Structural Characterization of Biological Macromolecules by Random Conical Tilt Pair Experiments

Abstract

Structural biology deals with determining the structure of biological macromolecules especially proteins, DNA, RNA. The conformational changes in the structure of a macromolecule helps to decipher its function. This finally paves the way for structure-based drug designing and mutational studies to assess key residues involved in the macromolecule. X-ray crystallography has been the most common technique for structural elucidation since almost last century and has contributed to almost 85 per cent of the structures deposited in Protein Data Bank (PDB). However, crystallising proteins such as those associated with membrane remains a major bottleneck till date though methods like lipid cubic phase (LCP) have somewhat circumvented this. It is also important to realize that imperfect crystals sometimes formed may not depict true physiological state of the protein in the cellular context and hence drug design based on that may turn out to be futile. NMR (Nuclear Magnetic Resonance) is a powerful technique to study the protein structure at atomic resolution in solution. It has also been used to study kinetics and dynamics of the protein. The major limitation of NMR is the size limit that it poses which is around 5-25 kDa and huge amount of protein that it requires. On the contrary, cryo-electron microscopy (cryo-EM) has emerged as a versatile tool for studying structure of proteins and macromolecular complexes. Recent “resolution revolution” has empowered cryo-EM in terms of resolution achieved due to better DED (Direct Electron Detector) cameras, stable microscopes and new algorithms for data processing. This has resulted in the surge of EM map deposition in the EMDB (Electron Microscopy Data Bank) (Fig. 1). The number of depositions of EM map per year has been depicted in Fig. 2.