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W2106436735 abstract "The structure of the cytochromeb 6 f complex has been investigated by electron microscopy and image analysis of thin three-dimensional crystals. Electron micrographs of negatively stained specimens were recorded and showed optical diffraction peaks to 10 Å resolution. A projection map was calculated at 8 Å resolution and showed the presence of cytochrome b 6 fdimers. The extramembrane part of each monomer featured a C shape, with mean external diameter ∼of 53 Å and an internal groove ∼14 Å long and ∼9 Å wide. Within each monomer, strong features were clearly resolved and tentatively attributed to some of the subunits of the cytochrome b 6 f complex. The data are consistent with the Rieske iron-sulfur protein lying close to the monomer-monomer interface and the heme-bearing domain of cytochromef far from it. The structure of the cytochromeb 6 f complex has been investigated by electron microscopy and image analysis of thin three-dimensional crystals. Electron micrographs of negatively stained specimens were recorded and showed optical diffraction peaks to 10 Å resolution. A projection map was calculated at 8 Å resolution and showed the presence of cytochrome b 6 fdimers. The extramembrane part of each monomer featured a C shape, with mean external diameter ∼of 53 Å and an internal groove ∼14 Å long and ∼9 Å wide. Within each monomer, strong features were clearly resolved and tentatively attributed to some of the subunits of the cytochrome b 6 f complex. The data are consistent with the Rieske iron-sulfur protein lying close to the monomer-monomer interface and the heme-bearing domain of cytochromef far from it. Cytochrome b 6 f(plastoquinol:plastocyanin oxidoreductase) is an integral membrane protein complex that participates in electron transfer and generation of an electrochemical proton gradient in oxygenic photosynthesis. It is homologous to the cytochrome bc 1 complex (ubiquinol:cytochrome c oxidoreductase) of the respiratory chains of the mitochondrion and many bacteria. Theb 6 f complexes from higher plants (1Cramer W.A. Soriano G.M. Ponomarev M. Huang D. Zhang H. Martinez S.E. Smith J.L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 477-508Crossref PubMed Scopus (163) Google Scholar) and from the unicellular green alga Chlamydomonas reinhardtii (2Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) are highly similar and comprise four subunits with a molecular mass of >17 kDa. Three of them, cytochromeb 6, cytochrome f, and the Rieske protein, contain redox prosthetic groups. The fourth, subunit IV, is involved together with cytochrome b 6 and the Rieske protein in forming the oxidizing plastoquinol binding site Qo (1Cramer W.A. Soriano G.M. Ponomarev M. Huang D. Zhang H. Martinez S.E. Smith J.L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 477-508Crossref PubMed Scopus (163) Google Scholar). There are at least three additional small hydrophobic polypeptides (∼4 kDa). The number of transmembrane helices in the seven-subunit monomeric complex (105 kDa) is probably 11 (3Breyton C. de Vitry C. Popot J.-L. J. Biol. Chem. 1994; 269: 7597-7602Abstract Full Text PDF PubMed Google Scholar, 4Popot J.-L. Pierre Y. Breyton C. Lemoine Y. Takahashi Y. Rochaix J.-D. Mathis P. Photosynthesis: From Light to Biosphere. Proceedings of the Xth International Congress on Photosynthesis. II. Kluwer Academic Publishers, Dordrecht, the Netherlands1995: 507-512Google Scholar). A dimeric form is believed to be the native state both in higher plants (5Huang D. Everly R.M. Cheng R.H. Hetmann J.B. Schägger H. Sled V. Ohnishi T. Baker T.S. Cramer W.A. Biochemistry. 1994; 33: 4401-4409Crossref PubMed Scopus (105) Google Scholar) and in C. reinhardtii (6Breyton, C., Tribet, C., Olive, J., Dubacq, J.-P. & Popot, J.-L. (1997)J. Biol. Chem., 272, in press.Google Scholar). Spectroscopic, biochemical, genetic, and electron microscopy studies of cytochrome b 6 f (reviewed in Ref. 1Cramer W.A. Soriano G.M. Ponomarev M. Huang D. Zhang H. Martinez S.E. Smith J.L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 477-508Crossref PubMed Scopus (163) Google Scholar) have yielded only sparse information about the three-dimensional structure of this complex. Huang et al. (5Huang D. Everly R.M. Cheng R.H. Hetmann J.B. Schägger H. Sled V. Ohnishi T. Baker T.S. Cramer W.A. Biochemistry. 