteriochlorophyll b molecules that form the ‘special pair’ are designated PA and PB. The other cofactors involved are two ‘accessory’ bacteriochlorophyll b molecules (BChlA and BChlB), bacteriopheophytin b molecules (BPheA and BPheB), one non-haem iron (Fe2+), a single carotenoid molecule (Car) and two quinones (menaquinone QA and ubiquinone QB). The haem groups are embedded in the cytochrome c subunit. The phytyl tails of BChls, BPhes and the quinone isoprenoid tails have been omitted for clarity.

Figure 1
Arrangement of cofactors in the Blc. viridis RC

The ‘active branch’, designated by subscript A, and the ‘inactive branch’, designated by subscript B that are predominantly associated with the L and M polypeptides respectively. The two bacteriochlorophyll b molecules that form the ‘special pair’ are designated PA and PB. The other cofactors involved are two ‘accessory’ bacteriochlorophyll b molecules (BChlA and BChlB), bacteriopheophytin b molecules (BPheA and BPheB), one non-haem iron (Fe2+), a single carotenoid molecule (Car) and two quinones (menaquinone QA and ubiquinone QB). The haem groups are embedded in the cytochrome c subunit. The phytyl tails of BChls, BPhes and the quinone isoprenoid tails have been omitted for clarity.

A single molecule of non-covalently-bound carotenoid is located next to the monomeric accessory BChlB (bacteriochlorophyll B), on the ‘inactive’ branch B. It has an essential role in the rapid quenching of the triplet state of the RC primary donor to prevent cell damage [1417]. The two almost identical major carotenoids found in membranes and in RCs from Blc. viridis are NS5 (1,2-dihydroneurosporene; 50–70%) and LY6 (1,2-dihydrolycopene; 30–40%) [18,19], and are shown in Figure 2. In the first RC crystal structure, the electron density for carotenoid was suggestive of a long curved molecule into which the central portion of the cis-isomer of NS5 could be successfully fitted [6]. Both ends of the carotenoid (nine carbon atoms at the 1,2-dihydro-end and five carbon atoms at the other end) were also fitted, but left with the atomic occupancies equal to 0.0 (PDB code 1PRC) [6]. This suggests a non-satisfactory fit and/or excessively high temperature factors for these atoms in that refinement. Subsequent RC structure determinations (PDB codes 1DXR [20] and 2JBL [21]) reported a similar situation. In all of these structures a significant ‘bend’ of the NS5 molecules was modelled at C-8 at the end of the fully conjugated central fragment (this atom is labelled C9 in these crystal structures). A similar bend was also modelled in more recent structures of the wild-type RC from Blc. viridis from different laboratories (PDB codes 1VRN [22], 2I5N [23], 2WJN [10] and 2WJM [10]), although in these cases all of the carotenoid atoms were reported to have an occupancy of 1.0 and the conformations of the saturated tail fragment were different from each other and from the earlier ones (see Figure 3). As described below, this ‘bend’ in the structure of the carotenoid is not seen in the RCs from other species of purple bacteria.

Chemical structures of the major carotenoids in RCs

Figure 2
Chemical structures of the major carotenoids in RCs

Structures are from Blc. viridis (1,2-dihydroneurosporene/NS5 and 1,2-dihydrolycopene/LY6), Tch. tepidum (spirilloxanthin/CRT) and wild-type strain 2.4.1 Rba. sphaeroides (spheroidene/SPO).

Figure 2
Chemical structures of the major carotenoids in RCs

Structures are from Blc. viridis (1,2-dihydroneurosporene/NS5 and 1,2-dihydrolycopene/LY6), Tch. tepidum (spirilloxanthin/CRT) and wild-type strain 2.4.1 Rba. sphaeroides (spheroidene/SPO).

Carotenoid electron density for Blc. viridis substrain-08 RC in disagreement with published models

Figure 3
Carotenoid electron density for Blc. viridis substrain-08 RC in disagreement with published models

(a) The 2FoFc electron density at 1σ level (in blue) and the FoFc electron density at 3σ level (positive density in green and negative in red) at the carotenoid-binding site of RC from Blc. viridis calculated in one of the first refinement cycles after molecular replacement for the 1.95 Å substrain-08 data. The preliminary RC model for substrain-08 is shown in yellow [with a shortened NS5 (labelled NS5* to avoid clashing with LeuM70)]. The two published models (PDB codes 1DXR [20] and 2I5N [23]) are shown with their carotenoids labelled NS5-1DXR (in cyan) and NS5-2I5N (in magenta) respectively. (b) Superposition of NS5 modelled in the Blc. viridis RC crystal structures (1DXR [20] in cyan, 2JBL [21] in red, 1VRN [22] in blue and 2I5N [23] in magenta) with molecules of CRT (in yellow) as in RC from Tch. tepidum (PDB code 1EYS [25]) and SPO (in green) as in wild-type RC strain 2.4.1 from Rba. sphaeroides (PDB code 3I4D). This overlap was obtained by superposition of M-subunit protein residues surrounding the carotenoid-binding site. NS5 molecules in the most recent crystal structures of Blc. viridis RC [10] (PDB codes 2WJN and 2WJM, not shown) were modelled similarly to the structure PDB code 1DXR [20].

Figure 3
Carotenoid electron density for Blc. viridis substrain-08 RC in disagreement with published models

(a) The 2FoFc electron density at 1σ level (in blue) and the FoFc electron density at 3σ level (positive density in green and negative in red) at the carotenoid-binding site of RC from Blc. viridis calculated in one of the first refinement cycles after molecular replacement for the 1.95 Å substrain-08 data. The preliminary RC model for substrain-08 is shown in yellow [with a shortened NS5 (labelled NS5* to avoid clashing with LeuM70)]. The two published models (PDB codes 1DXR [20] and 2I5N [23]) are shown with their carotenoids labelled NS5-1DXR (in cyan) and NS5-2I5N (in magenta) respectively. (b) Superposition of NS5 modelled in the Blc. viridis RC crystal structures (1DXR [20] in cyan, 2JBL [21] in red, 1VRN [22] in blue and 2I5N [23] in magenta) with molecules of CRT (in yellow) as in RC from Tch. tepidum (PDB code 1EYS [25]) and SPO (in green) as in wild-type RC strain 2.4.1 from Rba. sphaeroides (PDB code 3I4D). This overlap was obtained by superposition of M-subunit protein residues surrounding the carotenoid-binding site. NS5 molecules in the most recent crystal structures of Blc. viridis RC [10] (PDB codes 2WJN and 2WJM, not shown) were modelled similarly to the structure PDB code 1DXR [20].

