DOI: 10.1021/cg101282z
Published as part of the Crystal Growth & Design virtual special issue on the 13th International Conference on the Crystallization of Biological Macromolecules (ICCBM13).
2011, Vol. 11 627–631
Structure Determination without Crystals: The Ribosome, 1970-2000 Peter B. Moore* Department of Chemistry, Yale University, P.O. Box 208107, New Haven, Connecticut 06520-8107, United States Received September 29, 2010; Revised Manuscript Received December 6, 2010
ABSTRACT: From ∼1970 to 2000, the year its crystal structure was solved, no biological structure was investigated more intensely than the ribosome. Some of the structural experiments done in the precrystallographic era produced information that significantly advanced our understanding of that enzyme. Other approaches yielded little that would be regarded as useful today, even if crystal structures had never been obtained. That experience is reviewed here to provide guidance for those faced with similar challenges in the future.
Introduction This essay conveys the essence of a short talk that was given by the author at the 13th International Conference on the Crystallization of Biological Macromolecules in Dublin, Ireland, in September, 2010. That meeting’s organizers wanted someone to review the work done to elucidate the three-dimensional structure of the ribosome prior to the announcement of its crystal structure in 2000. They were of the opinion that those struggling with similarly difficult macromolecular structures today might benefit from finding out which of the many such experiments that were done on the ribosome proved useful, and which did not. It is left to the reader to decide whether the organizers were right about this. Ribosomes were identified as the sites of protein synthesis in all organisms in the 1950s, and by ∼1962, their physiological role was properly understood. Ribosomes are ribonucleoproteins, and had they been named on the basis of their function rather than their chemical compositions, the ribosome might be known today as “polypeptide polymerase”. The substrates ribosomes act on are aminoacyl tRNAs, and ribosomes assemble polypeptides from them, the sequences of which are determined by base-pairing interactions between aminoacyl tRNAs and mRNA templates that ribsomes mediate. From the late 1960s until the late 1990s, when it became clear that high resolution crystals structures would shortly appear, more than half the research done on ribosomes was directed at the elucidation of their three-dimensional structures by methods other than whole-particle crystallography. Every approach that could be thought of was tried, and that work continued unabated for 20 years after ribosome crystals were first reported because, for most of that time, it was not not clear that high resolution structures would ever be obtained from them. Thus, it remained possible for a long time that the information provided by noncrystallographic experiments would be
all that we would ever know about the three-dimensional organization of the ribosome. The ribosome community has held conferences at odd intervals over the years, and the one that took place in Denmark in the summer of 1999 marked a watershed in the field because it was at that meeting that the community finally realized that atomic resolution crystal structures would soon appear. The dismay of those who had devoted their lives to other methods of structure determination was palpable. By the time the meeting ended, there were a lot of people considering major career changes (see ref 1). What appears below is a brief summary of the work those scientists did and my assessment of its ultimate value. This commentary is by no means a comprehensive guide to the relevant literature; only a few references are provided. Those interested in a more complete account should consult Rheinberger’s recent history of the ribosome field, which is excellent.2 Taken as a whole, experience with the ribosome field confirms that crystallography remains the method of choice for determining the structures of biological macromolecules at atomic resolution, which will come as no surprise. However, some readers may be surprised to learn that the ribosome experience strongly suggests that electron microscopy will soon become a viable alternative. Physical Approaches to Ribosome Structure
*To whom correspondence should be addressed. E-mail: peter.moore@ yale.edu.
Ultracentrifugation. Both physical and biochemical experiments were done to elucidate the structure of the ribosome prior to 2000, and for historical reasons it makes sense to start by discussing what physical investigations contributed to our understanding of the ribosome prior to the determination of their crystal structures. In the 1950s, the ultracentrifuge was the most powerful tool available to the physical biochemist, and studies done with the ultracentrifuge played an important role in the discovery of the ribosome. Sedimentation studies done on cell lysates demonstrated that they contain a large population
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Figure 1. The locations of ribosomal protein in the small ribosomal subunit from E. coli as determined by neutron scattering.3 Proteins are shown as spheres having radii that correspond to their molecular weights and are identified using standard numbering. The protein array is shown from both its cytoplasmic side (left) and its subunit interface side (right).
