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W2104633154 abstract "Binding of stigmatellin, an inhibitor of the Qo site of the bc-type complexes, has been shown to induce large conformational changes of the Rieske protein in the respiratory bc 1 complex (Kim, H., Xia, D., Yu, C. A., Xia, J. Z., Kachurin, A. M., Zhang, L., Yu, L., and Deisenhofer, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8026–8033; Iwata, S., Lee, J. W., Okada, K., Lee, J. K., Iwata, M., Rasmussen, B., Link, T. A., Ramaswamy, S., and Jap, B. K. (1998) Science 281, 64–71; Zhang, Z., Huang, L., Shulmeister, V. M., Chi, Y. I., Kim, K. K., Hung, L. W., Crofts, A. R., Berry, E. A., and Kim, S. H. (1998) Nature 392, 677–684). Such a movement seems necessary to shuttle electrons from the membrane-soluble quinol to the extramembrane heme of cytochrome c 1. To see whether similar changes occur in the related photosyntheticb 6 f complex, we have studied the effect of the binding of stigmatellin to the eukaryoticb 6 f complex by electron crystallography. Comparison of projection maps of thin three-dimensional crystals prepared with or without stigmatellin, and either negatively stained or embedded in glucose, reveals a similar type of movement to that observed in the bc 1complex and suggests also the occurrence of conformational changes in the transmembrane region. Binding of stigmatellin, an inhibitor of the Qo site of the bc-type complexes, has been shown to induce large conformational changes of the Rieske protein in the respiratory bc 1 complex (Kim, H., Xia, D., Yu, C. A., Xia, J. Z., Kachurin, A. M., Zhang, L., Yu, L., and Deisenhofer, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8026–8033; Iwata, S., Lee, J. W., Okada, K., Lee, J. K., Iwata, M., Rasmussen, B., Link, T. A., Ramaswamy, S., and Jap, B. K. (1998) Science 281, 64–71; Zhang, Z., Huang, L., Shulmeister, V. M., Chi, Y. I., Kim, K. K., Hung, L. W., Crofts, A. R., Berry, E. A., and Kim, S. H. (1998) Nature 392, 677–684). Such a movement seems necessary to shuttle electrons from the membrane-soluble quinol to the extramembrane heme of cytochrome c 1. To see whether similar changes occur in the related photosyntheticb 6 f complex, we have studied the effect of the binding of stigmatellin to the eukaryoticb 6 f complex by electron crystallography. Comparison of projection maps of thin three-dimensional crystals prepared with or without stigmatellin, and either negatively stained or embedded in glucose, reveals a similar type of movement to that observed in the bc 1complex and suggests also the occurrence of conformational changes in the transmembrane region. 6-O-(N-heptylcarbamoyl)-methyl-α-d-glycopyranoside egg-l-α-phosphatidylcholine di-C18:1-phosphatidylglycerol contrast transfer function Most energy-transducing membranes contain a bc-type complex such as the cytochrome bc 1 complex or the cytochrome b 6 f complex. Members of this family couple electron transfer and proton translocation across the membrane. They also share some structural homologies, having three redox-active subunits in common: the Rieske iron-sulfur protein, a b-type cytochrome (cytochromeb or b 6), and a c-type cytochrome (cytochrome c 1 or f). Moreover, there is sequence homology between cytochromeb 6, subunit IV, and the Rieske protein of theb 6 f complex, and the N- and C-terminal parts of cytochrome b, and the Rieske protein of the bc 1 complex, respectively. Cytochromec 1 and cytochrome f, on the other hand, do not share any sequence homology. Spectroscopic and EPR properties of the hemes and of the iron-sulfur cluster are similar, although not identical, and some inhibitors affect both complexes equally (reviewed in Refs. 4.Hauska G. Nitschke W. Herrmann R.G. J. Bioenerget. Biomembr. 1988; 20: 211-228Crossref PubMed Scopus (131) Google Scholar, 5.Malkin R. Photosynth. Res. 1992; 33: 121-136Crossref PubMed Scopus (39) Google Scholar, 6.Furbacher P.N. Tae G.