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Crystallization and preliminary X-ray and optical spectroscopic characterization of the photochemical reaction center from Rhodobacter sphaeroides strain 2.4.1
,J. Mol. Biol. (1987) 198, 139-141
Crystallization and Preliminary X-ray and Optical Spectroscopic Characterization of the Photochemical Reaction Center from Rhodobacter sphaeroides Strain 2.4.1 The photochemical reaction center from Rhodobacter sphaeroides 2.4.1 has been crystallized. The crystals were obtained in a solution of B-octylglucoside by the vapor diffusion technique using polyethylene glycol 4000 as the precipitant at 22°C. The orthorhombic crystals (space group P2,2,2,) have cell constants a = 142.5 8, b = 136.1 A, c = 78.5 A, and diffract to 3.7 A. The crystals display pronounced linear dichroism in the carotenoid absorption spectral region.
et al., 1983), and electron spin resonance (Chadwick & Frank, 1986) the precise conformation and/or configuration of the reaction center-bound carohow this structure fulfils the tenoid and requirements for acting in a protective role are unknown. The crystal structures of the reaction from centers the carotenoidless mutant Rb. sphaeroides R26 and from Rps. viridis, where the carotenoid was either absent or somewhat disordered in the crystal, did not help in this regard. We present the details of the crystallization and preliminary X-ray characterization and optical linear dichroism of the photochemical reaction center complex from Rb. sphaeroides strain 2.4.1. The abundance of the biochemical, spectroscopic and dynamical information previously obtained (Parson & Monger, 1976; Cogdell et al., 1976; Lutz et al., 1978; Vermeglio et al., 1978; McGann & Frank, 1985; Chadwick & Frank, 1986; Gagliano et al., 1986) on the carotenoid spheroidene, which is known to be strongly bound in these reaction centers and to function as a trap of the triplet energy of the primary donor (Chadwick & Frank, 1986), makes the elucidation of the structure of this reaction center complex of particular importance. This is in contrast to the carotenoid 1,2-dihydroneurosporene in Rps. viridis, which is not strongly bound owing to the lack of a polar funct’ional group on the molecule and which does not trap t,riplet energy. Indeed, the Rb. sphaeroides st’rain 2.4.1 reaction center is one of the “laboratory standards” in photosynthetic carotenoid photochemical research. The reaction centers used herein were prepared according to the following procedure. Rb. sphaeroides 2.4.1 cells were grown anaerobically in a 3% yeast extract/3% Bactopeptone medium. Chromatophores were obtained by French pressure disruption at 1.4 x 10s Pa of whole cells followed by ultracentrifugation at 150,000 g for 90 minutes. The chromatophore membranes were solubilized by
The reaction centers of photosynthetic bacteria contain four bacteriochlorophyll molecules, two of which are closely associated and form the primary electron donor, two bacteriopheophytin molecules, and one iron-quinone complex (Okamura et al., 1982). All of these pigments are non-covalently bound to two polypeptides denoted L and M (Okamura et al., 1982). A third polypeptide denoted H is also isolated with the reaction center complex. An X-ray crystallographic analysis of the reaction center complexes from Rhodopseudomonas viridis 1982; Deisenhofer et al., 1985) and (Michel, Rhodobacter sphaeroides R26 (Allen et al., 1986; Chang et al., 1986) has revealed that the pigments are arranged with an approximate 2-fold rotation symmetry that relates the L and M subunits. Detailed studies in several laboratories are underway to relate the structure of the complex to its spectroscopic features and the dynamics of its photochemical events (Michel-Beyerle, 1985). In this manner, a complete pictupe of the mechanism of the reaction center photochemistry will unfold. Of particular interest are the structure and