Challenges of three-dimensional reconstruction of ribonucleoprotein complexes from electron spectroscopic images - Reconstructing ribosomal RNA

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Transmission electron microscopy has been dominant in structural studies of the ribosome and its constituent ribonucleic acids and proteins. Ribosomal RNA (rRNA) has central importance in the architecture of this complex and in protein synthesis. Our work has entailed using electron spectroscopic imaging (ESI) to probe the tertiary structure of rRNAin situin a prokaryote (Escherichia coli) and in a eukaryote (Thermomyces lanuginosus). ESI uses only electrons which have lost a specific amount of energy due to specific inner-shell ionisation interactions with the specimen to form an elemental map. In nucleoprotein complexes, a map of the phosphorus distribution represents primarily a projection of the phosphate backbone of the nucleic acid component. The visualisation of rRNAin situin the intact ribosomal subunit by ESI was demonstrated almost a decade ago to be feasible. Our work on quantitative image analysis of ES images ofE. coliandTh. lanuginosusribosomal subunits has presented unique challenges and has resulted in new algorithmic developments generally applicable to such images. These innovations include a singular pretreatment procedure, the use of mutual correlation functions rather than cross correlation functions to reduce the effect of low spatial frequency components, and angular determination using iterative quaternion-assisted angular reconstitution to compute a three-dimensional reconstruction. These investigations have produced direct information regarding ribosomal rRNA localisation in the ribosomal subunits ofE. coliandTh. lanuginosus, and the position of non-conserved sequences.