-S. Cramer W.A. Baltcheffsky H. Origin and Evolution of Biological Energy Conservation. VCH Publishers, Inc., New York1996: 221-253Google Scholar, 7.Cramer W.A. Black M.T. Widger W.R. Girvin M.E. Barber J. The Light Reactions. Elsevier Science Publishers B.V., Amsterdam1987: 447-493Google Scholar). The b 6 f complex is found in the thylakoid membrane of higher plants and algae and in the membrane of some cyanobacteria (for reviews, see Refs. 8.Widger W.R. Cramer W.A. Vasil I.K. Bogorad L. Molecular Biology of Plastids and the Photosynthesis Apparatus. Academic Press, Orlando, FL1991: 149-176Google Scholar, 9.Cramer 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, 10.Hope A.B. Biochim. Biophys. Acta. 1993; 1143: 1-22Crossref PubMed Scopus (132) Google Scholar, 11.Hauska G. Schütz M. Büttner M. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reactions. Kluwer Academic Publishers Group, Drodrecht, Netherlands1996: 377-398Google Scholar, 12.Wollman F.-A. Rochaix J.-D. Goldschmidt-Clermont M. Merchant S. Molecular Biology of Chlamydomonas reinhardtii: Chloroplasts and Mitochondria. Kluwer Academic Publishers Group, Dordrecht, Netherlands1998: 459-476Google Scholar). Besides the four high molecular weight subunits mentioned above, theb 6 f complex comprises four smaller subunits, PetG, PetL, PetM, and PetN (13.Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 14.Pierre Y. Popot J.-L. C. R. Acad. Sci. (Paris) Ser. III. 1993; 316: 1404-1409PubMed Google Scholar, 15.Haley J. Bogorad L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1534-1538Crossref PubMed Scopus (60) Google Scholar, 16.Takahashi Y. Rahire M. Breyton C. Popot J.-L. Joliot P. Rochaix J.-D. EMBO J. 1996; 15: 3498-3506Crossref PubMed Scopus (75) Google Scholar, 17.Hager M. Biehler K. Illerhaus J. Ruf S. Bock R. EMBO J. 1999; 18: 5834-5842Crossref PubMed Scopus (111) Google Scholar), that have no direct counterpart in the bc 1 complex. The cytochrome b 6 f is the middle component of the photosynthetic chain, coupling the electron transfer from photosystem II (via plastoquinol) to photosystem I (via plastocyanin or a c-type cytochrome). The most widely accepted mechanism for the bc-type complexes is the so-called “Q-cycle” mechanism proposed by Mitchell (18.Mitchell P. FEBS Lett. 1975; 57: 137-139Crossref Scopus (432) Google Scholar) and modified by Crofts et al. (19.Crofts A.R. Meinhardt S.W. Jones K.R. Snozzi M. Biochim. Biophys. Acta. 1983; 723: 202-218Crossref PubMed Scopus (323) Google Scholar). According to this model, two electron paths are distinguished within the complex as follows: a high potential chain, composed of the iron-sulfur cluster of the Rieske protein and the heme of the c-type heme, and a low potential chain, composed of the two hemes of the b-type cytochrome, the low potential b L and high potentialb H hemes. Two quinol/quinone-binding sites, located on opposite sides of the membrane, are also predicted: the Qo site, on the intermembrane space/lumenal side of the membrane, and the Qi site, on the matrix/stromal side of the membrane. A turnover of the Qo site comprises the oxidation of a two-electron carrier quinol, one electron reducing the Rieske protein, which in turn reduces the c-type cytochrome and which ultimately reduces plastocyanin or cytochrome c, and the other electron reducing b L, which in turn is oxidized by b H. These events are associated with the release of two protons in the intermembrane space/lumen. A second turnover reduces another plastocyanine molecule and places both hemes of the b-type cytochrome in a reduced state. The reduction of a quinone at the Qi site results in the oxidation of the two b hemes and an uptake of two protons from the matrix/stroma. Thus, these complexes contribute to generating the proton gradient that drives ATP synthesis in respiration and photosynthesis. Recently, the structure of several bc 1 complexes from mitochondria have been determined by x-ray crystallography (1.Kim H. Xia D., Yu, C.A. Xia J.Z. Kachurin A.M. Zhang L., Yu, L. Deisenhofer J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8026-8033Crossref PubMed Scopus (259) Google Scholar, 2.Iwata S. Lee J.W. Okada K. Lee J.K. Iwata M. Rasmussen B. Link T.A. Ramaswamy S. Jap B.K. Science. 1998; 281: 64-71Crossref PubMed Scopus (1068) Google Scholar, 3.Zhang Z. Huang L. Shulmeister V.M. Chi Y.I. Kim K.K. Hung L.W. Crofts A.R. Berry E.A. Kim S.H. Nature. 