function of the non-covalently bound carotenoid, which is co-isolated in reaction centers from carotenoid-containing strains of photosynthetic bacteria (Chadwick & Frank, 1986). This carotenoid, which generally occurs in a 1 : 1 stoichiometric ratio with the primary donor, is known not to be involved in the primary photochemical events (Parson & Monger, 1976: Chadwick & Frank, 1986). Its importance lies in the fact that it performs a protect.ive function by quenching the excited triplet state of the primary donor before it can sensitize the formation of singlet oxygen, a known oxidizing agent of bacteriochlorophyll (Krinsky, 197 1). Despite extensive spectroscopic experimentation using linear dichroism (Gagliano et al., 1986), photoselection (Vermeglio et aE., 1978), flash absorpt)ion (Parson & Monger, 1976; Cogdell et al., 1976), magnetophotoselection (McCann & Frank, 1985), resonance Raman (Lutz et al., 1978; Koyama 0022-2836/87/21013943
$03.00/O
139
0
1987 Academic
Press Limitsed
140
H. A. Frank
incubation in a 0.3 y. LDAOf/lO mM-Tris . HCl buffer (pH 8.0) at 22°C for 20 minutes. The mixture was then centrifuged at 15,OOOg for 10 minutes to remove the membrane debris. The supernatant was then loaded onto a DEAE-Sephacel (anion exchange) column, which was previously equilibrated with 0.2% LDAO/lO mM-Tris * HCl buffer (pH 8-O). The protein fractions were eluted from the column by a step-gradient elution using 0.04 M to 0.20 MNaCl/O+2% LDAO/lO mM-Tris. HCl buffer (pH 8.0) solutions in 0.02 M-NaCl concentration steps. The reaction center protein fractions were obtained at 0.18 M-NaCl concentration. The fractions (absorbance of 2.5 at 800 nm) were then diluted with 10 mw-Tris * HCl buffer (pH 8.0) and loaded onto a second DEAE-Sephacel column (30 ml bed volume). The purified reaction centers (absorbance of 2.1 at 800 nm) were obtained by elution with 0.4 iw-NaCl/ 0.2% LDAOjlO mM-Tris. HCl buffer (pH 8.0). Finally, the purified reaction centers were loaded on a third Sephacel column (10 ml bed volume) and washed with 10 mM-Tris. HCl buffer (pH 8.0) containing O*8o/o b-octylglucoside to exchange the detergent. Crystallization was accomplished by vapor diffusion at 22°C in the dark: 50-~1 droplets containing 30 PM-reaCtiOn centers, 0.8% /?-octylglucoside, 10% (w/v) PEG 4000, 1 y. heptanetriol, 0.15 M-NaCl, 10 mM-Tris. HCl (pH 8.0), 1 mMEDTA and 0.1 y. NaN3 were equilibrated against 22% (w/v) PEG 4000, 0.25 M-NaCl, 10 mMTris . HCl (pH 8.0), 1 mM-EDTA, 0.1 y. NaN,. After one week at 22”C, long prismatic crystals were observed. After about four weeks, crystal growth was complete, and crystals with typical dimensions of 1.2 mm x 0.05 mm x 0.05 mm (Fig. 1) were obtained. Absorption spectrum of several crystals dissolved in 0.1 y. LDAO/lO mM-Tris * HCl (pH 8.0) showed the same absorption spectral features as those of the original reaction center solution. Under a slightly different set of conditions, in which 20 PMreaction center protein in O-8 o/o B-octylglucoside, 8% (w/v) PEG 4000, 1 y. heptanetriol, O-25 M-NaCl at pH 8.3 was equilibrated against 25% PEG and having dimensions 1.0 M-NaCl, square plates 0.15 mm x 0.15 mm x 0.05 mm were obtained. For the X-ray analysis, a crystal of the larger crystalline form was placed in a glass capillary and kept in contact with a droplet of solution containing 22% PEG 4000, 0.25 M-NaCl, 1 y. heptanetriol, 0.8% p-octylglucoside, 1 mM-EDTA, O-1 y. NaN, and 10 mw-Tris . HCl buffer (pH 8.0) at 25°C. Nickel-filtered copper radiation (A = 1.542 d) from a Rigaku RU-100 rotating anode operated at 70 mA and 40 kV was used. Precession photography revealed a primitive orthorhombic lattice with cell constants a = 142.5 8, b = 136.1 8, c = 78.5 8, where a is the needle direction. The space group is P2,2,2,. Asssuming a molecular weight of 102,000>
t Abbreviations used: LDAO, lauryldimethylamine oxide; PEG, polyethyleneglycol; LD, linear dichroism.
et al.