This issue was brought to our attention during a study of the interactions of lipids and detergent molecules bound to the surface of membrane proteins, using RC crystals from Rhodobacter sphaeroides [24] and Blc. viridis as model membrane proteins. The structure of the RC from Blc. viridis obtained during that work revealed electron density for the carotenoid that was not in agreement with the published NS5 models (see Figure 3). The electron density of the carotenoid in this new structure extended roughly in the same plane as the central portion of the molecule described in the previous structures, but did not show the bend discussed above (Figure 3a). If carotenoid was modelled within this density its saturated tail fragment would clash with the side chain of LeuM70, of which side-chain atoms Cγ, Cδ1 and Cδ2 were located within the ‘carotenoid’ density. At the same time a negative FoFc density appeared in the position of the Cβ atom of leucine. These features suggested that residue M70 in our Blc. viridis RC could not be a leucine. It was clear that in the previously determined crystal structures of the Blc. viridis RC [6,10,2023], where the M70 residue was a leucine, the carotenoid molecule is bent (Figure 3b) to avoid clashing with the protruding side chain of this residue, and the bent tail fragment of the carotenoid is strongly disordered.

The equivalent residue in the Rba. sphaeroides RC (M71) is a glycine. In the RC from Thermochromatium tepidum, for which a crystal structure has also been determined (PDB code 1EYS) [25], this residue is also a glycine. In addition, when the M polypeptide sequences from other purple bacterial RCs are aligned (Figure 4), GlyM70 is conserved in most of the cases with or without the bound cytochrome c subunit. Strikingly, the conformation of the carotenoid RTC (spirilloxanthin) in the Tch. tepidum RC and conformations of three carotenoids [SPO (spheroidene), 3,4-dihydrospheroidene and spheroidenone) in Rba. sphaeroides RCs [26] are all similar and consistent with the features of carotenoid electron density for our new Blc. viridis RC structure. Finally, a thorough examination of the electron density maps for all RC protein chains has revealed potential disagreements in several other sequence positions, with four of the most apparent cases illustrated in Figure 5. All these results indicated that the protein sequences in our newly determined structure of the Blc. viridis RC are not identical with those originally published [79].

Multiple sequence alignment of a fragment from the M-subunit aligned by the ClustalW2

Figure 4
Multiple sequence alignment of a fragment from the M-subunit aligned by the ClustalW2

The sequence differences for the Blc. viridis substrains-94 and -08 (top two rows) are highlighted in yellow and green respectively. The red numbering of highlighted residues for Blc. viridis refers to the RC crystal structure in which the first residue, a methionine, is not present (owing to post-translational modification), however, the numbering shown in the right-hand column is based on full sequences. Amino acid residues in contact with the carotenoid are marked below with a letter ‘c’. The PheM89 residue in a π sandwich-binding site for the head group of the ubiquinone-like molecule (as shown in Figure 10) is highlighted in purple. DNA sequences with the following accession numbers were obtained from GenBank®: Rhodospirillum (Rdv.) centenum CP000613, Rhodopseudomonas (Rps.) palustris NC_005296, Ectothiorhodospira (Ect.) shaposhnikovii AF018955, Rhodovulum (Rdv.) sulfidophilum AB020784, Allochromatium (Alc.) phaeobacterium AM944091, Rps. lichen AB241419, Erythrobacter (Erb.) sp. strain NAP1 AAMW01000003, Citromicrobium (Cmi.) sp. strain CV44 DQ080991, Roseobacter (Rsb.) sp. strain BS110 EU009369, Rhodocyclus (Rcy.) tenuis D50651, Blastochloris (Blc.) viridis (strain DSM 133 substrain-94, the present study) HQ009849, Blc. viridis (strain DSM 133, substrain-08, the present study) FJ483785, Rsb. denitrificans NC_008209, Rsp. molischianum D50654, Rsp. rubrum NC_007643, Rubrivivax (Rvi.) gelatinosus AB034704, Rhodobacter (Rba.) sphaeroides NC_007493, Rba. capsulatus Z11165, Alc. vinosum AB011811 and Thermochromatium (Tch.) tepidum, D85518.

Figure 4
Multiple sequence alignment of a fragment from the M-subunit aligned by the ClustalW2

The sequence differences for the Blc. viridis substrains-94 and -08 (top two rows) are highlighted in yellow and green respectively. The red numbering of highlighted residues for Blc. viridis refers to the RC crystal structure in which the first residue, a methionine, is not present (owing to post-translational modification), however, the numbering shown in the right-hand column is based on full sequences. Amino acid residues in contact with the carotenoid are marked below with a letter ‘c’. The PheM89 residue in a π sandwich-binding site for the head group of the ubiquinone-like molecule (as shown in Figure 10) is highlighted in purple. DNA sequences with the following accession numbers were obtained from GenBank®: Rhodospirillum (Rdv.) centenum CP000613, Rhodopseudomonas (Rps.) palustris NC_005296, Ectothiorhodospira (Ect.) shaposhnikovii AF018955, Rhodovulum (Rdv.) sulfidophilum AB020784, Allochromatium (Alc.) phaeobacterium AM944091, Rps. lichen AB241419, Erythrobacter (Erb.) sp. strain NAP1 AAMW01000003, Citromicrobium (Cmi.) sp. strain CV44 DQ080991, Roseobacter (Rsb.) sp. strain BS110 EU009369, Rhodocyclus (Rcy.) tenuis D50651, Blastochloris (Blc.) viridis (strain DSM 133 substrain-94, the present study) HQ009849, Blc. viridis (strain DSM 133, substrain-08, the present study) FJ483785, Rsb. denitrificans NC_008209, Rsp. molischianum D50654, Rsp. rubrum NC_007643, Rubrivivax (Rvi.) gelatinosus AB034704, Rhodobacter (Rba.) sphaeroides NC_007493, Rba. capsulatus Z11165, Alc. vinosum AB011811 and Thermochromatium (Tch.) tepidum, D85518.

