Development of macromolecular phasing methods

Abstract

X-ray crystallography is a powerful method in determining the structure of both small molecules and macromolecules and is now routinely applied in many scientific fields. However, to apply this method, there is an unavoidable problem to tackle: the Phase Problem, which arises because the phases of a scattered x-ray cannot be measured in diffraction experiment and the original structure cannot be retrieved only with the measurable amplitudes. This thesis presents two approaches in the development of macromolecular phasing methods.

One approach presented here utilizes molecular envelope of NMR structures for molecular replacement (MR) phasing with the program FSEARCH at low resolution (about 6 Å). X-ray crystallography and NMR are complementary tools in structural biology. However, it is often difficult to use NMR structures as search models in MR to phase crystallographic data. For this purpose, in our study, several targets with both crystallographic and NMR structures available have been tested. The test protocol involves four steps: (1) Model preparation, NMR structures were processed into averaged polyalanine model, and centroid NMR models have also been tested; (2) Six-dimensional low resolution search were carried out by FSEARCH to find the best match between observed and calculated structure factors; (3) Apply the solution (4) Model building and refinement. In our tests, FSEARCH was able to find the correct translation and orientation of the search model in the crystallographic unit cell, while conventional MR procedures were unsuccessful.

The other approach presented in this thesis is protein complex structure completion using IPCAS (Iterative Protein Crystal structure Automatic Solution). Protein complexes have been concerned as essential components in almost every cellular process. X-ray crystallography method is quite useful in studying the nature of protein complexes. In this study, we demonstrated a protein complex completion procedure from a partial molecular replacement (MR) solution using IPCAS. IPCAS is a direct-method aided dual-space iterative phasing and model-building procedure. The test cases were carefully selected from a practical perspective and IPCAS could build the whole complex from one or less than one subunit once molecular replacement method could give a partial solution. Before delivering to IPCAS, MR solution model examination and improvement might be necessary. The IPCAS iteration procedure involves (1) real-space model building and refinement; (2) direct-method aided reciprocal-space phase refinement; and (3) phase improvement through density modification. In our tests, IPCAS is able to extend the full length complex from a less than 30% starting model while conventional model building procedure were unsuccessful.