1998; 392: 677-684Crossref PubMed Scopus (938) Google Scholar), supporting the Q-cycle model. Depending on the inhibitor binding at the Qo site, different conformations of the extramembrane domain of the Rieske protein are observed: in the presence of stigmatellin, the iron-sulfur cluster is within electron transfer distances of the Qo site (the “proximal” position), whereas in the presence of myxothiazol or methoxyacrylate stilbene, it is closer to the heme of cytochrome c 1 (the “distal” position); in the native structure (i.e. in the absence of inhibitor) it is found either in an intermediate position (1.Kim H. Xia D., Yu, C.A. Xia J.Z. Kachurin A.M. Zhang L., Yu, L. Deisenhofer J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8026-8033Crossref PubMed Scopus (259) Google Scholar, 2.Iwata S. Lee J.W. Okada K. Lee J.K. Iwata M. Rasmussen B. Link T.A. Ramaswamy S. Jap B.K. Science. 1998; 281: 64-71Crossref PubMed Scopus (1068) Google Scholar, 20.Xia D., Yu, C.A. Kim H. Xia J.Z. Kachurin A.M. Zhang L., Yu, L. Deisenhofer J. Science. 1997; 277: 60-66Crossref PubMed Scopus (873) Google Scholar) or in a distal position (3.Zhang Z. Huang L. Shulmeister V.M. Chi Y.I. Kim K.K. Hung L.W. Crofts A.R. Berry E.A. Kim S.H. Nature. 1998; 392: 677-684Crossref PubMed Scopus (938) Google Scholar). It is deduced from these data that the extramembrane domain of the Rieske protein undergoes a large scale movement to shuttle the electron from the quinol to the cytochrome c 1. This hypothesis has been confirmed by fluorescence quenching as a function of the redox potential between two fluorescent probes attached to the Rieske protein and cytochrome b of the bc 1 complex of Rhodobacter sphaeroides (21.Crofts A.R. Berry E.A. Kuras R. Guergova-Kuras M. Hong S. Ugulava N. Garab G. XIth International Congress on Photosynthesis. 3. Kluwer Academic Publishers, Budapest, Hungary1998: 1481-1486Google Scholar). EPR studies on theb6f complex of spinach suggest different conformations for the extramembrane domain of the Rieske protein as well (68.Schoepp B. Brugna M. Riedel A. Nitschke W. Kramer D.M. FEBS Lett. 1999; 450: 245-250Crossref PubMed Scopus (52) Google Scholar). Although bc 1 andb 6 f complexes are similar with regard to function and structure, the differences that occur do not allow direct transposition of structural information from thebc 1 to theb 6 f (see e.g.Refs. 22.Kuras R. Guergova-Kuras M. Crofts A.R. Biochemistry. 1998; 37: 16280-16288Crossref PubMed Scopus (19) Google Scholar, 24.Schoepp B. Chabaud E. Breyton C. Verméglio A. Popot J.-L. J. Biol. Chem. 2000; 275: 5275-5283Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, and 25.Joliot P. Joliot A. Biochemistry. 1998; 37: 10404-10410Crossref PubMed Scopus (27) Google Scholar for recent work on structural, spectroscopic, and functional differences 1Y. Pierre, P. Hervé, and J.-L. Popot, submitted for publication. and Refs. 4.Hauska G. Nitschke W. Herrmann R.G. J. Bioenerget. Biomembr. 1988; 20: 211-228Crossref PubMed Scopus (131) Google Scholar, 5.Malkin R. Photosynth. Res. 1992; 33: 121-136Crossref PubMed Scopus (39) Google Scholar, 6.Furbacher P.N. Tae G.-S. Cramer W.A. Baltcheffsky H. Origin and Evolution of Biological Energy Conservation. VCH Publishers, Inc., New York1996: 221-253Google Scholar, 7.Cramer W.A. Black M.T. Widger W.R. Girvin M.E. Barber J. The Light Reactions. Elsevier Science Publishers B.V., Amsterdam1987: 447-493Google Scholar for reviews). In contrast to the bc 1complex, no good three-dimensional crystals of theb 6 f complex could be grown to date to allow the calculation of a detailed structure of the complex. Atomic models of the soluble domains of the Rieske protein from spinach (26.Carrell C.J. Zhang H. Cramer W.A. Smith J.L. Structure. 1997; 5: 1613-1625Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) and of cytochrome f from turnip and Phormidium laminosum (27.Martinez 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, 28.Martinez S.E. Huang D. Ponomarev M. Cramer W.A. Smith J.L. Protein Sci. 1996; 5: 1081-1092Crossref PubMed Scopus (132) Google Scholar, 29.Carrell C.J. Schlarb B.G. Bendall D.S. Howe C.J. Cramer W.A. Smith J.L. Biochemistry. 