1994; 33: 4401-4409Crossref PubMed Scopus (105) Google Scholar) reported that negatively stained monomers and dimers of theb 6 f complex from spinach both appeared as round particles with clefts with diameters of 77 ± 10 and 91 ± 9 Å, respectively. Boekema et al. (7Boekema E.J. Boonstra A.F. Dekker J.P. Rögner M. J. Bioenerg. Biomembr. 1994; 26: 17-29Crossref PubMed Scopus (64) Google Scholar) have reported, also from single particle analysis, an elongated shape of the complex from Synechocystis PCC 6803 with dimensions of 83 × 44 × 60 Å, which they interpreted as monomers. Freeze-fractured vesicles reconstituted with purified spinachb 6 f featured particles 83 Å in diameter and 110 Å in height, which Mörschel and Staehelin interpreted as dimers (8Mörschel E. Staehelin L.A. J. Cell Biol. 1983; 97: 301-310Crossref PubMed Scopus (45) Google Scholar), whereas reconstituted monomers and dimers from C. reinhardtii appeared as particles with diameters of ∼80 and ∼100–110 Å, respectively (6Breyton, C., Tribet, C., Olive, J., Dubacq, J.-P. & Popot, J.-L. (1997)J. Biol. Chem., 272, in press.Google Scholar). Finally, Mosser et al. (9Mosser G. Dorr C. Hauska G. Kühlbrandt W. International Congress on Electron Microscopy, Electron Microscopy 1994, Les Editions de Physique. 1994; 3: 609-610Google Scholar) obtained tubular crystals and two types of thin three-dimensional crystals of spinachb 6 f. The projection map that was calculated did not give unambiguous limits of the molecule. Thus, these studies have not led so far to a clear description of the size and shape of the complex. Most interestingly, the structure of the cleaved extramembrane domain of turnip cytochrome f (10Martinez S.E. Huang D. Szczepaniak A. Cramer W.A. Smith J.L. Structure. 1994; 2: 95-105Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar) and that of the catalytic domain of the mitochondrial Rieske protein (11Iwata S. Saynovits M. Link T.A. Michel H. Structure. 1996; 4: 567-579Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) have both been solved to high resolution by x-ray crystallography. On the basis of these data, Link and Iwata have proposed a model for the association of cytochrome f with the photosynthetic Rieske protein (12Link T.A. Iwata S. Biochim. Biophys. Acta. 1996; 1275: 54-60Crossref PubMed Scopus (37) Google Scholar). X-ray crystallography is expected to lead rapidly to a detailed structural model of the entire mitochondrial bc 1complex (13Yu C.-A. Xia J.-Z. Kachurin A.M. Yu L. Xia D. Kim H. Deisenhofer J. Biochim. Biophys. Acta. 1996; 1275: 47-53Crossref PubMed Scopus (86) Google Scholar). However, due to the important differences between the two complexes (different subunit compositions, low sequence similarities of homologous subunits, presence of photosynthetic pigments inb 6 f, sensitivity to antimycin limited to bc 1, and possible functional differences), high resolution structural data on one complex might not be easily transposable to the other. Understanding the functional mechanism of cytochromeb 6 f hangs on the knowledge of its detailed structure. Growing well ordered two-dimensional crystals suitable for analysis by electron crystallography is one approach toward determining the structure of proteins at high resolution (14Unwin P.N.T. Henderson R. J. Mol. Biol. 1975; 94: 425-440Crossref PubMed Scopus (827) Google Scholar,15Kühlbrandt W. Quart. Rev. Biophys. 1992; 25: 1-49Crossref PubMed Scopus (238) Google Scholar). The yield, purity, and stability of theb 6 f preparations from C. reinhardtii (2Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 6Breyton, C., Tribet, C., Olive, J., Dubacq, J.-P. & Popot, J.-L. (1997)J. Biol. Chem., 272, in press.Google Scholar) fulfill the prerequisites for crystallization attempts, whereas enzymatic activity, the presence of the Rieske protein, the spectral properties, and the dimeric state provide various checks on the native state of the complex (6Breyton, C., Tribet, C., Olive, J., Dubacq, J.