Four examples of obvious incompatibilities between the published amino acid sequences and the electron density calculated for the substrain-08 Blc. viridis RC data

Figure 5
Four examples of obvious incompatibilities between the published amino acid sequences and the electron density calculated for the substrain-08 Blc. viridis RC data

The colour scheme for electron density maps is as in Figure 3; ThrM164 (a), IleC77 (b), LeuC277 (c) and AlaC43 (d).

Figure 5
Four examples of obvious incompatibilities between the published amino acid sequences and the electron density calculated for the substrain-08 Blc. viridis RC data

The colour scheme for electron density maps is as in Figure 3; ThrM164 (a), IleC77 (b), LeuC277 (c) and AlaC43 (d).

There could be three reasons for these differences between our present RC structure and those described previously for Blc. viridis. The first is a mistake in the original sequencing of the RC genes, which is unlikely and is not supported by electron density maps for the published structures that provide structure factors [10,2123]. The second is that we are working with a different strain of Blc. viridis. The structures and sequences published previously [210,2023] were all for the Blc. viridis strain DSM 133 (also designated as A.T.C.C. 19567; even though the Blc. viridis strains DSM 133 and A.T.C.C. 19567 from the two different culture collections are thought to be identical, the isolates from these two culture collections do show a few sequence differences in their 16S rRNA genes. We discuss this in the Results and discussion section and show the alignment of these two 16S rRNA sequences in Supplementary Figure S1 at http://www.BiochemJ.org/bj/442/bj4420027add.htm). The third is that our culture of Blc. viridis has accumulated a series of mutations during its cultivation in Glasgow. To clarify this issue it was decided to determine the gene sequences of the RC polypeptides L, M, H and C, and for the 16S rRNA gene, for the Blc. viridis working culture (subsequently called substrain-08), from which the RCs used in this analysis were purified. We went back to a glycerol stock culture that had been stored at −80°C since 1994 (subsequently called substrain-94) and determined its RC gene sequences and the 16S rRNA gene sequence. These two sets of sequences were then compared with each other and with the published ones. The crystal structure for RC of substrain-94 has also been determined. The results described below show that our working culture of Blc. viridis (substrain-08) is of the same strain as that used by other researchers [210,2023], but has accumulated a set of point mutations over the time it has been continuously cultured. There has been genetic drift over time and as a result the strain has evolved in the laboratory.

EXPERIMENTAL

DNA amplification and sequencing

Total DNA from cells of both substrains -08 and -94 of the Blc. viridis strain DSM 133 was isolated by standard phenol/chloroform treatment. Seven genes were chosen for amplification and sequencing: the five genes comprising the puf operon pufBALMC, the gene puhA and a gene for 16S rRNA. Primers for amplifying the genes encoding the photosynthetic proteins were designed according to known gene sequences obtained from GenBank® using the following accession numbers: pufBA, M55261 [27]; pufLM, X03915 [8]; pufC, X05768 [9]; and puhA, X02659 [7]. In order to obtain a full sequence of the puf operon, four primer pairs were employed to amplify four overlapping DNA fragments; ‘pufBAL’, ‘pufLM’, ‘pufMC’ and ‘pufC’ (primer pairs: pufBvirF and pufL504R, pufLvirF and pufMvirR, pufM478F and pufCvirR, and pufC138F and pufCvirR). The puhA gene was amplified as a single DNA fragment (primers puhAvirF and puhAvirR). For amplification of the 16S rRNA genes, a standard primer set of 27F and 1525R primers was used [28] and sequence obtained by using three internal primers, 440F, 608R and 970F. The sequences of all of the primers used are shown in the Supplementary Table S1 (at http://www.BiochemJ.org/bj/442/bj4420027add.htm). Phusion™ DNA polymerase (Finnzymes, Thermo Scientific) was used for all amplifications with the PCR protocol optimized for each fragment, and resulting DNA fragments were purified with QIAquick PCR Purification Kit (Qiagen). For sequencing, the Sanger dideoxy method was used on a MegaBACE 1000 sequencer (GE Healthcare). Three DNA fragments (‘pufBAL’, ‘pufLM’ and ‘pufMC’) were sequenced directly using both external and internal primers (pufBvirF, pufL104R, pufLvirF, pufL504R, pufL429F, pufM47F and pufC541R). DNA fragments ‘pufC’ and ‘puhA’ were cloned into the pGEM T-Easy vector, expressed in Escherichia coli strain JM109, isolated (QIAprep Spin Miniprep Kit, Qiagen) and sequenced with vector primers T7 and SP3. The DNA sequences obtained were then translated into the amino acid sequences. ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and BioEdit [29] software were used for all sequence alignments.

RC purification and crystallization

RCs were prepared from membranes of both substrains, -08 and -94, essentially using the method detailed previously [30] with the addition of a final gel-filtration step (Superdex 200) with 20 mM Tris/HCl and 0.1% LDAO (N,N-dimethyldodecylamine-N-oxide) (pH 8.0) to remove any free pigments and improve the quality of the final preparation. The final RC pool used for crystallization had an absorbance value of 5 at 830 nm in 20 mM sodium phosphate buffer (pH 6.0) with an optical purity ratio of A280/A830<2.4. Crystals of RCs were obtained by vapour diffusion from 20 μl sitting drops in Cryschem™ 24-well plates using ammonium sulfate (1.5 M) as the precipitant and heptanetriol (3%) as the amphiphile. The reservoir contained 2.2–2.4 M ammonium sulfate (pH 6.0).

X-ray diffraction studies

X-ray diffraction data was obtained for crystals of the RC from substrain-08 and the analysis led to an understanding that the observed electron density maps were in some cases not compatible with the published protein sequences [79]. The sequences of the RC proteins from substrain-08 were then determined and applied in the structure refinement. At this point the same analysis was then performed for the crystals of RC from substrain-94.

