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Pumping Ions © 1997 Nature Publishing Group http://www.nature.com/nsmb • picture story Pumping ions Membrane proteins present a particular chal­ tive to the charge distribution in the crystals. chrotron Radiation Facility (ESRF) and have lenge to structural biologists. Good three or Atoms carrying negative charges have nega­ yielded a structure of BR at 2.5 A resolution two-dimensional crystals are the most impor­ tive electron scattering factors at low resolu­ which has just been published 7• The X-ray tant prerequisite for any direct insight into their tion. Therefore negatively charged aspartates structure was solved by molecular replace­ atomic structure and molecular mechanisms, and glutamates should in principle show up ment, using the atomic coordinates deter­ yet membrane proteins are notoriously difficult less well than other side chains. This effect mined by electron crystallography3 as a to crystallize. One of the few exceptions is had already been noted in the 3.4 A map of search model. It is therefore not surprising bacteriorhodopsin (BR) which forms near-per­ the plant light harvesting complex5 which that the two structures are very similar overall, fect two-dimensional crystals already in the cell although the X-ray map contains small membrane of a salt-loving bacterium where it regions of density in the proton-pumping pumps protons out of the cell, using sunlight as channel which have been interpreted as water a source of energy. These 2D crystals are ideally molecules 7• It is surprising, however, that suited for structure analysis by electron some of the loops which show up clearly in microscopy and image analysis. Accordingly BR the 3 A structure reported by Kimura et al.4 was the first membrane protein for which a are less well defined or completely absent in three-dimensional structure was determined the X-ray map 7• Since it is unlikely that these originally at 7 A (ref. 1). Throughout the past loops are more highly ordered in two-dimen­ 20 years, BR has fueled the development of sional crystals, this observation suggests that new and better electron crystallographic tech­ a 3 A map with good phases, determined niques at ever higher resolution. Today, it is one directly by electron crystallography, contains of the most-investigated and best-understood as much, or more structural detail than a 2.5 A of all membrane proteins. An atomic model X-ray map. based on a 3.5 A 3D map was published in Electron crystallography of two-dimension­ 19902, followed by the refined structure3 and al crystals is now an established method. now by a 3 A map•. Although it does not yet produce structures at The ground-breaking work by Henderson the same alarming rate as X-ray crystallogra­ and colleagues2 had shown the structure of phy of soluble proteins, it has a firm place in the seven trans-membrane helices very clearly the arsenal of structural biologists when it and resulted in a detailed model of proton Fig. 1 A ribbon diagram of the 3 A structure o f comes to tackling macromolecules that do pumping. However, the loops connecting the bacteri orhodopsin, determined by electron not form well-ordered 3D crystals easily, in microscopy of two-dimensional crystals'. helices on the membrane surface were not Charged residues on the inner (top) and outer particular filamentous and membrane pro­ resolved and were thought to be disordered. surfaces and in the proton channel are circled. teins. In the near future we will see several In the paper now published by Kimura et al.4, The likely pathway of protons being pumped more exciting and unique structures deter­ through the membrane (light blue) is indicated the loops are almost as clearly seen as the by green arrows. The retinal bound in the cen­ mined by this powerful technique. trans-membrane regions. This is due to two tre o f the a-helical bundle is purple, and the technical advances. For one, Kimura et al. pre­ newly visualized 13-sheet structure on the extra­ cellular side is light green. This figure was kind­ Werner KOhlbrandt pared very flat specimens of the two-dimen­ ly provided by Y. Kimura (ref. 4). Max-Planck-lnstitut tor Biophysik, Heinrich-Hoffmann­ sional BR crystals which enabled them to Str. 7, D-60528 Frankfurt/ Main, Germany. collect data at higher tilt angles than before. They also used an electron microscope capa­ was determined by the same method. How­ Correspondence should be addressed to WK. e-mail: ble of recording higher resolution detail, by a ever, some of the asparates and glutamates kueh/[email protected] combination of low specimen temperature on the BR surface which have well-defined 1. Henderson R., & Unwin, P. N. T. Three-dimensional (close to 4K), high voltage (300 kV) and the densities in the 3 A map and are tentatively model of purple membrane obtained by electron Nature, 28-32 (1975). extra benefit of a highly coherent electron presented as uncharged by Kimura et al., microscopy. 257, 2. Henderson R. et al. A model for the structure of beam produced by a field emission gun. seem to be exposed to the aqueous medium bacteriorhodopsin based on high resolution Together these factors account for the greater and are unlikely to be protonated at neutral electron cryo-microscopy. J. Mal.Biol., 213, 89'l-929 (1990). completeness of the data and thus for the pH . It is therefore not yet clear whether elec­ 3. Grigorieff, N., Ceska, T.A., Downing, K.H., Baldwin, better definition of their map. tron crystallography can actually tell the J.M., & Henderson, R. Electron-crystallographic refinement of the structure of bacteriorhodopsin. /. Both membrane surfaces show the strate­ charge of an amino acid side chain, or Mot. Biol. 259, 393-421 (1996). gic location of charged residues around the whether the effect is partly obscured by noise. 4. Kimura, Y. et al. Surface of bacteriorhodopsin revealed by high-resolution electron crystallography. entrance and exit of the proton channel. It is Bacteriorhodopsin, probably the most sim­ Nature 389, 206-211 . likely that these side chains help to conduct ple and one of the most ancient ion pumps in 5. KGhlbrandt, W., Wang, D. N., & Fujiyoshi, Y. Atomic cytoplasmic living cells, continues to fascinate structural model of plant light-haivesting complex by electron protons to the channel on the crystallography. Nature , 367 61,i....;21 (1994) , side and disperse them on the exoplasmic biologists and to inspire new technological 6. Landau, E. M. , & Rosenbusch, J.P. lipidic cubic side (Fig. 1). The authors draw attention to a developments. Very recently, tiny but well­ phases: A novel concept for the crystallization o1 membrane proteins. Proc. Natl. Acad. Sci., 93, potential feature of their map which may ordered three-dimensional crystals of BR were 14532-14535 (1996). enable them to distinguish negatively grown in a cubic lipid phase6• These crystals 7. Pebay-Peyroula, E., Rummel, G., Rosenbusch, J., & Landau, E.M. X-ray structure of bactiorhodopsin at charged residues from uncharged ones: since were found to diffract X-rays to 2 A in the 2.5 A from microcrystals grown in lipidic cubic electrons are charged particles, they are sensi- microfocus beamline at the European Syn- phases, Science 277, 1676--1681(1997}. nature structural biology • volume 4 number 1O • october 1997 773 .
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