-P. & Popot, J.-L. (1997)J. Biol. Chem., 272, in press.Google Scholar, 16Pierre, Y., Breyton, C., Lemoine, Y., Robert, B., Vernotte, C. & Popot, J.-L. (1997) J. Biol. Chem., 272, in press.Google Scholar). In the present article, we describe the crystallization of theb 6 f complex from C. reinhardtii in very thin three-dimensional crystals. For this purpose, we have adapted and improved a reconstitution strategy that uses Bio-Beads as a detergent-removing agent and has been demonstrated successful for two-dimensional crystallization of different membrane proteins (17Rigaud J.-L. Mosser G. Lacapère J.-J. Olofsson A. Lévy D. Ranck J.-L. J. Struct. Biol. 1997; 118: 226-235Crossref PubMed Scopus (176) Google Scholar). Optimizing the pre-reconstitution conditions and combining the use of Bio-Beads with freeze-thaw cycles led to the formation of large and highly ordered thin three-dimensional crystals that diffract to better than 10 Å resolution in negative stain. After correction of lattice distortions, crystallographic analysis has yielded a projection map of cytochromeb 6 f at 8 Å resolution. The map is discussed in the light of available evidence on the organization of the subunits in the complex. SM2 Bio-Beads were obtained from Bio-Rad and di-C18:1-phosphatidylglycerol from Avanti Polars Lipids Inc. Sources for the other chemicals have been described in Ref. 2Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar. Cytochromeb 6 f complex was purified fromC. reinhardtii thylakoid membranes in the presence of 6-O-(N-heptylcarbamoyl)-methyl-α-d-glucopyranoside (Hecameg) 1The abbreviations used are: Hecameg, (6-O-(N-heptylcarbamoyl)-methyl-α-d-glucopyranoside; Tricine,N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (N-tris(hydroxymethyl)methylglycine). as described previously (2Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Briefly, the purification protocol comprises three steps: selective solubilization from thylakoid membranes, sucrose gradient sedimentation, and hydroxylapatite chromatography. Following solubilization, all media are supplemented with egg phosphatidylcholine to prevent the loss of the Rieske protein from the complex that follows delipidation (2Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 6Breyton, C., Tribet, C., Olive, J., Dubacq, J.-P. & Popot, J.-L. (1997)J. Biol. Chem., 272, in press.Google Scholar). Purified cytochromeb 6 f complex was resuspended in 20 mm Hecameg, 2 mm CaCl2, 0.3% glycerol, 0.3 mm NaN3, 5.6 mmε-aminocaproic acid, 1.1 mm benzamidine, 0.2 mm phenylmethylsulfonyl fluoride, 245 mmammonium phosphate, 6.8 mm Tricine, pH 8.0, and supplemented with a mixture of egg phosphatidylcholine and di-C18:1-phosphatidylglycerol (1:1 to 1.4:1, w/w). The protein concentration in the final reconstitution mixture was adjusted to 0.5 g/liter and that of the lipid to a lipid/protein ratio of 0.2 w/w. The samples were preincubated overnight in the cold room under gentle stirring and subsequently treated with 200 g/liter SM2 Bio-Beads according to the batch procedure previously described (17Rigaud J.-L. Mosser G. Lacapère J.-J. Olofsson A. Lévy D. Ranck J.-L. J. Struct. Biol. 1997; 118: 226-235Crossref PubMed Scopus (176) Google Scholar). After 6–12 h of incubation with beads, the reconstituted material was pipetted off and kept for 24 h at 4 °C before three cycles of freezing (−190 °C) and thawing (37 °C). Aliquots were taken daily and examined by electron microscopy. Samples, negatively stained with 1% uranyl acetate, were observed on a Philips CM120 transmission electron microscope operating at 120 kV. Low dose electron micrographs were recorded at magnifications of 45,000× and 60,000×. The best images were selected by optical diffraction, and areas exhibiting strong coherent diffraction spots were digitized on a Leafscan 45 CCD-array microdensitometer with a 5 μm scan spot size. Areas ranging up to 3200 × 3200 pixels in size, corresponding to 0.34 × 0.34 μm at the specimen level, were subjected to analysis using the Spectra program package (18Schmid M.F. Dargahi R. Tam M.W. Ultramicroscopy. 