Figure 1. The orthorhombic crystal of the reaction center of Rb. spheroides strain 2.4.1 obtained as described in the text. The picture was taken with the long axis of the crystal (top) perpendicular and (bottom) parallel to the direction of polarization of monochromatic (488 nm) incident light.
the volume-to-mass ratio is 3.7 A3/dalton, so that one reaction center complex is contained in the asymmetric unit (Chang et al., 1985). The crystals diffract to a resolution of 3.7 A with an eight hour exposure. Optical analysis of the crystal was accomplished using a Leitz polarizing microscope, equipped with a Leitz Polaroid camera as a detector. The light source (Leitz tungsten lamp) was polarized with a Calcite polarizer (Nicol). Upon selective excitation into the carotenoid absorption band using a 488 nm interference filter (Biared-Atomic), incident light is is polarized when it absorbed much more perpendicular to the long axis of the, crystal than when it is polarized parallel to this axis (see Fig. 1). This observation of maximum absorption at 90” to the long axis of the crystal indicates that the carotenoid transition moment makes an angle perpendicular to the long axis of the crystal. It should be emphasized that a pronounced linear dichroism for the carotenoid as observed here does not imply molecular order at the level of an X-ray diffraction analysis. However, the pronounced dichroism coupled with the facts that this cargtenoid is strongly bound to the protein via its polar functional group and that these crystals diffract to 3.7 A resolution is extremely promising for obtaining more precise structural information using X-ray the crystalline form Furthermore, methods. described here is rather isomorphous (same space group with unit cell dimensions a and c within and h within 2.6%) of those of 0.3 y. Rb. spheroides R26 (Chang et al.. 1986). SO, it should be amenable to solving its structure by molecular replacement and difference Fourier methods to locate the bound carotenoid molecule.
Letters to the Editor The authors thank Dr J. Groeger, MS C. Blouin and MS T. McNickle for their assistance in setting up the optical experiment. This work was supported by grants from the National Science Foundation (PCM-8408201) and the Competitive Research Grant Office of the C.S. Department of Agriculture (86~CRCR-1-2016) to H.A.F. and the National Institutes of Health (GM-37742) to .J.R.K.
Chang, C.-H., Tiede, D., Tang, J., Smith: U.. Norris. ,J. & Schiffer, M. (1986). FEBS Letters, 205, 82-86. Cogdell, R. J., Parson, W. W. & Kerr, M. A. (1976). Biochim.
Departments of ‘Chemistry and ‘Molecular and Cell Biology. University Storrs, CT 06268. U.S.A.
of Connecticut
Received 7 July 1987
References Allen. J. P., Feher, G.. Yeates, T. O., Rees, D. C., Deisenhofer, J., Michel, H. & Huber, R. (1986). Proc. Nat. Acud. Sci., U.S.A.
Chadwick,
83, 8589-8593.
B. W. & Frank, H. A. (1986). Biochim. Biophys. Acta, 851, 257-266. Chang, C.-H.. Schiffer, M., Tiede, D., Smith, U. & Norris, CT.(1985). .L Mol. Biol. 186, 201-203.
Biophys.
Acta, 430, 83-93.
Deisenhofer, J., Epp, O., Miki, K., Huber, R. & Michel. H. (1985). Nature (London), 318, 618-624. Gagliano, A. G., Breton, J. & Geacintov, N. E. (1986). Photobiochem.
Koyama,
Harry A. Frank’ Shahriar S. Taremi’ James R. Knox’
141
Photobiophys.
10, 213-221.
M., Saiki, K. & Tsukida. K. (1983). Photobiochem. Photobiophys. 5, 139-150. Krinsky, N. I. (1971). In Carotenoids (Isler. O., Gutmann. H. & Solms, U., eds), pp. 48-51, Birkhauser. Basel. Lutz, M., Agalidis, I., Hervo, G.. Codgell, R. J. $ ReissHusson, F. (1978). Biochim. Biophys. Acta. 503, 287 303. McGann, W. J. & Frank, H. A. (1985). Biochim. Riophys. Acta, 807, 101-109. Michel, H. (1982). J. Mol. Biol. 158, 567-572. Michel-Beyerle, M. (1985). Editor of Springer Series in Berlin. Chemical Physics, vol. 42, Springer-Verlag, Okamura, M. Y., Feher, G. & Nelson, N. (1982). In Photosynthesis: Energy Conversion by Plants and Bacteria, vol. 1, pp. 195-272. Academic Press, New York. Parson, W. W. 6 Monger, T. G. (1976). Brookhaven Aymp. Biol. 28, 196-212. Vermeglio, A., Breton, J., Paillotin. G. & Cogdell. R. (1978). Biochim. Biophys. Acta, 501, 514 530.
