A 9 Angstrom Resolution X-Ray Crystallography Map of the Large Ribosomal Subunit
Cell, Vol. 93, 1105-1115, June 26, 1998
Nenad Ban1, Betty Freeborn2, Poul Nissen1, Pawel Penczek4,5, Robert A. Grassucci4, Robert Sweet6, Joachim Frank4,5, Peter B. Moore1,2, and Thomas A. Steitz1,2,3,7
1Department of Molecular Biophysics and Biochemistry
2Department of Chemistry
3Howard Hughes Medical Institute, Yale University
4Wadworth Center, New York State Department of Health
5Department of Biomedical Sciences, State University of New York
6Department of Biology, Brookhaven National Laboratory
Abstract
The 50S subunit of the ribosome catalyzes the peptidyl-transferase reaction of protein synthesis. The X-ray crystallographic structure of the large subunit purified from Haloarcula marismortui was determined up to 9 Angstrom. The 20 Angstrom resolution EM image reconstruction derived three dimensional structure was used as an initial phase determination to locate the positions of heavy atom cluster in three derivatives by putting it into the unit cell. The resulting structure was in agreement with the EM-derived structure at 20 Angstrom resolution and high resolution fragment structures derived from NMR and X-ray method indicated right-handed twist of ribosomal RNA and a novel RNA-protein interaction was also found.
Introduction
In all living organisms, protein synthesis and elongation are proceeded during messenger RNA (mRNA) ¡V peptide translation . This catalysis function undergoes in ribonecleoprotein particles called ribosomes. Ribosome has a sedimentation coefficient of about 70S and consisits of two subunits: a small, 30S subunit and a large, 50S subunit. Ribosomal subunits are complexes of ribosomal RNA, rRNA and proteins. The latter has a molecular weight of about 1.5*106 Da and functions as a peptide bond formation machinery. The 50S subunit contains a 2900 nt RNA and a 120 nt RNA and about 33 different proteins. Atomic resolution structures of these assembled complexes can help us to understand the mechanism of peptide bond formation and RNA-protein interactions.
Up to date, many structural studies have been derived by physical and chemical methods, such as electron microscopy (EM), neutron scattering, and X-ray crystallography. The shape and the composition of the ribosomal subunits were determined roughly by EM, the sequences and secondary such as hammer-head and loop structures of rRNA of different subunits were also determined by molecular biochemistry assay. The composition and the three dimensional spatial relation of the proteins were determined by both cryo-EM image reconstruction (Fig.1) and neutron scattering. However, due to the limitation of resolution and technical restriction, these results were not precise enough at atomic level.
Although many high resolution crystal structures of spherical viruses, which are larger than the ribosome, have been determined during the last two decades, the structure of the ribosome is still a formidable problem to many crystallographers because of its large size and asymmetry. High symmetry of spherical viruses helped their structure determination by complexity reduction into single asymmetric unit, and thus helped the phase determination, which is the key to X-ray structures. As an approach to solve the phase problem in the ribosomal subunit, three heavy atom clusters (Fig. 2) were used: W18 ((AsW9O33)2), W11 (Cs5(PW11O39{Rh2(CH3COO)2}), and Ta (Ta6Br12+2). These heavy atom clusters gave dramatically increased scattering power at low resolution, but no significant improvement at high resolution. Putting the EM-map of the H. marismortui large ribosomal subunit into the crystal unit cell, the locations of these heavy atom clusters were determiner by difference Patterson and difference Fourier maps (Fig. 3).
Combination of heavy atom clusters and EM-map structure phase determination, the X-ray diffraction 50S ribosomal subunit structure was determined up to 9 Angstrom resolution by multiple isomorphous replacement and anomalous scattering (MIRAS) method. The structure revealed an atomic view of RNA helical structure and ribosomal protein L1. This result provides an insight to the structure and function of this super complex, and higher resolution will provide more detail information in the future.
Result and Discussion
The electron microscopic re-construction of large ribosomal subunits of different species were put into the crystal unit cell ( Fig. 4 ). The reconstruction was derived from 13,170 images of individual particles taken at two levels of defocus. The unit cell is orthorhombic with a space group of C221 and with unit cell dimensions of : a = 210, b = 300, c = 570 Angstrom. The data was collected at Brookhaven National Laboratory synchrotron. After molecular replacement and rigid body refinement, the EM reconstruction of H. marismortui showed a better R-factor (0.39) and correlation coefficient (0.51) than E. coli ( Table 1 ). The heavy atom derivative W18 was prepared by soaking crystals in W18 containing solution, and its difference Fourier peak increased from 13 s to 14 s while the R factor decrease from 44 % to 41 % for the 1322 reflections between 100 and 20 Angstrom resolution. After MIRAS analysis for three heavy atom cluster derivatives, the major and minor occupancy sites were determined, and the quality of the phasing was excellent to 12.5 Angstrom but dropped significantly beyond 9 Angstrom. Therefore, a 9 Angstrom resolution electron density map was calculated using MIRAS phasing from all three derivatives [ Data collection & phasing ].
The overall X-ray crystallo-graphically derived electron density map of the H. marismortui large ribosomal subunit at 9 Angstrom had a spherical radius of about 250 A and its rendering surface was very similar with the EM derived 20 Angstrom resolution reconstruction ( Fig. 5a). In a higher resolution , the surface structure provided more structural details, yet, the characteristics of the protruding arms, proteins (L1 and L7/L12) and rRNA segments, and mRNA penetrating cleft were almost identical as identified biochemically by immunoelectron microscopy and cryo-EM reconstruction in the early studies. Even though the 9 Angstrom resolution is not high enough to illustrate the atomic structure clearly, previous fragment X-ray and NMR structures, in another way, can greatly help picturing the huge puzzle. One example is a 5S RNA fragment solved ( PDB ID =1A4D) by X-ray diffraction. The backbone structure of this RNA duplex is a standard A-form Waston-Crick base pairing, rod like structure and it can be fit in the L1 arm region attached with the L1 protein at its terminal ( Fig. 6 ). Unlike the known nucleic acid-protein interaction, RNA and protein are intermingled, rather than the nucleic acid being wrapped around a protein core or encased in a protein shell. This model appears that the 50S NA ribosome structure is formed by struts of RNA rods whose branching cross-links the struts and protein acts as a stabilizing factor.
This X-ray crystallographic map of a ribosomal subunit shows electron density map expected in the previous studied. Although the quality of phasing and resolution are not good enough to identify the detail atomic coordinate, a 9 Angstrom resolution allows a accurate heavy atom localization and structural feature determination. As shown earlier, combining with the fragment structure results, we can still obtain a high resolution atomic structure. The further question is the mechanism and interaction of how the protein synthesis and elongation proceed among large 50S subunit, 30S small subunit and mRNA. This will rely on a higher resolution insight into it.
Reference
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