1999; 38: 9590-9599Crossref PubMed Scopus (72) Google Scholar) are available, but the only crystallographic information available from the whole complex are projection maps in negative stain (30.Mosser G. Breyton C. Olofsson A. Popot J.L. Rigaud J.L. J. Biol. Chem. 1997; 272: 20263-20268Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and of frozen hydrated samples at ∼9 Å resolution (31.Bron P. Lacapère J.J. Breyton C. Mosser G. J. Mol. Biol. 1999; 287: 117-126Crossref PubMed Scopus (21) Google Scholar) of the b 6 fcomplex of Chlamydomonas reinhardtii. These maps are derived from electron crystallography of assimilated two-dimensional crystals. This technique allows us to calculate density maps, i.e. to visualize the protein either in projection or in three dimensions (see Refs. 32.Yeager M. Unger V.M. Mitra A.K. Methods Enzymol. 1999; 294: 135-180Crossref PubMed Scopus (35) Google Scholar and 33.Walz T. Grigorieff N. J. Struct. Biol. 1998; 121: 142-161Crossref PubMed Scopus (75) Google Scholar for recent reviews and Ref. 34.Amos L.A. Henderson R. Unwin P.N. Prog. Biophys. Mol. Biol. 1982; 39: 183-231Crossref PubMed Scopus (409) Google Scholar for more details on the technique). Depending on the quality of the crystals, low (∼15 Å) to high (4–3 Å) resolution data can be obtain. Low to medium resolution maps will outline domains of the protein, whereas atomic models can be built into higher resolution data (35.Mitsuoka K. Hirai T. Murata K. Miyazawa A. Kidera A. Kimura Y. Fujiyoshi Y. J. Mol. Biol. 1999; 286: 861-882Crossref PubMed Scopus (238) Google Scholar, 36.Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1798) Google Scholar, 37.Kühlbrandt W. Wang D.N. Fujiyoshi Y. Nature. 1994; 367: 614-621Crossref PubMed Scopus (1538) Google Scholar). In this work, we have looked for inhibitor-induced conformational changes on the b 6 f complex using electron crystallography. We show that the binding of stigmatellin to the b 6 fcomplex either prior to crystallization or on the preformed crystals induces changes in the projected structure of the complex. A conformational change following stigmatellin binding is seen both in negatively stained samples, where the information comes mainly from the extramembrane part of the protein, and in samples embedded in glucose, where the whole molecule contributes to the signal. We conclude that a similar movement of the Rieske protein occurs in theb 6 f complex as in thebc 1 complex to shuttle the electron from the plastoquinol to the cytochrome f. However, our data also suggest that this movement is accompanied by conformational changes of the transmembrane region that would be specific to theb 6 f complex. The cytochromeb 6 f complex from C. reinhardtii was purified as described in Ref. 13.Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar. Briefly, specific solubilization of the thylakoid membranes by 6-O-(N-heptylcarbamoyl)-methyl-α-d-glycopyranoside (Hecameg)2 is followed by sucrose gradient and hydroxylapatite chromatography. The two last steps of purification must be performed in the presence of lipids (eggl-α-phosphatidylcholine (PC)) and at the critical micellar concentration of the detergent to prevent dissociation of the complex (38.Breyton C. Tribet C. Olive J. Dubacq J.P. Popot J.L. J. Biol. Chem. 1997; 272: 21892-21900Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The crystallization of the complex was performed as described in Refs.30.Mosser G. Breyton C. Olofsson A. Popot J.L. Rigaud J.L. J. Biol. Chem. 1997; 272: 20263-20268Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar and 31.Bron P. Lacapère J.J. Breyton C. Mosser G. J. Mol. Biol. 1999; 287: 117-126Crossref PubMed Scopus (21) Google Scholar, except that no calcium was added to the crystallization medium. Briefly, PC and di-C18:1-phosphatidylglycerol (DOPG) solubilized in Hecameg were added (PC/DOPG = 1) to the purified protein (10 μm) at a protein/lipid ratio of 0.3 to 1.2 (w/w). The mixture was allowed to equilibrate overnight, and the detergent was removed by the addition of 25 g/100 ml of SM2-Bio-Beads for 6 h at 4 °C. After overnight sedimentation of the reconstituted material, samples were frozen (−196 °C) and thawed (4 °C) three times, and crystals were harvested 1–5 days later. C13-stigmatellin, kindly synthesized by P. Hervé and P. Fellmann (CNRS UPR 9052, IBPC, Paris, from Ref. 39.Höfle G. Kunze B. Zorzin C. Reichenbach H. Liebigs Ann. Chem. 