| Summary: | Electron cryomicroscopy (cryo-EM) techniques allow for the determination of cellular structures in three dimensions. The single-particle reconstruction (SPR) technique uses images of biological macromolecular complexes in a thin vitreous ice layer to generate three-dimensional (3D) reconstructions of them, and is capable of yielding structures of complexes ranging in size from hundreds of kilodaltons to tens of megadaltons. Beam-induced motion (BIM) is one of the major causes of a decrease in the signal-to-noise ratio (SNR) in images obtained in electron cryomicroscopy. BIM is shown to be due to a physical doming movement of the ice sheet which can lead to translations of particles of up to several nanometers in the image plane. We show that computational correction of BIM using movie data can generate high resolution reconstructions with a fraction of the images previously required. SPR density maps also suffer from artifacts if images averaged into reconstructions represent a heterogeneous mixture. An algorithm capable of rapid classification of heterogeneous image data is presented and characterized. The algorithm is capable of similar performance as previously characterized algorithms. The ribosome synthesizes mRNA encoded polypeptides. During its catalytic cycle, after peptidyl transfer, tRNA must move through the ribosome from the A and P sites to the P and E sites, respectively, in a process called translocation. This activity is accelerated 1000-fold by the GTPase EF-G. The structure of a transient early translocation intermediate bound with EF-G was determined using the new classification algorithm. The structure provides insight into the conformational changes that occur on the ribosome, tRNA and EF-G during translocation. Ribosomal cap-dependent initiation is a tightly regulated process directing initiation of polypeptide synthesis in eukaryotes. Internal ribosomal entry sites (IRESs) are RNA structures which allow the translation of downstream encoded elements to bypass some or all of the molecular machinery in cap-dependent initiation. The structure of the TSV IRES bound to the yeast ribosome is described. It shows that the IRES tRNA mimic is bound at the A site, and must undergo two rounds of translation prior to the formation of the first peptide bond.
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