of (apparently) homogeneous high molecular weight particles that turned out to be ribonucleoproteins. We call them ribosomes today. By the middle of the decade, it was realized that ribosomes invariably consist of two unequal subunits that associate when the magnesium ion concentration is high, and dissociate when it is low. In bacteria, their molecular weights are about 750 000 and 1 500 000, respectively, and their hydrodynamic properties suggested that the complex the two subunits form is roughly spherical, which is so. Solution Scattering. Starting in the late 1960s, a lot of experiments were done on the ribosome using small-angle X-ray (SAXS) and neutron scattering (SANS) techniques. These studies provided a somewhat more refined sense of the overall shapes of the ribosome and its two subunits, and certainly influenced those who subsequently developed the programs now widely used to extract molecular shape information from small angle solution scattering data. However, on the whole, they provided little that was of lasting value to the ribosome field. The most complicated solution scattering experiment ever done on the ribosome was the SANS experiments carried out by Donald Engelman and me at Yale. The bacterial ribosome can be reconstituted from its macromolecular components, all of which can be produced in (nearly) perdeuterated form by growing bacteria on appropriately labeled media. Thus, it was possible to prepare small ribosomal subunits in which pairs of ribosomal proteins were selectively deuterated so that the scattering from those proteins could be measured directly, and the distances between their centers of mass estimated from the interference ripple all such two-protein scattering curves include. The end result was a map showing the relative location of the 21 proteins in the small ribosome subunit from Escherichia coli (Figure 1).4 After the crystal structure of the small subunit was determined, we could find out how well we had done. Figure 2 is correlation plot that compares SANS-determined distances with crystallographic distances. While the correlation as a whole is quite convincing, it is clear that “mistakes were made”. Furthermore, we never solved a problem fundamental to this kind of distance-based structure determination, namely, how to find all the arrangements of objects in space that are plausibly compatible with a given set of distances, given their associated errors.
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Figure 2. Comparison of the distances between proteins centers of gravity in the small ribosomal subunit obtained by neutron scattering with crystallographically determined center of gravity distances. The error bars shown are those estimated for each of the neutrondetermined distances plotted. Reprinted from ref 5 with permission. Copyright 2001 Cold Spring Harbor Laboratory Press.
In those same years, a second attempt was made to use the reconstitutability of the ribosome as the means for determining the arrangement of proteins in the small ribosomal subunit. The difference between this approach, which was carried out by Cantor and his colleagues, and the SANs approach just described is that the distance data required was to be obtained by fluorescence resonance energy transfer (FRET) measurements, rather than neutron scattering.6 Technically, this approach was even more difficult to execute than its SANs equivalent, not the least of its drawbacks being the fact that many ribosomal proteins have long, extended “tails”. Purified proteins were labeled covalently with chromophores in the denatured state, and in that era there was no way to control which residues of a protein reacted. If the tail of some protein got labeled, the FRET data that emerged when that molecule was used to measure distances might have little to say about the location of its center of mass. Electron Microscopy. Elecron microsocopy is by far the most important of the noncrystallographic physical techniques that has been applied to the ribosome, and the contributions it made to our understanding of these particles prior to 2000 were substantial. For example, electron micrographs of sectioned tissue and cell extract preparations taken in the 1950s by investigators such as Palade and Siekevitz played an important role in the discovery of the ribosome. Electron microscopy is still making major contibutions to the field. A remarkably accurate sense of the three-dimensional shape of the ribosome emerged from the analysis of negative stained images of purified ribosomes, the first of which were produced by Zubay and Huxley in 19607 (Figure 3). By the mid1970s, guided by intuition, James Lake had arrived at a sound understanding of the shapes of the two ribosomal subunits and their relationship in the intact ribosome (Figure 4).8 Along with Georg Stoeffler, Lake also determined the locations on the surface of the ribosome where the epitopes of specific ribosomal proteins are found by examining the complexes ribosomes form with the appropriate antibodies in negative stain. (It is surprising to me how little this technique has been used by others interested in the quaternary structures of other macromolecular complexes.) The ribosome has been the favorite object of those concerned with the development of single particle reconstruction techniques for the determination of macromolecular structures by
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electron microscopy. The technical challenges in this area are huge, but Joachim Frank and others working in this field have made remarkable progress over the last 15 years. Figure 5 is a view of a 5.5 A˚ resolution image of a bacterial ribosome that agrees very well with the crystal structures now available for these particles. As a method for determining structures at high resolution, single particle reconstruction has several significant advantages over X-ray crystallography. The amounts of material it requires are several orders of magnitude smaller, and not only can it cope with conformationally heterogeneous samples, it can be used to characterize the nature of that heterogeneity. Since 2000, intermediate resolution electron density maps produced this way have provided a lot of valuable information about the conformational changes that occur in the ribosome during protein synthesis. It seems inevitable that within a few years, it will yield electron density maps that have resolutions high enough so that they can be interpreted
at atomic resolution without reference to any existing X-ray structure. The Ribosome in Parts. The bacterial ribosome is composed of about 60 different macromolecules, and shortly after methods were developed for purifying its protein components, work began on determining their structures in isolation. Over the years, roughly a dozen crystal structures and NMR structures were obtained for proteins from the bacterial ribosome. In addition, the structures of a number of rRNA fragments and ribosomal protein/RNA fragments were also determined. In retrospect, the RNA-related parts of this activity make some sense because at the time those structures were solved very little was known about RNA structure, let alone about the way RNA interacts with protein. Some useful general lessons were learned, but otherwise, in my estimation, little of lasting value emerged from this work, even though most of the structures obtained of isolated ribosome components turned out to be relevant to the conformations those components assume when they are embedded in the ribosome.