Diffraction data for RC crystals of substrain-08 were collected at stations ID14-1 (2.3 Å data) and BM14 (1.95 Å data) of the ESRF (European Synchrotron Radiation Facility) and for substrain-94 crystal at station I02 of the Diamond Light Source, Oxfordshire. All crystals were gradually cryoprotected against freezing in mother liquor solutions containing increasing amounts of glycerol from 5 to 35%. Crystals of both substrains belong to the space group P43212 with one RC complex in the asymmetric unit. Table 1 presents the data collection, processing and refinement statistics for the highest resolution data sets for each substrain used in the present study. The data were indexed, integrated and scaled using the program d*TREK [31]. Structure solution was performed by molecular replacement with programs AMoRe [32] and Phaser [33] using the RC structure PDB code 1DXR [20] as a search model to phase the 2.3 Å substrain-08 data (results not shown), then using the partially refined 2.3 Å model to phase the 1.95 Å substrain-08 data, and finally using the substrain-08 model to phase the 1.92 Å substrain-94 data. Further computations were carried out using programs from the CCP4 package [34]. Refinement by the maximum likelihood method was performed by the program REFMAC [35]. The TLS (Translation/Libration/Screw) thermal mode was used, which allows a separation of the overall lattice vibrations before the standard restrained refinement of atomic co-ordinates and the individual atom isotropic B-factors [36]. The whole atomic content of the asymmetric unit was treated as a single TLS group and that led to significant lowering of the R factors and improvement of RC surface features in electron density maps. Manual rebuilding of the RC protein chains and fitting of the carotenoid molecules, NS5 or LY6, the DAG (diacyglycerol) molecule covalently bound to cytochrome N-terminal cysteine residue, and of the detergent (LDAO) and solvent molecules (glycerol, heptanetriol, sulfate ions and water) into the electron density was carried out using the program COOT [37]. In the substrain-94 structure three closely located pairs of LDAO molecules were replaced by three more DAG molecules. Occupancies of the fitted molecules (except for water) were adjusted manually to approximately equalize their atomic B-factors to the overall Wilson plot B-factor and/or to the B-factors of the adjacent protein atoms. The occupancies were not refined. Substrain-94 RC electron density for CysM160 suggests that this residue has been oxidized probably due to X-ray irradiation [38], and so it was modelled as a cysteine sulfenic acid. A similar change of electron density for residue CysM160 was observed for the Blc. viridis RC structure PDB code 2WJN [10]. This structure was refined using data obtained when the crystal was intentionally exposed with a 16-times-higher dose of X-rays in comparison with the lower-dose structure PDB code 2WJM [10]. It should be noted, however, that no other cysteine residue in these structures has been affected this way.

Table 1
Data collection and refinement statistics for the Blc. viridis RCs of the two substrains DSM 133-94 and DSM 133-08

CCD, charge-couple device; RMSD, root mean square deviations.

Substrain Substrain-08 Substrain-94 
PDB code 3T6D 3T6E 
Data collection   
 Space group P4321P4321
 Cell dimensions   
  a=b, c (Å) 220.44, 113.10 221.58, 113.42 
  α=β=γ (°) 90 90 
 Solvent contents (%) ~70 ~70 
 Beamline BM14 at ESRF I02 at DLS 
 CCD detector Marmosaic 225 ADSC Q315 
 Wavelength (Å) 0.97108 0.9194 
 Resolution range (Å) 48.34–1.95 43.46–1.92 
 Highest resolution shell (Å) 2.02–1.95 1.99–1.92 
 Number of unique reflections* 193323 (19553) 212134 (20895) 
 Redundancy* 4.6 (4.8) 19.4 (18.9) 
 Completeness (%)* 96.2 (98.6) 99.5 (99.1) 
 Mean I/σ(I)* 7.1 (1.9) 9.4 (1.6) 
Rmerge† (%)* 8.6 (59.2) 11.5 (82.3) 
Refinement   
 Refinement resolution (Å) 48.34–1.95 43.46–1.92 
Rwork/Rfree‡ (%) 18.17/21.63 15.48/17.80 
 Number of non-hydrogen atoms refined 12112 12022 
 Co-ordinate error§ (Å) 0.109 0.085 
 RMSD for bond lengths (Å) 0.019 0.019 
 RMSD for bond angles (°) 2.24 2.23 
 Mean atomic/Wilson plot B-factor (Å243.73/33.9 46.9/36.0 
 Ramachandran plot features¶ (%) 97.6/2.3/0.1 98.0/1.9/0.1 
Substrain Substrain-08 Substrain-94 
PDB code 3T6D 3T6E 
Data collection   
 Space group P4321P4321
 Cell dimensions   
  a=b, c (Å) 220.44, 113.10 221.58, 113.42 
  α=β=γ (°) 90 90 
 Solvent contents (%) ~70 ~70 
 Beamline BM14 at ESRF I02 at DLS 
 CCD detector Marmosaic 225 ADSC Q315 
 Wavelength (Å) 0.97108 0.9194 
 Resolution range (Å) 48.34–1.95 43.46–1.92 
 Highest resolution shell (Å) 2.02–1.95 1.99–1.92 
 Number of unique reflections* 193323 (19553) 212134 (20895) 
 Redundancy* 4.6 (4.8) 19.4 (18.9) 
 Completeness (%)* 96.2 (98.6) 99.5 (99.1) 
 Mean I/σ(I)* 7.1 (1.9) 9.4 (1.6) 
Rmerge† (%)* 8.6 (59.2) 11.5 (82.3) 
Refinement   
 Refinement resolution (Å) 48.34–1.95 43.46–1.92 
Rwork/Rfree‡ (%) 18.17/21.63 15.48/17.80 
 Number of non-hydrogen atoms refined 12112 12022 
 Co-ordinate error§ (Å) 0.109 0.085 
 RMSD for bond lengths (Å) 0.019 0.019 
 RMSD for bond angles (°) 2.24 2.23 
 Mean atomic/Wilson plot B-factor (Å243.73/33.9 46.9/36.0 
 Ramachandran plot features¶ (%) 97.6/2.3/0.1 98.0/1.9/0.1 
*

Values in parentheses refer to the highest resolution shell.

Rmerge=

Rwork and Rfree=Rwork was calculated for all data except for 5% that was used for the Rfree calculations.

§

Estimated standard uncertainty calculated by the Cruickshank method [40].

Percentages of residues in favoured/allowed/outlier regions calculated by program RAMPAGE [39].

A weak electron density for the loop H45–54 located in the region of a crystal lattice contact between adjacent RC complexes does not follow the conformations presented in structures PDB codes 1PRC [6] 1DXR [20], 2JBL [21] and 1VRN [22]. An alternative modelling of the loop (both substrains show similar density) has been attempted but atoms of the residues H45–54 were left with 0.0 occupancies in the final refinements.

