Duncan Sousa | PubMed Health

PubMed2006

Ab initio resolution measurement for single particle structures.

A computational method is described that allows the measurement of the signal-to-noise ratio and resolution of a three-dimensional structure obtained by single particle electron microscopy and reconstruction. The method does not rely on the availability of the original image data or the calculation of several structures from different parts of the data that are needed for the commonly used Fourier Shell Correlation criterion. Instead, the correlation between neighboring Fourier pixels is calculated and used to distinguish signal from noise. The new method has been conveniently implemented in a computer program called RMEASURE and is available to the microscopy community.

Escherichia coli↗

PubMed2004

Three-dimensional structure of C complex spliceosomes by electron microscopy.

The spliceosome is a multimegadalton RNA-protein machine that removes noncoding sequences from nascent pre-mRNAs. Recruitment of the spliceosome to splice sites and subsequent splicing require a series of dynamic interactions among the spliceosome's component U snRNPs and many additional protein factors. These dynamics present several challenges for structural analyses, including purification of stable complexes to compositional homogeneity 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 a resolution of 30 A. The structure reveals a particle of dimensions 27 x 22 x 24 nm with a relatively open arrangement of three primary domains.

Cryoelectron Microscopy↗