Ribosome crystallization

Khulbrandt W, Unwin PNT. Structural analysis of stained and unstained two-dimensional ribosome crystals, in Electron Microscopy at Molecular Dimensions, State of the Art and Strategies for the Future (Baumeister W, Vogell W, eds.), Springer-Verlag, Berlin, Germany, 1980, pp. 108-116. 

Peptide bond formation is the essential reaction catalyzed by the ribosome. Despite its importance, it was for a long time not the focus of ribosomal research, for several reasons. First, before the determination of the high-resolution ribosome crystal structures almost nothing was known about the active site. Second, under most experimental conditions accommodation of the incoming aminoacyl-tRNA is rate limiting for peptide bond... 

Experiments towards growing ribosomal crystals in vitro were challenged by the observations that ribosomes may self-organize into ordered aggregates in the living cell two-dimensional arrays have been found under special conditions, such as hibernation or lack of oxygen (e.g. ). However, the complex structure, the enormous size and the flexibility of ribosomal particles render their crystallization in vitro extremely difficult. Therefore, only a few successful efforts to produce three-dimensional crystals have been reported (e.g. >). 

Rib-X Pharmaceuticals was founded to capitalize on the antibacterial cocrystal structures determined by Steitz and coworkers cited above. Use of the ribosome crystal structures has proven fruitful for the company as will be described in the two published case studies detailed herein. 

Wimberly, B.T. The use of ribosomal crystal structures in antibiotic drug design. Curr. Opin. Investig. Drugs 2009, 10(8), 750. 

On the technical side, synchrotron X-ray radiation is necessary for most protein complexes as they have large unit cells and weak diffraction patterns. It may be necessary to protect the crystds from the beam by freezing them this was particularly valuable for obtaining data from ribosome crystals. ... 

Another important point is radiation damage. Thermal neutrons appear to destroy protein crystal structures much less than X-rays do. This is so at room temperature. Recently, it has been found that cooling of ribosome crystals to liquid nitrogen temperature makes the crystal structure extremely resistant to the intense synchrotron... 

Spectacular use of cryocrystallography has been made to avoid an impasse reached in data collection at or near room temperature from ribosome crystals (Hope et al 1989). At room temperature these samples decay in the X-ray beam (monochromatic X in the range 0.9-1.5A) dramatically making data collection beyond 18 A essentially impossible. At liquid nitrogen temperatures an initial resolution of 5 A is preserved essentially indefinitely. Mosaic spreads are unchanged between room temperature and cryotemperatures but are quite large anyway (—3°). The intensity of the synchrotron beam is used to make the data collection time manageable. 

Byers, B., 1971, Chick embryo ribosome crystals analysis of bonding and functional activity in vitro, Proc. Nat. Acad. Sci. USA, 68 440. 

Unwin, N., 1979, Attachment of ribosome crystal to intracellular membranes, J. Mol. Biol., 132 69. 

The atomic structure of this subunit and its complexes with substrate analogs revealed the enzymatic activity of the rRNA backbone. Thus, the ribosome is in fact a ribozyme P Nissen, J Hansen, N Ban, PB Moore, TA Steitz. Science 289 920-930, 2000. Atomic structure of the ribosome s small 30S subunit, resolved at 5 A WM Clemons Jr, JL May, BT Wimberly, JP McCutcheon, MS Capel, V Ramakrishnan. Nature 400 833-840, 1999. The 8-A crystal structure of the 70S ribosome reveals a double-helical RNA bridge between the 50S and the 30S subunit GM Culver, JH Cate, GZ Yusupova, MM Yusupov, HF Noller. Science 285 2133-2136, 1999. 

Since the X-ray structural analysis of crystallized proteins yields the most direct information on the tertiary structure, many attempts have been made in the last decade to crystallize individual ribosomal proteins. However, it was many years before any progress in this field was made. The N- and C-terminal fragments of the . coU protein L7/L12 have been crystallized, and the crystals diffract to 4 and 2.6 A, respectively (Liljas et ai, 1978). According to the X-ray analysis, the C-terminal fragment (positions 53-120) has a compact, plum-shaped tertiary structure with three a helices and three p sheets (Leijonmarck et ai, 1980). 

The only other E. coli ribosomal protein whose crystallization has so far been reported is L29 (Appelt et al., 1981). On the other hand, attempts to crystallize ribosomal proteins from the thermophilic Bacillus stearothermophilus have been more successful. Protein BL17, which according to its amino acid sequence (Kimura et al., 1980) corresponds to protein L9 from the E. coli ribosome (Kimura et al., 1982), was the first intact ribosomal protein to give crystals useful for X-ray structural analysis (Appelt et al., 1979). Several other B. stearothermophUus ribosomal proteins, namely BL6 and BL30 (Appelt eteU., 1981,1983) from the large and BS5 (Appelt et al., 1983) from the small subunit have been crystallized, and the determination of their three-dimensional structure at a resolution of better than 3 A is now in progress. Furthermore, crystals of aB. stearothermophilus ribosomal protein complex, which corresponds to the complex (L7/L12)4 LIO from E. coli ribosome, have been obtained (Liljas and Newcomer, 1981). 

Three-dimensional crystals have been obtained with 50 S ribosomal subunits from B. stearothermophilus (Yonath et al, 1980, 1982a,b) and with 70 S ribosomes from E. coli (Wittmann et al, 1982) as shown in Fig. 7. 

Fig. 7. (a) Crystals of E. colt 70 S ribosomes, (b) and (c) Electron micrographs of sections through three-dimensional crystals shown in (a) in two orthogonal directions (Wittmann et al., 1982). (d) and (e) Crystals and computed filtered image of a section through a crystal of Bacillus stearothermophilus 50 S ribosomal subunits (Yonath et al., 1982a,b Leonard et al., 1983). (d) and (e) are related to two different crystal forms. Reproduced with permission from Wittmann (1983). 

Of great interest is the fact that ribosomal subunits and ribosomes themselves have now been crystallized, and low-resolution structural maps have already been obtained. However, to grow suitable crystals and to resolve the ribosomal structure at a sufficiently high resolution remains a great challenge and task to biochemists and crystallographers. 

The ribosome is a unique cellular machine in that its main functional component is RNA whereas proteins seem to play only a structural role. For a long time, it has been debated whether RNA or proteins contribute most to the ribosome s function. With the determination of high-resolution crystal structures, this question could finally be answered. Clearly, these structures have revolutionized the field of ribosome studies. Already in the 1980s, Yonath and coworkers had grown crystals of active ribosomes that diffracted to about 0.6 nm (6 A) (1 A = 0.1nm) resolution. However, owing to the large size of the ribosome of about 2 500 000 Da (lDa=lgmoP), the ribosome structure was not solved to atomic resolution until tbe year 2000. 

RF3)" " with the ribosome. However, the maximum resolution that can currently be obtained by cryo-EM is about 10 nm (8-12 A), far from the desired atomic resolution. Therefore, the crystal structures of the 30S subunit with initiation factors 1 and 3 (IFl IF3 ) and of the 70S subunit with release factors 1 and 2 (RFl/2 " ) as well as RRF have been important milestones toward understanding the interaction of the ribosome with protein factors. 

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