During the refinement, the models of the RCs were monitored for geometrical quality using the program RAMPAGE [39] and the validation features of the program COOT [37]. The Ramachandran plot calculated for the final protein co-ordinates by RAMPAGE [39] places only one residue, AsnM193, in the outlier region. Electron density for this residue does not indicate any problem with its geometry nor refinement. Figures were prepared using COOT [37] and PyMOL (http://www.pymol.org).

RESULTS AND DISCUSSION

Gene sequencing

Our original culture of Blc. viridis strain DSM 133 was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen in the early 1990s. These Blc. viridis cells were laid down as a parent culture in glycerol stocks during 1994 and the culture has been used for experiments in the laboratory since that time employing standard passaging methods. Two cultures of Blc. viridis were used in the present study: the working culture and the 1994 parent culture resuscitated in 2010. In order to check the strain designation of these cultures, a standard sequencing of the 16S rRNA gene was carried out for both. The alignment of the parent and working strain gene with the two other 16S rRNA gene sequences of this bacterium published previously showed very high similarity (99% and higher), which excluded the possibility that we were working with a different strain (alignment of all four genes is shown in Supplementary Figure S1). The variability between our parent and working strain (only three different nucleotides at positions 181, 1273 and 1309) fell well into the intrastrain variability of two previously published 16S rRNA gene sequences from the same strain of Blc. viridis. These genes, isolated from Blc. viridis designated DSM 133 (GenBank® accession number AF084495) [41] and A.T.C.C. 19567 (GenBank® accession number D25314) [1], contained eight different nucleotides when compared with each other; however, they are still considered to be from identical strains. Moreover, the three nucleotides that were different between our working and parent cultures mentioned above were not different between our parent culture and both of the previously published genes. This suggests that these point mutations arose during the 14 years of culturing and can be associated with natural intra-strain evolutionary changes occurring under laboratory cultivation conditions. In order to distinguish between these two cultures for the purpose of the present study we have designated them substrain-08, for the working culture (RC proteins were purified from this culture for crystallization and all genes were sequenced in 2008), and substrain-94, for the parent culture. For consistency, this nomenclature has been used throughout the present paper and for depositing the new sequences into GenBank®.

In order to sequence the genes encoding the RC polypeptides from both cultures, primers were designed based on the previously published gene sequences [79]. The protein subunits L, M, C and H are encoded by the pufL, pufM, pufC and puhA genes respectively. Isolated genomic DNA was used as a template for the amplification of the puhA gene and a part of the puf operon containing the pufL, pufM and pufC genes. The pufA and pufB genes encoding the α- and β-apoproteins of the LH1 (light harvesting 1) complex were also amplified and sequenced to complete the puf operon sequence. The nucleotide sequences obtained were compared with the previously published sequences [79,27] (Supplementary Figure S2 at http://www.BiochemJ.org/bj/442/bj4420027add.htm).

The new sequence data obtained for the genes pufB and pufA from both substrains revealed exactly the same nucleotide sequences and, moreover, were found to be identical with the sequences published earlier [27]. The situation was different in all the other sequenced genes. In the pufL gene sequence of substrain-08, 32 single nucleotide differences were found within codons in comparison with substrain-94, whereas the equivalent substrain-94 sequence was identical with the previously described sequence for the strain DSM 133 [8]. In this case, none of these mutations resulted in an amino acid change in the L subunit (Supplementary Figures S2a and S3 at http://www.BiochemJ.org/bj/442/bj4420027add.htm). For the puhA, pufC and pufM genes the results were more striking. In total 101 nucleotide differences between substrain-94 and substrain-08 were found in these genes, and 24 of them produced changes in 16 amino acids. All of the amino acid changes in RC polypeptides of substrains-94 and -08 are summarized in Table 2. In detail, in the puhA genes 18 nucleotide differences were identified (Supplementary Figure S2b), of which two resulted in amino acid changes. The changed residues, H216 and H256, are not conserved residues when compared with the corresponding sequences from other purple bacteria (Supplementary Figure S4 at http://www.BiochemJ.org/bj/442/bj4420027add.htm). In the pufC genes 45 nucleotide differences were found (Supplementary Figure S2a). Thirteen of them resulted in changes of nine amino acids (Table 2). The change at position C43 converts alanine into proline, which is present at this position in many bacterial species. The change at position C323 from lysine to glutamine is from conserved to not conserved. All of the other changes are in non–conserved positions (Supplementary Figure S5 at http://www.BiochemJ.org/bj/442/bj4420027add.htm). None of these changes involve residues that are in a direct contact with the cytochrome haem groups. In the pufM genes 38 nucleotide differences were identified (Supplementary Figure S2a), of which nine produced changes of five amino acids (Table 2). Residues at positions M29, M63 and M89 are not involved in any protein–cofactor interactions (Supplementary Figure S6 at http://www.BiochemJ.org/bj/442/bj4420027add.htm), however, two other changes, at positions M70 and M74, are different because these residues are part of the binding pocket for the carotenoid. The glycine at position M70 in substrain-08 appears to be critical for a satisfactory fit of the RC carotenoid into the electron density map (see discussion below). As mentioned in the Introduction section, GlyM70 is a highly conserved residue across most purple bacterial species (partial alignment is shown in Figure 4 and complete alignment in Supplementary Figure S6). Considering position M74, purple bacteria can be divided into two groups (also shown in Figure 4) with M74 being leucine or tryptophan respectively. An alanine residue at this position (found in DSM 133 strain and in substrain-94) is not a common residue at this position.

Table 2
All amino acid differences in the Blc. viridis RC polypeptides between the two substrains DSM 133-94 and DSM 133-08

The other mutations in the DNA did not result in changes in amino acid sequences.

