Cryo-EM combined with single-particle reconstruction, a method pioneered in the Pi's lab, is capable of visualizing macromolecular machines such as transcription complexes, chaperones, or ribosomes under native conditions in different functional states. The goal of attaining 10 A for the ribosome, a structure without symmetry, has driven development of data collection and image processing techniques in our lab. This goal has been achieved, thanks to consistent funding of this grant from NIH. The dissemination of software (SPIDER) has benefited the research of other groups on numerous other biological structures. In the study of translation, density maps for a broad range of functional complexes in the resolution range of 9-13 A, interpreted by fitting of X-ray structures, have greatly advanced our knowledge of factor binding and our understanding of the dynamics of translation. A new resolution goal is set in Specific Aim #1 of the present proposal: the achievement of atomic (~3A) resolution for certain ribosomal complexes known to be highly stable. This goal requires simultaneous efforts in several areas: (i) exploration of grid preparation parameters that lead to uniform layers of ice with optimum thickness; (ii) large increase in data collected (~10-fold, for a yield of 1,000,000 """"""""good particles""""""""), requiring an increased level of automation and computational resources; (iii) strategies for carrying out CTF correction; (iv) angular refinement methods, which must be accelerated and improved through design and implementation of more efficient algorithms and computational strategies; (v) methods for fitting and docking of X-ray structures into density maps must be explored.
Specific Aim #2 concerns the elucidation, at near-atomic resolution, of a pivotal step (GTP hydrolysis) in mRNA-tRNA translocation both in eubacteria and eukaryotes, to be achieved by application of the technology developed and implemented in Specific Aim #1 to specific complexes with proven stability. Already along the way toward the goal of 3 A, we can expect that new discoveries will be made due to the improved definition of rRNA structure, as well as protein subdomains and structural motifs such as a-helices and -?-sheets, allowing conformational changes and molecular interactions during translocation to be modeled and described with much higher precision. To achieve these goals, we collaborate with key experts in the areas of reconstruction algorithms, ribosome biochemistry, X-ray crystallography, and computational modeling. ? ? ?
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