1993; 48: 251-264Crossref PubMed Scopus (73) Google Scholar) and the MRC image processing system (19Crowther R.A. Henderson R. Smith J.M. J. Struct. Biol. 1996; 116: 9-16Crossref PubMed Scopus (667) Google Scholar). A total of 14 crystalline areas from nine images were analyzed with unbending methods. The underfocus level was determined by multiple measurements on the Thon-rings from the Fourier transform of the raw image, and the reflections corrected for the overall transfer function. The reflections from the processed images were centered and averaged, utilizing spots up to IQ 7. An image scale factor and the reflection peak height were used as a weighing factor during averaging. A refinement step on the positions of the zeroes in the contrast transfer function was performed, using the preliminary average as a reference. An optimal position of the lattice extraction, centered around the best crystalline area of each image, was achieved by extracting a few sets of reflections and testing for phase quality. The data set was phase-minimized with a small step size and merged, in several rounds, to yield a new average. A cut-off selection of maximally 45 ° phase deviation from the real values, in addition to IQ 7 for the amplitudes, was applied for acceptance of a reflection prior to calculation of the map. An error weighing factor was included by multiplying the amplitudes by the cosine of the phase deviation. The symmetry was imposed, and the projection map was displayed using histogram equalization and the plot program Pluto. Equidistant line levels were generally employed to the maximum positive density (i.e. stain-excluding region) in the map. Thin three-dimensional crystallization of cytochrome b 6 f was achieved by reconstituting the solubilized complex into phospholipid bilayers containing egg phosphatidylcholine and an anionic unsaturated phospholipid, dioleoyl-phosphatidylglycerol, in the presence of calcium ions and protease inhibitors. Reconstitution was performed by removing detergent from the lipid-Hecameg-protein micellar solution by adsorption onto Bio-Beads SM2. Previous systematic studies have determined precisely the amount of beads to be added to remove the detergent initially present in about 6 h at 4 °C while avoiding protein adsorption and limiting lipid adsorption (17Rigaud J.-L. Mosser G. Lacapère J.-J. Olofsson A. Lévy D. Ranck J.-L. J. Struct. Biol. 1997; 118: 226-235Crossref PubMed Scopus (176) Google Scholar). Provided the amount of beads was carefully controlled, as well as the lipid to protein ratio, crystallization of cytochromeb 6 f was reproducible. Another important parameter was the duration of the preincubation period before detergent removal, which had to be sufficiently long. Although the reason for this requirement is not definitely identified, it may be related to the time needed for full equilibration of the initial populations of detergent-lipid and detergent-lipid-protein micelles. Finally, the presence of calcium was found to be essential for crystal formation. Following detergent removal, proteoliposomes with densely packed proteins were observed by electron microscopy. They tended to aggregate upon further incubation at 4 °C with concomittant formation of crystalline areas. Growth of large crystals from these aggregates was improved by treating the samples through freeze-thaw cycles. Possible explanations would be that such a treatment not only induces fusion of proteoliposomes but also creates some defects in the bilayer or some protein aggregation, which would favor crystal growth (20Young H.S. Rigaud J.-L. Lacapère J.