Figure 3. An electron microscopic image of 70S ribosomes from E. coli embedded in negative stain. Reprinted with permission from ref 7. Copyright 1960 Elsevier.
Figure 5. A view of a single particle reconstruction of the 70S ribosome from E. coli determined at a resolution of 5.5 A˚. Reprinted from 11 with permission. Copyright 2009 AAAS.
Figure 4. Comparison of the shape of the prokaryotic ribosome deduced from negative stained images8 (left) with the crystallographically determined structure of the same object (right). The structure on the right was generated by docking the crytal structure of the small subunit from Thermus thermophilus9 with the crystal structure of the large ribosomal subunit from Haloarcula marismortui.10 In the large subunit, proteins are dark blue, and RNA is brown. In the small subunit, proteins are light purple, and RNA is tan. The lateral protuberance of the large subunit, which is shown extending toward the reader in the EM image, is not visible in most ribosome crystal structures because of its high degree of mobility.
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The reason I am so hard on this body of work is that the ultimate objective of the structural biologist is to elucidate the function of the object he or she is investigating, not to determine structures per se. Inevitably, the determination of the structure of a component of some macromolecular assembly that accounts for less than, say, 5%, of its total mass is unlikely to shed much light on how that assembly works. If you had never seen an internal combustion engine, for example, how much would you learn about how it works from studying the blueprints for its carburetor? Unfortunately, work of this sort is being done on other complicated macromolecular structures today, and I am guessing that much of that effort will also turn out to have been wasted. Not everything that can be done should be done, and those who dole out research support should be more sensitive to that fact than they seem to be. Biochemical Approaches to Ribosome Structure The Inventory. Although biochemists cannot determine the three-dimensional structures of macromolecules, the information they gather can be invaluable to those who are able to do so. From the structural biologist’s point of view, the first major contribution the biochemists made to the ribosome field was the inventory of components they produced between roughly 1965 and 1975, which, in effect, defined the scope of the structural problem that had to be solved. The bacterial ribosome is (very nearly) a stoichiometric complex of 3 different RNA molecules, which make up two-thirds of its mass, and roughly 55 different proteins, which account for the rest. That fact alone tells you that the ribosome is asymmetric, and thus quite different from spherical viruses or multienzyme complexes that have similar molecular weights. Eukaryotic ribosomes are organized the same way, but they are bigger and there are more proteins and more RNAs. Sequences. The components of the ribosome began being sequenced even before the inventory was complete. For the first decade or so, ribosomal protein sequences were determined the old-fashioned way, which is to say at the protein level. Only at the end of the 1970s did it become common to sequence them at the DNA level, which is much easier. Wittmann-Liebold and her colleagues in Berlin produced a remarkably large number of bacterial ribosomal protein sequences, and Wool and his co-workers in Chicago took care of eukaryotes by sequencing rat ribosomal proteins. These sequences provided some interesting information about the phylogenetic relationships between species, but protein sequences are not easy to read in this regard. Perhaps the biggest contribution this heroic effort made to progress in the field came when the sequences it produced were used to interpret crystallographic electron density maps of the ribosome. The first rRNA sequence to appear was that of 5S rRNA from E. coli, which was determined at the RNA level by Brownlee at the MRC in Cambridge in 1968.12 The methods Brownlee used were not powerful enough to sequence the large rRNAs, and so the first sequences for those rRNAs did not appear until the late 1970s, when DNA sequencing methods became available.13,14 Quite apart for their importance for the ultimate determination of the three-dimensional structure of the ribosome, these rRNA sequences had a huge impact on biology. A few years after Brownlee’s 5S sequence appeared, Fox and Woese showed that the secondary structure of 5S rRNA could be worked out by comparing 5S rRNA sequences from different species.15 In
Figure 6. The secondary structure of 16S rRNA. This model was generated using the sequence comparison method by Gutell and his colleagues.17 The sequence is colored by domain. Adapted by permission from ref 9. Copyright 2000 MacMillan Publishers Ltd.