1984; 12: 1883-1904Crossref Scopus (41) Google Scholar), solubilized in 96% ethanol, was added (100 μm) either before or after crystallization. When added to preformed crystals, the incubation time ranged from 2 min to several days at 4 °C. It was checked that the presence of 2% ethanol in the crystallization medium did not alter nor modify crystallization. For negative stain studies, 2.5 μl of the crystal suspension were allowed to sit for 2 min on a 400-mesh carbon-coated copper grid. Excess of solution was blotted off, and the grid was washed 10 s with 2.5 μl of water to remove the ammonium phosphate present in the crystallization buffer, before being stained with two successive washes of 1% uranyl acetate (30 s each). Negative staining is mainly used to increase the contrast of the sample and to allow fast screening of crystallization conditions. The protein is embedded in a thin layer of a heavy metal salt outlining the protein, like a replica or a cast. As the stain is much denser than the protein, an image of a negatively stained crystal will be dominated by the information coming from the stain; the protein will be seen in negative. The resolution attained by such images is limited by the stain, and maps of negatively stained crystals are usually not calculated beyond ∼15 Å. At such a resolution, only the low resolution features as large domains of the protein are resolved. In the case of membrane proteins with large extramembrane domains, this method can give interesting data; as the stain does not usually penetrate the interior of the membrane (the heavy atoms are hydrophilic), only the extrinsic domains of the protein will be outlined. The projection map will thus be dominated by the extramembrane part of the protein. For electron cryo-microscopy, grids were prepared using the back injection method, i.e. 2.5 μl of sample were injected into a 2–6% glucose solution on a carbon-coated copper grid, blotted for 15 s face on the filter paper, and frozen in liquid nitrogen where samples were stored until examined. This method, rather than plunging the sample in liquid ethane as described previously (31.Bron P. Lacapère J.J. Breyton C. Mosser G. J. Mol. Biol. 1999; 287: 117-126Crossref PubMed Scopus (21) Google Scholar), improved the yield of diffracting crystals. In this case, it is now the whole protein that contributes to the information contained in the image, so that in a projection map, extramembrane features as well as transmembrane elements will overlap. Because of the presence of the glucose though, the low resolution contribution of the extramembrane part of the protein will be lessened due to contrast matching between the glucose and the protein (40.Kühlbrandt W. Ultramicroscopy. 1982; 7: 221-232Crossref PubMed Scopus (36) Google Scholar, 41.Grigorieff N. Beckmann E. Zemlin F. J. Mol. Biol. 1995; 254: 404-415Crossref PubMed Scopus (49) Google Scholar). Moreover, in the case of an α-helical membrane protein, helices perpendicular to the membrane will be projected as strong density peaks at 10–7 Å resolution, whereas tilted helices, loops, or β-sheets will contribute less strongly to the projection map. These projection maps will thus be dominated by the information coming from the transmembrane helices. Electron micrographs of stained or unstained crystals were recorded on a CM12 or a CM120 electron microscope at 120-kV acceleration and at magnifications of × 45,000–60,000 x. A GATAN cold stage was used for unstained specimens. Images were taken with a 1-s exposure time using the low dose facility of the microscopes, at a total electron dose of ∼10 e·Å−2 in flood beam mode. Other cryo-grids were mounted in a Leica KF80 apparatus for transfer into a JEOL 3000 SFF electron microscope operated at 300 kV, equipped with a top entry cryo-stage cooled to 4 K with liquid helium. Images were then taken using a spot-scan procedure with a total electron dose of 20–30 e·Å−2, at magnifications of × 53,000–70,000, on a 0° tilt holder. In all cases Kodak SO-163 films were used to record images and were developed for 12 min with full-strength Kodak D19. 