New Applications Of Direct-methods In Solving Protein Crystal Structures At Low-resolution

Exploring the structure and biological function from proteins is one of the most important research fields in life science.Accurate three-dimensional structure determination is beneficial to understand the life process at the molecular level and will greatly promote the development of pharmacology and drug design.In the field of protein crystallography,direct methods were initially used to solve the heavy-atom substructures and ab initio protein structure determinations at atomic resolution.By combining direct methods with other existing methods,more new applications could be proposed.Based on this,this paper had systematically explored different ways of direct methods in solving protein crystal structures at low-resolution.Within the framework of dual-space iteration,by combining direct methods with several methods in protein crystallography,the author has achieved:(i)The automated structure determinations using 16 sets of weak,single-wavelength anomalous scattering(SAD)data,(ii)Propose an iterative direct-methods MAD/MIR phasing procedure and complete two cases of automated phase extension and refinement starting from low resolution to high-resolution data(from 6.9 ? to 2.8 ? and from 6.8 ? to 3.0 ?)by using direct methods,and(iii)Propose a new protocol of de novo structure determination and automated model building for membrane proteins at low resolution(down to 4.5 ?).Those three applications of direct methods are all difficult projects in protein crystallography.This paper consists of five chapters.The first and the second chapters are about the basic knowledge of protein crystallography and direct methods.Chapters 3,4 and 5 introduce the new researches and important applications of direct methods in the low-resolution protein crystallography.Chapter 1: Introduction.The history of X-ray crystallography,the three-dimensional structure of proteins and the conventional steps of protein structure determination are introduced.Details of the three types of widely-used phasing methods in protein crystallography are also listed.Chapter 2: Former researches and applications of direct methods in protein crystallography.By combining with the conventional SAD/SIR techniques,direct methods had successfully broken the phase ambiguity intrinsic in SAD/SIR.Based on that,the direct-methods SAD/SIR phasing and fragments extension as well as the direct-methods aided partial-model were also developed.Those applications had gradually been integrated into an automated protein structure solution pipeline,named IPCAS with its 2.0 version newly released in 2018.Chapter 3: Automated structure determinations in IPCAS using 16 sets of weak,single anomalous scattering data.SAD phasing is the dominant method of experimental phasing in protein crystallography,but it can be challenging because of the intrinsic phase ambiguity,especially when the anomalous scattering signal is weak.In this chapter,16 sets of weak anomalous scattering SAD data were all automatically determined in the IPCAS pipeline and leading to satisfactory results.Comparison were carried out between IPCAS with the two popular packages PHENIX and CRANK2,results from the IPCAS were better.Chapter 4: The iterative direct-methods MAD/MIR phasing procedure and low-resolution phase extension.Multi-wavelength anomalous scattering and multi-isomorphous replacement methods(MAD/MIR)are both powerful tools for the structure determination of proteins at low resolution.In this chapter,the author proposed an iterative MAD/MIR phasing procedure.The results showed that this procedure had effectively improved the quality of low-resolution phases based on the conventional MAD/MIR methods,and two successful cases of automated phase extension using direct methods starting from low-resolution to high-resolution native data(6.9 ? to 2.8 ? and 6.8 ? to 3.0 ?)is also presented.Chapter 5: De novo structure determination and automated model building for membrane proteins at low resolution.Knowing the three-dimensional structure of membrane proteins is of great importance both in structural biology and pharmacology.In this chapter,new methods and a brief protocol for the SAD phasing and automated model building of membrane proteins at low resolution is proposed.Based on the dual-space direct-methods iterations,the innovative points of the methods are as following:(i)prior searching of the non-crystallographic symmetry(NCS)by resolution screening,(ii)alternative model building strategy,and(iii)feeding back the combined model/phases information within the iterations.Four challenging membrane protein cases at low-resolution(better than 4.5 ?)diffraction data were tested and finally obtained accurate structures,showing the feasibility and a potential universality of the methods.Chapter 6: Conclusion and prospection.