-L. Stokes D.L. Biophys. J. 1997; 72: 2545-2558Abstract Full Text PDF PubMed Scopus (57) Google Scholar). Samples were periodically checked by electron microscopy to monitor the growth of the crystals. The crystals presented a smooth homogeneous and continuous gray appearance (Fig.1 A) and were always composed of a stack of lamellae. The best time to collect them was generally between the third and the seventh day after three freeze-thaw cycles. After that, the crystals increased inhomogeneously in thickness and sometimes started to deteriorate. Our belief that the crystals observed in the reconstituted preparations are formed by the b 6 f in its native state is based on several lines of evidence. Neither visible absorption spectra nor SDS-polyacrylamide gel electrophoresis patterns changed during the 3-day to 1-week period required for crystallization. Two good indices of the native state of the complex are its dimeric nature and the stability of the spectrum of theb 6 f-associated chlorophylla; indeed, loss of the Rieske protein, which is the first step in the degradation of the complex (6Breyton, C., Tribet, C., Olive, J., Dubacq, J.-P. & Popot, J.-L. (1997)J. Biol. Chem., 272, in press.Google Scholar), is always accompanied by a red shift of the visible absorption peak of the chlorophyll (16Pierre, Y., Breyton, C., Lemoine, Y., Robert, B., Vernotte, C. & Popot, J.-L. (1997) J. Biol. Chem., 272, in press.Google Scholar) and almost invariably followed by monomerization (6Breyton, C., Tribet, C., Olive, J., Dubacq, J.-P. & Popot, J.-L. (1997)J. Biol. Chem., 272, in press.Google Scholar). The crystals (Fig.1 A) generally grew up to 5 μm in one direction (along theb direction) and up to 1.5 μm in the other (along thea direction). In some cases, they reached up to 10 μm ′ 3 μm. At high magnification, the array appeared clearly and spread over the whole crystal (Fig. 1 B). Although the crystals presented in this study are actually stacks of lamellae, they were selected such that they presented a small number of layers and were of extremely good crystallinity. The coherence of the lattice was confirmed by optical diffraction analysis of low dose negatives, which revealed sharp, coherent peaks out to 10 Å over the whole electron micrographs. This also strongly suggests that the different layers are in perfect register, making the stacks equivalent to very thin three-dimensional crystals. The diffraction pattern corresponded to a rectangular lattice with parameters a = 175 Å, b = 68 Å, and γ = 90 °. Systematic extinctions were observed for [h(odd),0] and [0,k(odd)]. Fourteen areas were selected by optical diffraction from nine of the best images and digitized, and the lattice distortions were corrected. Calculated phases indicated that the crystals belonged to plane groupp22 1 2 1. In this space group, rows of molecules alternatively face up and down with respect to the membrane plane. The results of the R value test for this symmetry are shown in Table I for four resolution ranges, indicating the high quality of the phases to 8 Å resolution.Table ICrystallographic dataPlane group symmetryp22121No. crystals9No. images14Unit cell parametersa = 175 Å, b = 68 Å, γ = 90 °Underfocus level1800–5500 ÅNo. unit cells averaged104No. reflections used in the map223Resolution rangeTwo-fold residual IQ 5 (45 degrees random)CompletnessUnweightedAmplitude-weightedIQ 5IQ 6Å % 175–209.64.2100.0100.0 20–1514.711.994.194.1 15–1118.315.477.8100.1 11–817.915.839.184.1Total range 175–815.47.865.191.8 Open table in a new tab Fig. 2 A shows the Fourier transform of the best distortion-corrected images. Reflections with IQ values of 4 are visible out to 8 Å resolution. Fig. 2 Bshows the phase deviations of reflections from the expected values of zero or π for all images. Excluding the phases of reflections having IQ values larger than 5, the root mean square phase error is about 16° at 8 Å resolution. Because plane groupp22 1 2 1 gave the best phase residual, it was used to calculate projection maps to 20 and 8 Å resolution (Fig. 3, A andB).Figure 3Projection maps after symmetry averaging (p22 1 2 1) of negatively stained cytochrome b 6 f. A, 20 Å resolution. The two main domains are labeledX and Y. B, 8 Å resolution. The four main domains are labeled I–IV; the central groove is labelled G. The unit cell (a = 175 Å,b = 68 Å, and γ = 90 °) is indicated bysolid lines; symmetry elements are marked. Positive density representing the protein is shown as solid lines, and negative density is shown as dotted lines.