1981, using a far less rich store of sequence information as input, Noller and Woese demonstrated that the same could be done with the large rRNAs16 (Figure 6). Ever since, rRNA sequences have been the data of choice for sorting out the phylogenetic relationships between organisms, and when ribosome crystal structures appeared in 2000, the accuracy of the descendents of the secondary structure models first elaborated around 1980 was resoundingly confirmed. Chemical Approaches to Ribosome Quaternary Structure. Starting in the 1970s, biochemists began applying a wide variety of cross-linking techniques to the ribosome in an effort to determine which components are neighbors, to elucidate rRNA quaternary structure, and to locate the protein binding sites on rRNAs. After the first atomic resolution crystal structures became available, Altman at Stanford18 and Alexey Bogdanov19 in Russia did a retrospective analysis of the relationship between those data and the underlying structures. Their general conclusion is that while some of the biochemical approaches used worked better than others, that is, produced more reliable data, on the whole, the proximity and binding site data produced chemically correlate reasonably well with the crystal structures. Nevertheless, just as is the case with the neutron scattering, some of the data these chemical experiments produced are clearly “outliers” in the sense that they are incompatible with what we now know about the structure of the ribosome. Altman’s group attempted to interpret these outliers by postulating that the ribosome is a dynamic object, which is indeed the case, arguing quite plausibly that if the ribosome were flexibile the cross-linking data obtained from it might not be compatible with a single structural model.20 However, much of the outlying data were produced by experiments that were very challenging to execute at the time they were done, and the identification of the components in crosslinked products was often problematic. Thus, it is highly likely that some (most?) of the outliers are simply the products of experimental error. The only way such errors could possibly be dealt with in this context is if the experimental process that generates them yields such large amounts of nontrivially redundant information about the structure being investigated that outliers can confidently be rejected on the grounds of their incompatibility with the rest of the data. None of the strategies we are talking about here produced data sets that were anywhere near that rich.
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The one approach developed to investigate ribosome structure chemically that did consistently yield valuable data was the chemical protection/footprinting approach that was worked out for diagnosing RNA secondary structure, and for tracking conformational changes in RNAs, for example, ref 21. These methods have yielded a lot of valuable information about the conformational states of the ribosome and are routinely used to today to study RNAs of all kinds. Conclusions In retrospect, I think that the noncrystallographic work done to elucidate ribosome quaternary structure, that is, the three-dimensional arrangement of protein and RNA in the ribosome, was of little ultimate value. In my estimation, that work was not so much wrong as it was misguided. You have only to ask what would have been learned if all the data had been perfect, which, of course, they were not. The product would have been a three-dimensional picture of the ribosome that was accurate, but low-resolution in some way that is horribly difficult to define, and because it was so, it would have provided little insight into ribosome function. Worse from the point of view of those engaged in such work, all “one distance at a time” approaches to structure determination involve soul-destroyingly hard work. It takes 4n - 10 pairwise measurements to locate n objects in space, which works out to a minimum of 78 nonredundant measurements to determine the quaternary structure of the small ribosomal subunit, and 134 for the large ribosomal subunit. Furthermore, all such processes are distressingly sensitive to error. The presence of even a single bad measurement in such a data set can have devastating consequences for the model that is derived from it, as NMR spectroscopists get reminded from time to time when they misassign NOE cross peaks. So what is the proper role of neutron scattering, FRET, cross-linking, chemical protection, and all the other noncrystallographic structure determining techniques ribosomologists worked so hard to develop between 1970 and 2000? I think the answer is that they are best used in contexts where a small number of measurements can confirm or deny a hypothesis about how a structure works, that is, to track and characterize specific conformational changes, or to adjudicate issues where the answer is long or short/yes or no. In these contexts, they can be invaluable, as the work being done today on many systems by single-molecule methods clearly demonstrates. In short, they
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are tools to be called on once a strucure has been solved at reasonably high resolution, not the tools to use to obtain such a structure to begin with.