2 (JEOL) + 5 (CM120) images were used to calculate the native cryo-map and 4 (JEOL) + 2 (CM120) were used to calculate the stigmatellin cryo-map. It was checked by comparing individual images that the differences observed did not come from the unequal number of images taken with each microscope. Optical diffraction was used to screen the micrographs, and areas giving rise to symmetrical distribution of sharp spots were digitized using a Zeiss SCAI scanner with a 7-μm step size over an area of 6,000 × 6,000 pixels. Images were then corrected for distortion of the crystal lattice and effects of the contrast transfer function (CTF) using the MRC image-processing programs (42.Crowther R.A. Henderson R. Smith J.M. J. Struct. Biol. 1996; 116: 9-16Crossref PubMed Scopus (665) Google Scholar). Amplitude correction for the CTF was not made, as the low resolution amplitudes are particularly strong with these crystals, probably due to their three-dimensional nature. Projection maps were calculated from merged data, imposing the p22121 symmetry revealed by the program ALLSPACE (43.Valpuesta J.M. Carrascosa J.L. Henderson R. J. Mol. Biol. 1994; 240: 281-287Crossref PubMed Scopus (145) Google Scholar) and already determined in previous work (30.Mosser G. Breyton C. Olofsson A. Popot J.L. Rigaud J.L. J. Biol. Chem. 1997; 272: 20263-20268Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 31.Bron P. Lacapère J.J. Breyton C. Mosser G. J. Mol. Biol. 1999; 287: 117-126Crossref PubMed Scopus (21) Google Scholar), using the standard crystallographic computer programs in the CCP4 package (44.Collaborative Computational Project No. 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19768) Google Scholar). Projection maps were scaled to a maximum density of 250 and contoured in steps of 0.25 × root mean square density. A negative temperature factor of −600 was applied in Fig. 3 to compensate partially for the resolution-dependent degradation of image-derived amplitudes. This value is within the range of values usually used at this resolution (45.Unger V.M. Schertler G.F.X. Biophys. J. 1995; 68: 1776-1786Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 46.Unger V.M. Kumar N.M. Gilula N.B. Yeager M. Nature Struct. Biol. 1997; 4: 39-43Crossref PubMed Scopus (107) Google Scholar). Difference maps were calculated from the vectorial difference between structure factors for the native crystals and the crystals with stigmatellin, after scaling the two merged data sets to the same total intensity. The background level was estimated by calculating a difference map between two sets of images resulting from halving the data set of the images of native crystals, and 2 × root mean square density value from this difference was used to contour the difference maps. Fig. 1 shows the projection maps in negative stain truncated at 15 Å resolution obtained from images of native crystals (Fig. 1 A) and of crystals grown in the presence of stigmatellin (Fig. 1 B). In both cases, theb 6 f is present as a dimer, the unit cell dimensions are similar, and the crystallographic statistics show that both maps are of comparable quality, allowing further comparisons to be made (TableI).Table ICrystallographic data for negatively stained images−stigmatellin+stigmatellinUnit cell parametersa = 178 Å ± 3a = 182 Å ± 3b = 72 Å ± 2b = 74 Å ± 2γ = 90.2° ± 0.4γ = 90.0° ± 0.3Number of images76Number of unique reflections (to IQ 7)4344Phase error to 15 Å after rounding to 0/180° (IQ 7 max, 45° is random)8.9°14.1° Open table in a new tab Within a monomer, two densities (labeled I and IIin Fig. 1) are resolved. Whereas density I does not show a major change in its position, density II appears to be shifted away from the 2-fold dimer axis and is more compact and round when stigmatellin is bound to the complex. The change in position is confirmed by the difference map (native − stigmatellin) shown in Fig. 1 C, where positive signal represents a density present in the native map but absent in the stigmatellin map. The most prominent signals (labeled a andb in Fig. 1 C) reflect the shift of density II away from the dimer axis induced by the binding of stigmatellin. The same behavior was observed when using phosphotungstic acid and ammonium molybdate as negative stains (data not shown), as well as when the stigmatellin was added on the preformed native crystals (data not