کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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5913839 | 1162704 | 2015 | 10 صفحه PDF | دانلود رایگان |
A single-particle cryoEM reconstruction of the large ribosomal subunit from Saccharomyces cerevisiae was obtained from a dataset of â¼75,000 particles. The gold-standard and frequency-limited approaches to single-particle refinement were each independently used to determine orientation parameters for the final reconstruction. Both approaches showed similar resolution curves and nominal resolution values for the 60S dataset, estimated at 2.9 à . The amount of over-fitting present during frequency-limited refinement was quantitatively analyzed using the high-resolution phase-randomization test, and the results showed no apparent over-fitting. The number of asymmetric subunits required to reach specific resolutions was subsequently analyzed by refining subsets of the data in an ab initio manner. With our data collection and processing strategies, sub-nanometer resolution was obtained with â¼200 asymmetric subunits (or, equivalently for the ribosomal subunit, particles). Resolutions of 5.6 à , 4.5 à , and 3.8 à were reached with â¼1000, â¼1600, and â¼5000 asymmetric subunits, respectively. At these resolutions, one would expect to detect alpha-helical pitch, separation of beta-strands, and separation of Cα atoms, respectively. Using this map, together with strategies for ab initio model building and model refinement, we built a region of the ribosomal protein eL6, which was missing in previous models of the yeast ribosome. The relevance for more routine high-resolution structure determination is discussed.
Journal: Journal of Structural Biology - Volume 192, Issue 2, November 2015, Pages 235-244