Edited by A. Klug
Y., Kito,
Crystallization and Preliminary X-ray and Optical Spectroscopic Characterization of the Photochemical Reaction Center from Rhodobacter sphaeroides Strain 2.4.1 The photochemical reaction center from Rhodobacter sphaeroides 2.4.1 has been crystallized. The crystals were obtained in a solution of B-octylglucoside by the vapor diffusion technique using polyethylene glycol 4000 as the precipitant at 22°C. The orthorhombic crystals (space group P2,2,2,) have cell constants a = 142.5 8, b = 136.1 A, c = 78.5 A, and diffract to 3.7 A. The crystals display pronounced linear dichroism in the carotenoid absorption spectral region.
et al., 1983), and electron spin resonance (Chadwick & Frank, 1986) the precise conformation and/or configuration of the reaction center-bound carohow this structure fulfils the tenoid and requirements for acting in a protective role are unknown. The crystal structures of the reaction from centers the carotenoidless mutant Rb. sphaeroides R26 and from Rps. viridis, where the carotenoid was either absent or somewhat disordered in the crystal, did not help in this regard. We present the details of the crystallization and preliminary X-ray characterization and optical linear dichroism of the photochemical reaction center complex from Rb. sphaeroides strain 2.4.1. The abundance of the biochemical, spectroscopic and dynamical information previously obtained (Parson & Monger, 1976; Cogdell et al., 1976; Lutz et al., 1978; Vermeglio et al., 1978; McGann & Frank, 1985; Chadwick & Frank, 1986; Gagliano et al., 1986) on the carotenoid spheroidene, which is known to be strongly bound in these reaction centers and to function as a trap of the triplet energy of the primary donor (Chadwick & Frank, 1986), makes the elucidation of the structure of this reaction center complex of particular importance. This is in contrast to the carotenoid 1,2-dihydroneurosporene in Rps. viridis, which is not strongly bound owing to the lack of a polar funct’ional group on the molecule and which does not trap t,riplet energy. Indeed, the Rb. sphaeroides st’rain 2.4.1 reaction center is one of the “laboratory standards” in photosynthetic carotenoid photochemical research. The reaction centers used herein were prepared according to the following procedure. Rb. sphaeroides 2.4.1 cells were grown anaerobically in a 3% yeast extract/3% Bactopeptone medium. Chromatophores were obtained by French pressure disruption at 1.4 x 10s Pa of whole cells followed by ultracentrifugation at 150,000 g for 90 minutes. The chromatophore membranes were solubilized by
The reaction centers of photosynthetic bacteria contain four bacteriochlorophyll molecules, two of which are closely associated and form the primary electron donor, two bacteriopheophytin molecules, and one iron-quinone complex (Okamura et al., 1982). All of these pigments are non-covalently bound to two polypeptides denoted L and M (Okamura et al., 1982). A third polypeptide denoted H is also isolated with the reaction center complex. An X-ray crystallographic analysis of the reaction center complexes from Rhodopseudomonas viridis 1982; Deisenhofer et al., 1985) and (Michel, Rhodobacter sphaeroides R26 (Allen et al., 1986; Chang et al., 1986) has revealed that the pigments are arranged with an approximate 2-fold rotation symmetry that relates the L and M subunits. Detailed studies in several laboratories are underway to relate the structure of the complex to its spectroscopic features and the dynamics of its photochemical events (Michel-Beyerle, 1985). In this manner, a complete pictupe of the mechanism of the reaction center photochemistry will unfold. Of particular interest are the structure and function of the non-covalently bound carotenoid, which is co-isolated in reaction centers from carotenoid-containing