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The 20 Å density map reveals the dimeric organization of theb 6 f complex. The dimer features an elongated S shape, ∼88 Å long and ∼53 Å wide. The monomer has a C-like shape with two major domains denoted X and Y. The 8 Å resolution map reveals within the extramembrane part of each monomer a ring of densities surrounding a deep groove (G). The external diameter of the monomer is ∼53 Å, whereas the internal groove is ∼14 Å long and ∼9 Å wide. The main goal of this study was to generate a projection map of cytochrome b 6 f to provide information about the localization of its different subunits. The purity and reproducibility of theb 6 f preparations from C. reinhardtii facilitated the identification of conditions favoring the growth of large and coherent thin three-dimensional crystals suitable for structural analysis by electron microscopy. In addition to the usual parameters that affect the formation and quality of crystals, we believe that two key factors in the success of our procedure are the rapid and total removal of detergent by Bio-Beads (see also Ref. 17Rigaud J.-L. Mosser G. Lacapère J.-J. Olofsson A. Lévy D. Ranck J.-L. J. Struct. Biol. 1997; 118: 226-235Crossref PubMed Scopus (176) Google Scholar) and the use of freeze-thaw cycles to increase the size and ordering of the crystals (see also Ref. 20Young H.S. Rigaud J.-L. Lacapère J.-L. Stokes D.L. Biophys. J. 1997; 72: 2545-2558Abstract Full Text PDF PubMed Scopus (57) Google Scholar). The excellent quality of the crystals allowed us to record low dose images of negatively stained specimens that diffracted out to 10 Å resolution prior to the correction of lattice distortions and to better than 8 Å following it. The IQ plot of reflection intensities (Fig.2 A) and the crowding of calculated phases around the values of 0 ° and 180 ° expected from thep22 1 2 1 lattice symmetry (Fig. 2 B) are indicative of the quality of the data. Such a high resolution has never been reported before for negatively stained crystals of any membrane protein. The multi-layered nature of the crystals cannot by itself account for this result, because similar resolutions have already been observed with negatively stained single-layered crystals such as those of annexin V (21Olofsson A. Mallouh V. Brisson A. J. Struct. Biol. 1994; 113: 199-205Crossref PubMed Scopus (44) Google Scholar) and of subunit B of cholera toxin. 2G. Mosser, unpublished observations. Thus, our observations suggest that the resolution attainable following negative staining may be higher than usually accepted (∼15 Å). The projection maps show a dimeric organization of cytochromeb 6 f, in keeping with biochemical determinations on the solubilized complex (6Breyton, C., Tribet, C., Olive, J., Dubacq, J.-P. & Popot, J.-L. (1997)J. Biol. Chem., 272, in press.Google Scholar). Each monomer presents a C-like shape, ∼53 Å in diameter, covering a total area of about 2,000 Å2. By comparison with the dimensions of the transmembrane regions in bacteriorhodopsin (14Unwin P.N.T. Henderson R. J. Mol. Biol. 1975; 94: 425-440Crossref PubMed Scopus (827) Google Scholar) or cytochromec oxidase (22Iwata S. Ostermeier C. Ludwig B. Michel H. Nature. 1995; 376: 660-669Crossref PubMed Scopus (1985) Google Scholar), this area significantly exceeds that needed to accommodate 11 transmembrane α-helices per monomer. In the 20 Å projection map, each monomer features two main domains (labeledX and Y in Fig. 3), surrounding a deep central groove (G in Fig. 3). Domain X, which is near the 2-fold axis of symmetry, is less bulky than domain Y, suggesting that much of the extramembrane mass of the complex lies away from this axis. The dimensions and overall appearance of cytochromeb 6 f at 20 Å resolution are not dissimilar to those of the subcomplex of Neurospora crassacytochromes b and c 1 (lacking the Rieske and core proteins), except that in the latter case domain X appeared stronger than domain Y (23Hovmöller S. Leonard K. Weiss H. FEBS Lett. 1981; 123: 118-122Crossref PubMed Scopus (31) Google Scholar), at variance with theb 6 f map. A three-dimensional reconstruction of bovine heart mitochondrial bc 1at 16 Å resolution has recently been calculated from electron micrographs of frozen tubes (24Akiba T. Toyoshima C. Matsunaga T. Kawamoto M. Kubota T. Fukuyama K. Namba K. Matsuhara H. Nat. Struct. Biol. 1996; 3: 553-561Crossref PubMed Scopus (38) Google Scholar). It shows, protruding into the intermembrane space, four proteic masses per dimer, reaching into the solvent and delineating a relatively empty space around the 2-fold axis of symmetry. A depression about the C2 axis is also apparent in the preliminary x-ray map of beef heart bc 1(13Yu C.