Forum 04/06/2006

“EM Structural Studies of the Human Spliceosome and Resolution Measurement”

Duncan Sousa – Brandeis University

The spliceosome is a multimegadalton RNA-protein machine that removes noncoding sequences from nascent pre-mRNAs.  Splicing requires a series of dynamic interactions among the spliceosome’s many components.  These dynamics present several challenges for structural analyses, including purification of stable complexes and assessment of conformational heterogeneity.  We have isolated spliceosomes arrested before the second chemical step of splicing (C complex) in which U2, U5 and U6 snRNAs are stably associated.  Using electron microscopy, we obtained images of C complex spliceosomes under cryogenic conditions and determined a three-dimensional structure of a core complex to an estimated resolution of 30.  The resolution estimate is based on the commonly used Fourier Shell Correlation (FSC) criterion.  To evaluate the FSC, the original image data is required for calculation of two reconstructions, each from one half of the data.  Since assessment of the resolution is an important indicator of the quality of reconstruction, we developed a new resolution measurement method which does not require the original data for evaluation.  It can, therefore, be used to evaluate a structure after reconstruction.  The method is based on the correlation between neighboring terms in the Fourier transform of the structure that arise from the fact that the structure occupies only part of a volume.

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