strains of photosynthetic bacteria (Chadwick & Frank, 1986). This carotenoid, which generally occurs in a 1 : 1 stoichiometric ratio with the primary donor, is known not to be involved in the primary photochemical events (Parson & Monger, 1976: Chadwick & Frank, 1986). Its importance lies in the fact that it performs a protect.ive function by quenching the excited triplet state of the primary donor before it can sensitize the formation of singlet oxygen, a known oxidizing agent of bacteriochlorophyll (Krinsky, 197 1). Despite extensive spectroscopic experimentation using linear dichroism (Gagliano et al., 1986), photoselection (Vermeglio et aE., 1978), flash absorpt)ion (Parson & Monger, 1976; Cogdell et al., 1976), magnetophotoselection (McCann & Frank, 1985), resonance Raman (Lutz et al., 1978; Koyama 0022-2836/87/21013943
$03.00/O
139
0
1987 Academic
Press Limitsed
140
H. A. Frank
incubation in a 0.3 y. LDAOf/lO mM-Tris . HCl buffer (pH 8.0) at 22°C for 20 minutes. The mixture was then centrifuged at 15,OOOg for 10 minutes to remove the membrane debris. The supernatant was then loaded onto a DEAE-Sephacel (anion exchange) column, which was previously equilibrated with 0.2% LDAO/lO mM-Tris * HCl buffer (pH 8-O). The protein fractions were eluted from the column by a step-gradient elution using 0.04 M to 0.20 MNaCl/O+2% LDAO/lO mM-Tris. HCl buffer (pH 8.0) solutions in 0.02 M-NaCl concentration steps. The reaction center protein fractions were obtained at 0.18 M-NaCl concentration. The fractions (absorbance of 2.5 at 800 nm) were then diluted with 10 mw-Tris * HCl buffer (pH 8.0) and loaded onto a second DEAE-Sephacel column (30 ml bed volume). The purified reaction centers (absorbance of 2.1 at 800 nm) were obtained by elution with 0.4 iw-NaCl/ 0.2% LDAOjlO mM-Tris. HCl buffer (pH 8.0). Finally, the purified reaction centers were loaded on a third Sephacel column (10 ml bed volume) and washed with 10 mM-Tris. HCl buffer (pH 8.0) containing O*8o/o b-octylglucoside to exchange the detergent. Crystallization was accomplished by vapor diffusion at 22°C in the dark: 50-~1 droplets containing 30 PM-reaCtiOn centers, 0.8% /?-octylglucoside, 10% (w/v) PEG 4000, 1 y. heptanetriol, 0.15 M-NaCl, 10 mM-Tris. HCl (pH 8.0), 1 mMEDTA and 0.1 y. NaN3 were equilibrated against 22% (w/v) PEG 4000, 0.25 M-NaCl, 10 mMTris . HCl (pH 8.0), 1 mM-EDTA, 0.1 y. NaN,. After one week at 22”C, long prismatic crystals were observed. After about four weeks, crystal growth was complete, and crystals with typical dimensions of 1.2 mm x 0.05 mm x 0.05 mm (Fig. 1) were obtained. Absorption spectrum of several crystals dissolved in 0.1 y. LDAO/lO mM-Tris * HCl (pH 8.0) showed the same absorption spectral features as those of the original reaction center solution. Under a slightly different set of conditions, in which 20 PMreaction center protein in O-8 o/o B-octylglucoside, 8% (w/v) PEG 4000, 1 y. heptanetriol, O-25 M-NaCl at pH 8.3 was equilibrated against 25% PEG and having dimensions 1.0 M-NaCl, square plates 0.15 mm x 0.15 mm x 0.05 mm were obtained. For the X-ray analysis, a crystal of the larger crystalline form was placed in a glass capillary and kept in contact with a droplet of solution containing 22% PEG 4000, 0.25 M-NaCl, 1 y. heptanetriol, 0.8% p-octylglucoside, 1 mM-EDTA, O-1 y. NaN, and 10 mw-Tris . HCl buffer (pH 8.0) at 25°C. Nickel-filtered copper radiation (A = 1.542 d) from a Rigaku RU-100 rotating anode operated at 70 mA and 40 kV was used. Precession photography revealed a primitive orthorhombic lattice with cell constants a = 142.5 8, b = 136.1 8, c = 78.5 8, where a is the needle direction. The space group is P2,2,2,. Asssuming a molecular weight of 102,000>
t Abbreviations used: LDAO, lauryldimethylamine oxide; PEG, polyethyleneglycol; LD, linear dichroism.
et al.