-A. Xia J.-Z. Kachurin A.M. Yu L. Xia D. Kim H. Deisenhofer J. Biochim. Biophys. Acta. 1996; 1275: 47-53Crossref PubMed Scopus (86) Google Scholar). The projection of the monomer obtained does not resemble any of the three possible projections (Y, Z, and L shapes) previously observed with spinach b 6 f (9Mosser G. Dorr C. Hauska G. Kühlbrandt W. International Congress on Electron Microscopy, Electron Microscopy 1994, Les Editions de Physique. 1994; 3: 609-610Google Scholar). Differences in the conditions of crystal formation may explain these dissimilarities. Whereas low calcium concentration (0.5 mm), Hecameg, and Bio-Beads were used to crystallize the complex from C. reinhardtii, comparatively high calcium concentration (10 mm), octylglucoside, and dialysis were employed for the crystallization of the spinach complex. These differences may have led to the monomerization of the complex from spinach, explaining why no tight dimers were observed. The location of the stain in the case of membrane proteins is not perfectly understood. It seems to vary from one protein to another, but it is usually accepted that the hydrophobic membrane core is largely stain-excluding. Therefore, we made the assumption that the structural information obtained, including the presence of the groove, primarily concerns the extramembrane parts of theb 6 f complex. Positive densities should be due mainly to the cytochrome f extramembrane domain (28 kDa; residues 1–250), the Rieske protein (19 kDa), the main loops of cytochrome b 6 (11 kDa; residues 1–33, 58–82, and 140–181) and subunit IV (8 kDa; residues 215–249 and 272–308), and the extramembrane extensions of the three 4-kDa subunits (∼1 kDa each). However, because of the smallness of 4-kDa subunits' extramembrane extensions, their contribution to the projection map will not be discussed in the following. The projection map at 8 Å resolution (Fig. 3 A) confirms the C shape of the monomer. Each monomer features four domains of variable importance (II > I ≫ III > IV), which can be tentatively allocated to some of theb 6 f subunits. The two major stain-excluding regions, I and II, are well individualized, indicating that they correspond to two independent proteic masses. Taking into account their respective importance (II > I) and the fact that the extramembrane part of cytochrome f and the Rieske protein fold into autonomous domains, one may tentatively assign density II to the largest of these subunits, the cytochromef, whereas domain I would correspond to the Rieske protein. The other densities (domains III and IV), which include that close to the 2-fold symmetry axis, would be due to the extramembrane loops of cytochrome b 6 and subunit IV. However, in this case, because the mass of the loops is small compared with the whole mass of these two subunits, it would be premature to assign individual subunits to specific features at this stage of the structural study. Domain IV appears to be separated from domain III by a gap and appears to connect more closely to domain I. This suggests that the extramembrane regions of the two monomers may partially engage into one another close to the symmetry axis, as proposed in Fig.4. Moreover, the continuity observed between the two densities IV of the dimer suggests that the subunits that contribute to it tightly interact through their extramembrane parts. It should also be stressed that domain IV appears small to fit either subunit IV or cytochrome b 6. However, this domain is rather flat, and it is reasonable to assume that the subunit that contributes to it may extend under the Rieske protein while contributing in a negligible way to domain I. This general arrangement would be consistent with biochemical data that have shown that cytochrome b 6 can be easily cross-linked to cytochrome b 6 and subunit IV to subunit IV within a dimer, indicating that regions of both subunits come close to the symmetry axis (25Chain R.K. Malkin R. Photosynth. Res. 1991; 28: 59-68PubMed Google Scholar, 26Vater J. Heinze K. Friedrich B. Kablitz B. Blokesch A. Irrgang K.