shown). Thus, upon binding of stigmatellin, one of the densities of theb 6 f complex projection map in negative stain undergoes a major change in its position. It should be mentioned that in the crystals grown in the presence of stigmatellin, two type of images could be distinguished as follows: a set of images that merged, resulting in the projection map shown in Fig. 1 B, and another set of images, with the same lattice parameters, that after merging resulted in an identical map except for an additional large density between the dimers along the bdirection. These images have not been taken into account in this analysis, for it was not visible in maps calculated from crystals stained with phosphotungstic acid or ammonium molybdate. To visualize further and to confirm the conformational change seen in the negative stain study, analysis of the crystals grown with or without stigmatellin was done on unstained crystals. Fig.2 A shows a computed Fourier transform of an image of a crystal grown in the presence of stigmatellin, embedded in glucose, and observed in a JEOL 3000 SFF microscope at liquid helium temperature. The transform shows significant reflections up to 9 Å resolution. The phase errors associated with each of the Fourier components after merging six images, plotted in Fig. 2 B, show that the data are complete and isotropic to 9 Å resolution. The overall phase residual to 9.0 Å resolution was 18.8° (Table II). Fig.3 B shows the projection map at 9 Å resolution of b 6 fcrystals grown in the presence of stigmatellin. As previously published (31.Bron P. Lacapère J.J. Breyton C. Mosser G. J. Mol. Biol. 1999; 287: 117-126Crossref PubMed Scopus (21) Google Scholar), the complex crystallizes as a dimer, and within the molecular boundaries, sharp and round densities surrounded by more elongated densities are resolved. These densities have diameters and spacings that could be compatible with the presence of transmembrane α-helices, but one should keep in mind that theb 6 f complex has two large extramembrane domains (of cytochrome f and of the Rieske protein) that will also contribute to the map (for examples of comparison between projection and three-dimensional maps of proteins that have extramembrane domains, see Refs. 47.Gohlke U. Warne A. Saraste M. EMBO J. 1997; 16: 1181-1188Crossref PubMed Scopus (29) Google Scholar and 48.Iwata S. Ostermeier C. Ludwig B. Michel H. Nature. 1995; 376: 660-669Crossref PubMed Scopus (1983) Google Scholar for cytochromec oxidase and Refs. 49.Cyrklaff M. Auer M. Kühlbrandt W. Scarborough G.A. EMBO J. 1995; 14: 1854-1857Crossref PubMed Scopus (55) Google Scholar and 50.Auer M. Scarborough G.A. Kühlbrandt W. Nature. 1998; 392: 840-843Crossref PubMed Scopus (185) Google Scholar for the H+-ATPase).Table IICrystallographic data for cryo images−Stigmatellin+StigmatellinUnit cell parametersa = 178 Å ± 6a = 180 Å ± 4b = 72 Å ± 2b = 74 Å ± 1γ = 89.8° ± 0.6γ = 90.2° ± 0.6Number of images76Range of defocus3,200–10,300 Å5,000–10,000 ÅNumber of unique reflectionsaTaking reflections to IQ 7. (to 10 Å)105105Overall phase residual to 10 Å after rounding to 0/180° (IQ 7 max, 45° is random)17.8°16.9°+Stigmatellin resolution rangeNo. of unique reflectionsaTaking reflections to IQ 7.Phase residual (IQ 7 max, 45° is random)Å200.0–18.0347.6°18.0–12.73118.1°12.7–10.43223.6°10.4–8.93029.0°8.9–8.02347.1°200–9.012518.8°a Taking reflections to IQ 7. Open table in a new tab A projection map of the native complex was calculated (Fig. 3 A). Even though the crystals were embedded in glucose and image-derived amplitudes were used to calculate the map rather than diffraction amplitudes, it compares well with the map previously published (31.Bron P. Lacapère J.J. Breyton C. Mosser G. J. Mol. Biol. 1999; 287: 117-126Crossref Pu" @default.
W2104633154 created "2016-06-24" @default.
W2104633154 creator A5088857591 @default.
W2104633154 date "2000-05-01" @default.
W2104633154 modified "2023-09-27" @default.
W2104633154 title "Conformational Changes in the Cytochromeb 6 f Complex Induced by Inhibitor Binding" @default.
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W2104633154 doi "https://doi.org/10.1074/jbc.275.18.13195" @default.
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