 Amino acids 
Gene (protein) Position Substrain-08 Substrain-94 
pufL (L subunit) No change No change No change 
pufM (M subunit) M29 Ile Val 
 M63 Ala Ser 
 M70 Gly Leu 
 M74 Leu Ala 
 M164 Ala Thr 
pufC (cytochrome c subunit) C43 Pro Ala 
 C77 Met Ile 
 C84 Glu Gln 
 C252 Ser Thr 
 C255 Thr Ser 
 C277 Met Leu 
 C287 Thr Ala 
 C288 Val Ser 
 C323 Gln Lys 
puhA (H subunit) H216 Asp Glu 
 H256 Ala Ser 
 Amino acids 
Gene (protein) Position Substrain-08 Substrain-94 
pufL (L subunit) No change No change No change 
pufM (M subunit) M29 Ile Val 
 M63 Ala Ser 
 M70 Gly Leu 
 M74 Leu Ala 
 M164 Ala Thr 
pufC (cytochrome c subunit) C43 Pro Ala 
 C77 Met Ile 
 C84 Glu Gln 
 C252 Ser Thr 
 C255 Thr Ser 
 C277 Met Leu 
 C287 Thr Ala 
 C288 Val Ser 
 C323 Gln Lys 
puhA (H subunit) H216 Asp Glu 
 H256 Ala Ser 

To make this comparison complete it should be noted that there were also three nucleotide differences identified between the substrain-94 gene sequences and the DSM 133 published sequences [79]; however, they did not produce amino acid changes (for details see Supplementary Figure S2).

Crystal structure of the Blc. viridis RC from strain DSM 133, substrain-08

The new sequences of subunits M, H and C of substrain-08 were applied to our working RC model, which was refined against the X-ray data and the refinement was finalized. All 16 amino acid changes introduced via the new sequence were in agreement with the observed electron density. In some cases the new amino acid assignments were critical for a perfect fit, including the four residues presented in Figure 5. Figure 6 shows these residues within the final 2FoFc electron density and their excellent fit is evident. Figure 7(a) illustrates the fit of the carotenoid in the electron density near GlyM70, plus the fit of the side chain of the mutated LeuM74. It is apparent that the replacement of LeuM70 with glycine, i.e. the removal of the LeuM70 side chain allows the almost planar carotenoid to fit the electron density very well.

Final refinement results for the substrain-08 RC for the residues presented in Figure 5 with the 2FoFc electron density shown in blue at 1σ level

Figure 6
Final refinement results for the substrain-08 RC for the residues presented in Figure 5 with the 2FoFc electron density shown in blue at 1σ level

AlaM164 (a) previously Thr, MetC77 (b) previously Ile, MetC277 (c) previously Leu and ProC43 (d) previously Ala.

Figure 6
Final refinement results for the substrain-08 RC for the residues presented in Figure 5 with the 2FoFc electron density shown in blue at 1σ level

AlaM164 (a) previously Thr, MetC77 (b) previously Ile, MetC277 (c) previously Leu and ProC43 (d) previously Ala.

Final 2FoFc electron density map at 0.7σ level at the carotenoid-binding site of the RC from Blc. viridis near residues M70–M74

Figure 7
Final 2FoFc electron density map at 0.7σ level at the carotenoid-binding site of the RC from Blc. viridis near residues M70–M74

(a) Substrain-08 RC with mutated GlyM70 and LeuM74. (b) Substrain-94 RC with LeuM70 and AlaM74.

Figure 7
Final 2FoFc electron density map at 0.7σ level at the carotenoid-binding site of the RC from Blc. viridis near residues M70–M74

(a) Substrain-08 RC with mutated GlyM70 and LeuM74. (b) Substrain-94 RC with LeuM70 and AlaM74.

The conformation of carotenoid in the Blc. viridis RC from strain DSM 133, substrain-08

The major carotenoid present in the Blc. viridis membranes and in the RC, NS5 (top molecule in Figure 2), was modelled in the 2FoFc electron density within the carotenoid-binding site calculated after omission of the carotenoid. The saturated end of NS5 was fitted near GlyM70 (see Figure 7a), which in published Blc. viridis RC structures from strain DSM 133 is a leucine [26,8,10,2023]. Steric hindrance between the carotenoid and the LeuM70 side chain causes the saturated end of the carotenoid to bend out of the plane of its central system of conjugated double bonds (see Figures 3 and 7b). In the RC from substrain-08 the electron density of the carotenoid is planar and, therefore, fits the 15,15′-cis-isomer of NS5 in a planar conformation, including its saturated tail. The fit of the NS5 in electron density after its final refinement is presented in Figure 8. Interestingly, in spite of such an extensive degree of conjugation and the overall planarity of this molecule, there is a significant left-handed twist observed along the length of the carotenoid, with the result that the torsion angle between methyl groups at the ends of the conjugated region in positions 9 and 5′ is 98°. One can best see this left-handed twist of the ‘orange’ carotenoid in Figure 9. The most likely reason for this twist are the steric interactions between the hydrophobic carotenoid and the surrounding protein. Fitting of the alternative carotenoid, LY6 (a minor component of the Blc. viridis membranes and the RC [18]), was also attempted and is described in Supplementary Table S1 and Supplementary Figure S7 at http://www.BiochemJ.org/bj/442/bj4420027add.htm.

The carotenoid NS5 within the final 2FoFc electron density at 0.7σ level for the RC from Blc. viridis substrain-08

Figure 8
The carotenoid NS5 within the final 2FoFc electron density at 0.7σ level for the RC from Blc. viridis substrain-08

The accessory bacteriochlorophyll, with which the carotenoid interacts, is labelled BChlB.

Figure 8
The carotenoid NS5 within the final 2FoFc electron density at 0.7σ level for the RC from Blc. viridis substrain-08

The accessory bacteriochlorophyll, with which the carotenoid interacts, is labelled BChlB.

Superposition of carotenoid NS5 of RC from Blc. viridis substrain-08 (the present study, in orange) with molecules of CRT as modelled in the RC from Tch. tepidum (CRT, 1EYS [25] in yellow) and SPO as in wild-type RC strain 2.4.1, from Rba. sphaeroides (SPO, 3I4D in green) presented in two perpendicular views

Figure 9
Superposition of carotenoid NS5 of RC from Blc. viridis substrain-08 (the present study, in orange) with molecules of CRT as modelled in the RC from Tch. tepidum (CRT, 1EYS [25] in yellow) and SPO as in wild-type RC strain 2.4.1, from Rba. sphaeroides (SPO, 3I4D in green) presented in two perpendicular views

This overlap was obtained by superposition of the M-subunit protein chains surrounding the carotenoid-binding site.

