Abstract
The crystallization of proteins remains a bottleneck in our fundamental understanding of their functions. Therefore, discovering tools that aid crystallization is crucial. In this review, the versatility of fragment-antigen binding domains (F(ab)s) as protein crystallization chaperones is discussed. F(ab)s have aided the crystallization of membrane-bound and soluble proteins as well as RNA. The ability to bind three F(ab)s onto a single protein target has demonstrated their potential for crystallization of challenging proteins. We describe a high-throughput workflow for identifying F(ab)s to aid the crystallization of a protein of interest (POI) by leveraging phage display technologies and differential scanning fluorimetry (DSF). This workflow has proven to be especially effective in our structural studies of assembly-line polyketide synthases (PKSs), which harbor flexible domains and assume transient conformations. PKSs are of interest to us due to their ability to synthesize an unusually broad range of medicinally relevant compounds. Despite years of research studying these megasynthases, their overall topology has remained elusive. One F(ab) in particular, 1B2, has successfully enabled X-ray crystallographic and single particle cryo-electron microscopic (cryoEM) analyses of multiple modules from distinct assembly-line PKSs. Its use has not only facilitated multidomain protein crystallization but has also enhanced particle quality via cryoEM, thereby enabling the visualization of intact PKS modules at near-atomic (3-5 Å) resolution. The identification of PKS-binding F(ab)s can be expected to continue playing a key role in furthering our knowledge of polyketide biosynthesis on assembly-line PKSs.