Figure 1. The orthorhombic crystal of the reaction center of Rb. spheroides strain 2.4.1 obtained as described in the text. The picture was taken with the long axis of the crystal (top) perpendicular and (bottom) parallel to the direction of polarization of monochromatic (488 nm) incident light.
the volume-to-mass ratio is 3.7 A3/dalton, so that one reaction center complex is contained in the asymmetric unit (Chang et al., 1985). The crystals diffract to a resolution of 3.7 A with an eight hour exposure. Optical analysis of the crystal was accomplished using a Leitz polarizing microscope, equipped with a Leitz Polaroid camera as a detector. The light source (Leitz tungsten lamp) was polarized with a Calcite polarizer (Nicol). Upon selective excitation into the carotenoid absorption band using a 488 nm interference filter (Biared-Atomic), incident light is is polarized when it absorbed much more perpendicular to the long axis of the, crystal than when it is polarized parallel to this axis (see Fig. 1). This observation of maximum absorption at 90” to the long axis of the crystal indicates that the carotenoid transition moment makes an angle perpendicular to the long axis of the crystal. It should be emphasized that a pronounced linear dichroism for the carotenoid as observed here does not imply molecular order at the level of an X-ray diffraction analysis. However, the pronounced dichroism coupled with the facts that this cargtenoid is strongly bound to the protein via its polar functional group and that these crystals diffract to 3.7 A resolution is extremely promising for obtaining more precise structural information using X-ray the crystalline form Furthermore, methods. described here is rather isomorphous (same space group with unit cell dimensions a and c within and h within 2.6%) of those of 0.3 y. Rb. spheroides R26 (Chang et al.. 1986). SO, it should be amenable to solving its structure by molecular replacement and difference Fourier methods to locate the bound carotenoid molecule.
Letters to the Editor The authors thank Dr J. Groeger, MS C. Blouin and MS T. McNickle for their assistance in setting up the optical experiment. This work was supported by grants from the National Science Foundation (PCM-8408201) and the Competitive Research Grant Office of the C.S. Department of Agriculture (86~CRCR-1-2016) to H.A.F. and the National Institutes of Health (GM-37742) to .J.R.K.
Chang, C.-H., Tiede, D., Tang, J., Smith: U.. Norris. ,J. & Schiffer, M. (1986). FEBS Letters, 205, 82-86. Cogdell, R. J., Parson, W. W. & Kerr, M. A. (1976). Biochim.
Departments of ‘Chemistry and ‘Molecular and Cell Biology. University Storrs, CT 06268. U.S.A.
of Connecticut
Received 7 July 1987
References Allen. J. P., Feher, G.. Yeates, T. O., Rees, D. C., Deisenhofer, J., Michel, H. & Huber, R. (1986). Proc. Nat. Acud. Sci., U.S.A.
Chadwick,
83, 8589-8593.
B. W. & Frank, H. A. (1986). Biochim. Biophys. Acta, 851, 257-266. Chang, C.-H.. Schiffer, M., Tiede, D., Smith, U. & Norris, CT.(1985). .L Mol. Biol. 186, 201-203.
Biophys.
Acta, 430, 83-93.
Deisenhofer, J., Epp, O., Miki, K., Huber, R. & Michel. H. (1985). Nature (London), 318, 618-624. Gagliano, A. G., Breton, J. & Geacintov, N. E. (1986). Photobiochem.
Koyama,
Harry A. Frank’ Shahriar S. Taremi’ James R. Knox’
141
Photobiophys.
10, 213-221.
M., Saiki, K. & Tsukida. K. (1983). Photobiochem. Photobiophys. 5, 139-150. Krinsky, N. I. (1971). In Carotenoids (Isler. O., Gutmann. H. & Solms, U., eds), pp. 48-51, Birkhauser. Basel. Lutz, M., Agalidis, I., Hervo, G.. Codgell, R. J. $ ReissHusson, F. (1978). Biochim. Biophys. Acta. 503, 287 303. McGann, W. J. & Frank, H. A. (1985). Biochim. Riophys. Acta, 807, 101-109. Michel, H. (1982). J. Mol. Biol. 158, 567-572. Michel-Beyerle, M. (1985). Editor of Springer Series in Berlin. Chemical Physics, vol. 42, Springer-Verlag, Okamura, M. Y., Feher, G. & Nelson, N. (1982). In Photosynthesis: Energy Conversion by Plants and Bacteria, vol. 1, pp. 195-272. Academic Press, New York. Parson, W. W. 6 Monger, T. G. (1976). Brookhaven Aymp. Biol. 28, 196-212. Vermeglio, A., Breton, J., Paillotin. G. & Cogdell. R. (1978). Biochim. Biophys. Acta, 501, 514 530.
Edited by A. Klug
Y., Kito,