Figure 9
Superposition of carotenoid NS5 of RC from Blc. viridis substrain-08 (the present study, in orange) with molecules of CRT as modelled in the RC from Tch. tepidum (CRT, 1EYS [25] in yellow) and SPO as in wild-type RC strain 2.4.1, from Rba. sphaeroides (SPO, 3I4D in green) presented in two perpendicular views

This overlap was obtained by superposition of the M-subunit protein chains surrounding the carotenoid-binding site.

Comparison of the carotenoid conformation in RCs of Blc. viridis with Tch. tepidum and Rba. sphaeroides (wild-type)

Figure 9 shows the superposition of the carotenoid molecules in the RC crystal structures from Blc. viridis substrain-08 of strain DSM 133 (NS5, orange), Tch. tepidum (CRT, yellow, PDB code 1EYS [25]) and Rba. sphaeroides wild-type 2.4.1 (SPO, green, PDB code 3I4D). This overlap was obtained by superposition of the M subunit protein chains surrounding the carotenoid-binding site and not by the direct match of the carotenoid molecules. All three carotenoids have a 15,15′-cis configuration and their overall topology is approximately planar, consistent with the long conjugated central portions of these molecules. The 15,15′-cis double bond of the carotenoid is in close proximity to the accessory BChlB, with an approximately parallel arrangement of this cis double bond and the BChlB-conjugated acetyl group (via the C3B–CAB bond). The distance between them is in the range 3.3–3.7 Å. It is believed that the close proximity between the carotenoid and the BChlB-conjugated bond systems facilitates the mechanism by which the photoprotective triplet–triplet energy transfer occurs [26,42].

Some differences between the geometries of these three carotenoids at their ends are apparent, a not altogether unexpected finding owing to the presence of different tail groups. The greatest difference in the structures of these three carotenoids and the largest deviations from planarity exist at the region of the saturated C3–C4 and C7–C8 bonds of NS5 equivalent to the saturated C3′–C4′ and C7′–C8′ bonds of SPO. Changes in these saturated regions of carotenoids are, however, not expected to affect their function because all of the photochemistry takes place in the conjugated bond system.

Crystal structure of the Blc. viridis RC from strain DSM 133, substrain-94

Crystals were grown and the X-ray diffraction data were collected for the Blc. viridis RC from the second substrain, substrain-94. The newly determined sequences of the RC polypeptides for this substrain, which were found to be identical with the original DSM 133 sequences at the amino acid level [79], were applied in the refinement of the RC model. Electron density maps obtained for the substrain–94 RC were found to be in complete agreement with these protein sequences at the amino acid level, including the presence of the critical residue LeuM70 and the sharp end of good electron density for the carotenoid at C-8 (using numbering from Figure 2, C9 in the crystal structure). These features resulted in modelling of carotenoid with bending in the saturated C-7–8 region (C8–C9 in the crystal structure) and, therefore, the structure of RC of substrain-94 was found to be in agreement with the previously published structures [6,10,2023]. Figure 7(b) shows the electron density for this bent carotenoid and its environment in the case of the Blc. viridis substrain-94 RC in order to compare it to the equivalent region in the structure of the substrain-08 RC (Figure 7a).

DAG lipid covalently bound to the N-terminus of the cytochrome c subunit

In the LSP crystal structure of Blc. viridis RC (PDB code 2WJM) [10] a covalently bound lipid was found on the periplasmic side of membrane attached to the N-terminal cysteine residue of the cytochrome c subunit. Based on previous MS studies [43] this lipid has been identified as a DAG and is covalently attached to the cysteine residue side chain through a thioether bond. It was suggested that this post-translational modification of the N-terminal cysteine residue functions as a membrane anchor for the cytochrome subunit prior to its association with the other protein subunits of the RC [43,44]. Interestingly, this cysteine-C1-bound lipid has not been modelled in the detergent-based Blc. viridis RC structures published till now, mainly due to the fact that only limited electron density was visible beyond the N-terminal cysteine. X-ray radiation damage was suggested to be responsible for breaking of the thioether bond, based on the differences between two crystal structure determinations for the LSP-grown crystals exposed to low and high doses of X-rays respectively [10].

In contrast to the earlier/published detergent-based Blc. viridis RC crystallizations, the crystal structures of RC from substrain 94 and -08 reported here do show strong electron density at the N-terminus of the cytochrome subunit. This feature allows the DAG lipid bound through a thioether bond to be modelled. Similarly to the RC from LSP-grown crystals [10] one of the fatty acid tails of DAG is modelled in the well-defined groove, which leads towards the poorly ordered space occupied by the tail of ubiquinone QB. The other fatty acid tail can be modelled into a relatively weaker section of density on the hydrophobic surface of the RC, interacting predominantly with the PheL246 side chain. The thioether bond (SGCys-C1–CG3DGA-D730) was modelled with a bond length of 1.82 Å. Figure 10 shows this DGA lipid within 2FoFc electron density for the substrain-08 RC.

DAG covalently attached through a thioether bond (represented by a broken green/yellow line) to the N–terminal cysteine (CysC1) of the cytochrome subunit

Figure 10
DAG covalently attached through a thioether bond (represented by a broken green/yellow line) to the N–terminal cysteine (CysC1) of the cytochrome subunit

The 2FoFc electron density for the RC from Blc. viridis substrain-08 is shown at 0.7σ level. The moiety UQ2 shown on the right is a tentative model of a ubiquinone molecule with its tail limited to only two isoprene units. Its head group is sandwiched between two aromatic side chains of highly conserved residues PheM89 and TrpL266 (see alignments in Supplementary Figures S3 and 6 at http://www.BiochemJ.org/bj/442/bj4420027add.htm) with relevant interplanar distances in the 3.4–3.6 Å range suggesting a reasonable π–π stacking interaction. The head group of UQ2 molecule is hydrogen bonded via water molecule to the main chain O and N atoms of residues ArgM86, PheM88 and PheM89 (not shown). The exact identity of this quinone is unknown as the remaining isoprene units are not bound to the RC surface, rather project out to the detergent micelle and are, therefore, disordered. The placement of the ubiquinone head group in this π-sandwich site has been suggested in structure 2I5N [23]. It has also been modelled in the RC from Rba. sphaeroides wild-type 2.4.1 (PDB code 3I4D). DGA, diacylglycerol (DAG).