-D. Thiede B. Salnikow J. Ber. Busenges. Phys. Chem. 1996; 100: 2107-2111Crossref Scopus (7) Google Scholar). On the contrary, homologous cross-linking between the large extramembrane domains is almost never observed. The effects of mutations on the functionality and sensitivity to inhibitors of site Qo (reviewed in Ref. 27Brasseur G. Saribas G.S. Daldal F. Biochim. Biophys. Acta. 1996; 1275: 61-69Crossref PubMed Scopus (156) Google Scholar) and on the strength of the Rieske protein's association with the complex (27Brasseur G. Saribas G.S. Daldal F. Biochim. Biophys. Acta. 1996; 1275: 61-69Crossref PubMed Scopus (156) Google Scholar, 28Finazzi G. Büschlen S. de Vitry C. Rappaport F. Joliot P. Wollman F.-A. Biochemistry. 1997; 36: 2867-2874Crossref PubMed Scopus (39) Google Scholar) suggest that at least part of the second (c-d) extramembrane loop of cytochrome b 6 and of the first loop of subunit IV is located close to the Rieske protein. The allocation of the Rieske protein to density I and of parts of cytochrome b 6 and subunit IV to less intense features near the monomer-monomer interface would imply that site Qo, which is formed by these three subunits, might lie relatively close to the 2-fold axis of the dimer. Such a location would be consistent with the close apposition of the twob L hemes in the bc 1dimer, as revealed by x-ray diffraction (13Yu C.-A. Xia J.-Z. Kachurin A.M. Yu L. Xia D. Kim H. Deisenhofer J. Biochim. Biophys. Acta. 1996; 1275: 47-53Crossref PubMed Scopus (86) Google Scholar). The relative proximity of the two Qo sites and the two b Lhemes within the bc 1 andb 6 ƒ dimers opens up interesting vistas regarding the possibility of a functional cooperation between the two monomers (cf. Ref. 13Yu C.-A. Xia J.-Z. Kachurin A.M. Yu L. Xia D. Kim H. Deisenhofer J. Biochim. Biophys. Acta. 1996; 1275: 47-53Crossref PubMed Scopus (86) Google Scholar). It is also worth noting that removal of the Rieske protein is generally, even though not always, accompanied by the monomerization of the complex and vice versa (6Breyton, C., Tribet, C., Olive, J., Dubacq, J.-P. & Popot, J.-L. (1997)J. Biol. Chem., 272, in press.Google Scholar). The proposed positioning of the Rieske protein close to the monomer-monomer interface suggests a possible origin for the frequent correlation between these two events: destabilization could result from the loss of interactions between the Rieske protein of one monomer and neighboring subunit(s) of its partner. In conclusion, our results provide the first crystallographic information on the structure of the cytochromeb 6 f complex in its intact form. The 8 Å projection map of the enzyme has permitted proposals to be made regarding the location cytochrome f and the Rieske protein in the b 6 f dimer. In future work, subunit assignment will be further examined using the same approach to crystallization associated with labeling methods or with the removal of specific subunits (cf. Ref. 6Breyton, C., Tribet, C., Olive, J., Dubacq, J.-P. & Popot, J.-L. (1997)J. Biol. Chem., 272, in press.Google Scholar). Obtaining highly ordered crystals opens the way to the determination of a three-dimensional model of cytochrome b 6 f using cryo-electron microscopy. Establishing a three-dimensional model of the complex, although facing the problem of analyzing multilayered crystals, can be attempted by appropriate treatment of the crystallographic data and/or by modifications of the crystallization protocol favoring the growth of monolayered crystals. It should be stressed that even a moderate resolution three-dimensional map would be extremely useful in helping to delineate structural differences between the bc 1 andb 6 f complexes. We are extremely grateful to D. Picot (Institut de Biologie Physico Chimigue, Paris) for useful discussions." @default.
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