References (1) Moore, P. B. In The Ribosome. Structure, Function, Antibiotics and Cellular Interactions; Garrett, R. A., Douthwaite, S., Liljas, A., Matheson, A. T., Moore, P. B., Noller, H. F., Eds.; American Society for Microbiology: Washington, D.C., 2000; pp 555-556. (2) Rheinberger, H.-J. In Protein Synthesis and Ribosome Structure; Nierhaus, K. H., Wilson, D. N., Eds.; Wiley-VCH Verlag: Weinheim, 2004; pp 1-51. (3) Capel, M. S.; Engelman, D. M.; Freeborn, B. R.; Kjeldgaard, M.; Langer, J. A.; Ramakrishnan, V.; Schindler, D. G.; Schneider, D. K.; Schoenborn, B. P.; Sillers, I.-Y.; Yabuki, S.; Moore, P. B. Science 1987, 238, 1403–1406. (4) Capel, M. S.; Engelman, D. M.; Freeborn, B. R.; Kjeldgaard, M.; Langer, J. A.; Ramakrishnan, V.; Schindler, D. G.; Schneider, D. K.; Schoenborn, B. P.; Sillers, I.-Y.; Yabuki, S.; Moore, P. B. Science 1987, 238, 1403–1406. (5) Moore, P. B. Cold Spring Harbor Symp. Quant. Biol. 2001, 66, 607–614. (6) Huang, K. H.; Fairclough, R. H.; Cantor, C. R. J. Mol. Biol. 1975, 97, 443–470. (7) Huxley, H. E.; Zubay, G. J. Mol. Biol. 1960, 2, 10–18. (8) Lake, J. A. J. Mol. Biol. 1976, 105, 131–159. (9) Wimberly, B. T.; Brodersen, D. E.; Clemons, W. M.; Morgan-Warren, R. J.; Carter, A. P.; Vonrhein, C.; Hartsch, T.; Ramakrishnan, V. Nature 2000, 407, 327–339. (10) Ban, N.; Nissen, P.; Hansen, J.; Moore, P. B.; Steitz, T. A. Science 2000, 289, 905–920. (11) Seidelt, B.; Innis, A.; Wilson, D. N.; Gartmann, M.; Armache, J.-P.; Villa, E.; Trabuco, L. G.; Becker, T.; Mielke, T.; Schulten, K.; Steitz, T. A.; Beckmann, R. Science 2009, 326, 1412–1415. (12) Brownlee, G. G.; Sanger, F.; Barrell, B. G. J. Mol. Biol. 1968, 34, 379–412. (13) Brosius, J.; Palmer, M. L.; Kennedy, P. J.; Noller, H. F. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 4801–4805. (14) Brosius, J.; Dull, T. J.; Noller, H. F. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, 201–204. (15) Fox, G. E.; Woese, C. R. Nature 1975, 256, 505–507. (16) Noller, H. F.; Woese, C. R. Science 1981, 212, 403–411. (17) Gutell, R. R. In Ribosomal RNA. Structure, Evolution, Processing and Function in Protein Biosynthesis; Dahlberg, A., Zimmerman, R., Eds.; CRC Press: Boca Raton, FL, 1996; pp 111-128. (18) Whirl-Carrillo, M.; Gabashvilli, I. S.; Bada, M.; Rey Banatao, D.; Altman, R. B. RNA 2002, 8, 279–289. (19) Sergiev, P. V.; Dontsova, O. A.; Bogdonov, A. A. Mol. Biol. 2001, 35, 472–495. (20) Gabashvilli, I. S.; Whirl-Carrillo, M.; Bada, M.; Rey Banatao, D.; Altman, R. B. RNA 2003, 9, 1301–1307. (21) Moazed, D.; Noller, H. F. Cell 1986, 47, 985–994.