Figure 10
DAG covalently attached through a thioether bond (represented by a broken green/yellow line) to the N–terminal cysteine (CysC1) of the cytochrome subunit

The 2FoFc electron density for the RC from Blc. viridis substrain-08 is shown at 0.7σ level. The moiety UQ2 shown on the right is a tentative model of a ubiquinone molecule with its tail limited to only two isoprene units. Its head group is sandwiched between two aromatic side chains of highly conserved residues PheM89 and TrpL266 (see alignments in Supplementary Figures S3 and 6 at http://www.BiochemJ.org/bj/442/bj4420027add.htm) with relevant interplanar distances in the 3.4–3.6 Å range suggesting a reasonable π–π stacking interaction. The head group of UQ2 molecule is hydrogen bonded via water molecule to the main chain O and N atoms of residues ArgM86, PheM88 and PheM89 (not shown). The exact identity of this quinone is unknown as the remaining isoprene units are not bound to the RC surface, rather project out to the detergent micelle and are, therefore, disordered. The placement of the ubiquinone head group in this π-sandwich site has been suggested in structure 2I5N [23]. It has also been modelled in the RC from Rba. sphaeroides wild-type 2.4.1 (PDB code 3I4D). DGA, diacylglycerol (DAG).

Conclusion

After 14 years of continuous passaging of Blc. viridis in our laboratory, a similar carotenoid environment and planar carotenoid conformation has arisen as observed in the other two known structures of purple bacteria RCs, including variety of carotenoids for Rba. sphaeroides RC [25,26]. It is not yet clear whether this apparent reversion to the more common conformation is just a chance event or whether it is suggestive that the fully planar conformation is somehow more favourable.

The results described above represent an interesting case study illustrating the care needed when continuously culturing a bacterial species. Blc. viridis during its continuous passaging in our laboratory has accumulated a series of point mutations. Whenever the culture became contaminated it was routinely re-purified by plating out and picking a pure single colony. If such a single colony harboured a neutral mutation that change would then be fixed in the subsequent pure culture. Clearly, since the cells still grow well photosynthetically, these mutations did not significantly reduce the fitness of the Blc. viridis culture. None of the 133 nucleotide changes affect residues that are in contact with any of the pigments on the active A branch. This may mean that mutations in these regions of the protein are more likely to be inherently detrimental and so have not been retained. The lack of a strong resultant phenotype makes the easy detection of the mutations found in the present study difficult. As a result of these findings we have changed our laboratory protocol when a culture becomes contaminated. Now we go back to the original frozen glycerol stock of the bacteria in question. If the species under study is one that cannot be easily regenerated from a frozen stock, then we would recommend picking at least 20 single pure colonies and mixing them together to regenerate a pure culture that has much less chance of fixing random neutral mutations.

This whole issue could be especially problematic for groups not well trained in microbiology who grow purple bacteria in order to purify photosynthetic complexes for sophisticated biophysical studies, where the effects of rather subtle neutral mutations may well be detected and give rise to confusion. Moreover, this could result in the unfortunate circumstance that different groups assume that they are working on the same protein when they in fact are not. Indeed this problem may be widespread as was suggested by Perfeito et al. [45] who have shown that mutation rates with bacterial laboratory cultures are much higher than previously thought.

Abbreviations

     
  • BChl

    bacteriochlorophyll

  •  
  • BPhe

    bacteriopheophytin

  •  
  • CRT

    spirilloxanthin

  •  
  • DAG

    diacyglycerol

  •  
  • LDAO

    N,N-dimethyldodecylamine-N-oxide

  •  
  • LSP

    lipidic sponge phase

  •  
  • LY6

    1,2-dihydrolycopene

  •  
  • NS5

    1,2-dihydroneurosporene

  •  
  • RC

    reaction centre

  •  
  • SPO

    spheroidene

  •  
  • TLS

    Translation/Libration/Screw

AUTHOR CONTRIBUTION

Alastair Gardiner was responsible for the protein purification, crystallization and participated in the data collection. Aleksander Roszak performed the data collection, analysed the data and carried out the structure solution, refinement and analysis. Vladimira Moulisova and Adhie Reksodipuro performed the DNA amplification and the sequencing analyses. Ritsuko Fujii and Hideki Hashimoto provided valuable contributions towards the carotenoid structure. Richard Cogdell and Neil Isaacs are the principal investigators involved.

We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and we thank Dr Gianluca Cioci for assistance in using beamline ID14-1 and Dr Martin Walsh for assistance in using beamline BM14. We also acknowledge the support of the Diamond Light Source for provision of beamtime at station I02 and thank Dr James Sandy for his assistance. We also thank Dr Michal Koblizek for providing the three primers, 440F, 608R and 970F, used in sequencing of the 16S rRNA genes.

FUNDING

This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC; to A.W.R., A.T.G., N.W.I. and R.J.); the Membrane Protein Structure Initiative (MPSi; to A.W.R., A.T.G., N.W.I. and R.J.C.); the EU BIMORE Program [grant number MRTN-CT-2006-035859 (to V.M.)]; the Beasiswa Unggulan Program from the Ministry of the National Education of Indonesia (to A.D.P.R.); the Grant-in-aid for Scientific Research [grant numbers 17204026 and 17654083 (to H.H.), and 18684016 (to R.F.)]; the Japanese Ministry of Education, Culture, Sports, Science, and Technology; the Nissan Science Foundation (to H.H.) and from the HFSP (Human Frontier Science Program; to R.J.C. and H.H.).

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Author notes

The new gene sequencing data for two Blastochloris viridis substrains of the strain DSM 133 have been deposited at GenBank® under the following accession numbers: FJ483784 (gene puhA, for protein subunit H, substrain-08); FJ483785 [puf operon containing the genes pufL, pufM, pufC, pufA and pufB, for reaction centre subunits L, M and C, and for LH1 (light harvesting 1) apoproteins α and β respectively, substrain-08]; HQ009852 (16S rRNA gene, substrain-08); HQ009850 (gene puhA, substrain-94); HQ009849 (puf operon, substrain-94); and HQ009851 (16S rRNA gene, substrain-94). The co-ordinates and structure factors have been deposited in the PDB under accession codes 3T6D (substrain-08) and 3T6E (substrain-94).

Supplementary data