Cryo-electron microscopy single-particle analysis of interphotoreceptor retinoid-binding protein and the peripherin/ROM1 complex, proteins necessary for the sensation of vision.

High-resolution structure determination is the necessary for understanding how protein fold affects function. Although x-ray crystallization is the gold-standard for determining high resolution maps of protein, cryo-electron microscopy has emerged with numerous benefits. The technique can be applied to bio-molecular complexes across a wide range of molecular mass, has the ability to visualize proteins which are resistant to crystallization, and image a variety of biological sample types including protein and their higher-ordered structural arrangements, entire cells, bacterial populations, and even tissue sections. Here we show how we can employ the technique to reconstruct structures of two proteins that are necessary for the sensation of vision, interphotoreceptor retinoid-binding protein (IRBP) and the peripherin-2/ROM1 (Prph2/ROM1) complex.IRBP is a highly expressed protein secreted by rod and cone photoreceptors that has roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Here we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo-electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N-linked carbohydrate post-translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three-dimensional architecture not seen among other retinoid-binding proteins.Peripherin-2 (Prph2) and its homologue ROM1 are photoreceptor-specific members of the tetraspanin family of membrane proteins that are required for proper formation and function of rod and cone outer segments of photoreceptors. The PRPH2 gene has been associated with over 150 individual pathogenic mutations (according to the Human Gene Mutation Database with a variable degree of severity and diagnoses, ranging from retinitis pigmentosa to macular degeneration. Prph2 and ROM1 associate in vivo and are predicted to form higher-ordered oligomers of heterotetramers. Here we show our strategy for purification, utilizing a nanobody specific for bovine Prph2/ROM1 as a tool for immunoprecipitation and solubilization of the complex. We show this route as a way for achieving high purity and stability, which will promote our ability to image the complex by cryo-EM or even promote crystallization.