Strategies to stabilize RNP complexes for structural determination by 3D cryo-electron microscopy
by Wen-ti Liu
Date of Examination:2013-10-30
Date of issue:2014-10-24
Advisor:Prof. Dr. Holger Stark
Referee:Prof. Dr. Holger Stark
Referee:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Kai Tittmann
Files in this item
Name:dissertation_wliu_2014_web.pdf
Size:66.2Mb
Format:PDF
Description:Main article
Abstract
English
The physiological reactions in a cell are generally not performed by single biological macromolecules, but by complexes of several molecules. They can be a complex of several proteins or can be composed of RNA and proteins as a ribonucleoprotein (RNP) complex. The RNP complexes perform their functions either through dynamic assembly and disassembly of components, such as the spliceosome, or through structural dynamics, such as the ribosome. Due to their dynamic nature and large size, single particle cryo-electron microscopy is an ideally suited method to study these RNP complexes. However, due to the high heterogeneity of samples, which can be a result of compositional difference or conformational flexibility, most of the structures thus far could only be obtained with restricted resolution. In this work, biochemical and computational methods were applied to reduce sample heterogeneity and to resolve heterogeneous sub-states of RNP complexes. In the first approach, the structure of the human spliceosomal C complex was analyzed. Different sub-states representing the heterogeneity were sorted in silico, and the regions with high heterogeneity were characterized. Unfortunately, the amount of heterogeneity exceeded the capacity which could be dealt with by image processing alone and has obstructed the improvement of resolution. Therefore, biochemical methods have been developed to stabilize samples and to decrease heterogeneity. Two approaches were pursued: (1) to stabilize the sample during purification, and (2) to prevent the macromolecules from disruption during the sample preparation for transmission electron microscopy (TEM). As a proof of concept, the optimization of purification was performed on an endogenous small nuclear ribonucleoprotein (snRNP). With the optimization of the crowding agent, as well as replacing chromatography with selective precipitation, the 3D model of the yeast snRNP could be reconstructed for the first time. To prevent macromolecules from disruption during sample preparation, p-maleimidophenyl isocyanate (PMPI) was evaluated as an RNA-protein crosslinker. PMPI was tested on the human 80S ribosome. In the reconstructed 3D model, a crosslink between the expansion segment ES7L and the ribosomal protein L7A was observed. Besides, the E-site tRNA showed higher occupancy. To further stabilize the ribosome during purification, the conventional "salt wash" step, which utilizes high salt concentration to remove salt-unstable proteins, was replaced by selective precipitation under low salt conditions. The L1 and P stalks in the reconstructed 3D models were significantly stabilized in the low salt purification procedure. Furthermore, a factor located next to the nascent chain exit tunnel was copurified. These biochemical approaches were shown to stabilize the ribosome and the spliceosome, and can be applicable in the future for cryo-EM studies on all RNP complexes.
Keywords: single particle cryo-electron microscopy; ribonucleoprotein complex