Objective
The objective of the proposed project is to solve a fundamental scientific problem of present-day structural molecular biology - the problem of determining the three-dimensional structure of the 70S ribosomal particle at a resolution of about 20 A. The proposed approach has no equivalents anywhere in the world and has never been used in structural studies.
This approach consists in the use of the data of X-ray diffraction in crystal combined with the data on small-angle X-ray and neutron scattering obtained on the same preparations of the 70S ribosomal particles. The method of spherical harmonics will be used to design a three-dimensional model of the 70S ribosomal particle on the basis of the data of small-angle scattering at a resolution of 40 x.. Then this model will be used for determining the packing function in crystal and the resolution will be increased by the diffraction set. The small-angle X-ray and neutron data in the range of scattering vectors 0.17-0.35 A will be used as a basis for refinement and interpretation of the reflections, corresponding to 40-20 A. The expected three-dimensional resolution (20 A) will be sufficient for determining an accurate three-dimensional arrangement of the two ribosomal subparticles relative to each other and for visualization of the spatial arrangement of the RNA within the 70S ribosomal particle. In case of getting a higher resolution it will be possible to localize individual protein and RNA domains. It should be also noted that the expected three-dimensional resolution should essentially specify the data obtained recently for the 70S ribosome using electron microscopy.
A novel contribution will be the information on the spatial structure of ribosomal RNAs within the 70S ribosomal particle which cannot be obtained from the electron microscopy data. To solve the task, the participants of the project will use recent achievements in crystal grown of 70S ribosomal particles, biosynthetic deuteration of microorganisms, and new contrast variation methods in neutron scattering based on studying the 70S particle with different percentage of deuterium incorportaion in the RNA and protein components of ribosomes that excludes the reconstitution process.
A successful realization of the proposed project can be expected taking into account the existence of suitable crystals of 70S ribosomes from Thermus thermophilus for a set of diffraction data both at room and lower temperatures (see the photo) as well as the carried out testing of the technique that allows to obtain particles with a significantly varying percentage of deuterium incorporation in the RNA and protein components of the 70S ribosome eliminating the reconstitution stage, and as a result, a possibility to obtain neutron scattering curves from such particles in 100% D2O in the range of scattering vectors from 0.007 to 0.35 A-1.
References
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2. Trakhanov S.D. Yusupov M.M. Agalarov S.C. Garber M.B. Ryazantsev S.N. Tischenko S.V. & Shirokov V.A. (1987) FEBS Lett. 20, 319-322.
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8. Stark H., Mueller F., Orlova E., Schatz M., Dube P., Erdemir T., Zemlin F., Brimacombe R. & van Heel M. (1995) Structure 3, 815-821.
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Coordinator
YO1 5DD York - Heslington
United Kingdom
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