Intrinsic fiducials for small membrane protein structure determination.

Abstract

Solute carrier proteins (SLC) are small membrane transport proteins that play a critical role in many physiological processes. Despite their critical importance, our understanding of how these proteins function is limited due to the difficulty of structure determination. This is particularly true for major facilitator superfamily (MFS) proteins where there are often limited protein features to drive computational alignment of particles. While tools like cryo-EM have greatly enhanced the ability to determine membrane protein structures, fiducial markers are needed to help promote particle alignment. Fragment antigen-binding antibodies (Fab) or nanobodies (Nb) are the most commonly used examples, but generating these is not a trivial task. Often this is the largest barrier for membrane protein structure determination, taking a significant amount of time and resources. In the end, the resulting fiducial may be too flexible to enable high resolution structure determination, requiring the whole process to be done again. A feature intrinsic to the protein sequence would eliminate this barrier as it would require no additional generation time, and can be purified concurrently with the transporter of interest. Here we investigate the use of an intrinsic fiducial consisting of GFP and anti-GFP Nb labeled at the each of the protein's termini to solve a novel structure of an MFS transporter. We have generated three fully functional constructs, each with varying termini lengths, and demonstrated successive improvements in fiducial rigidity and cryo-EM reconstructions. We believe this method will not only enable us to solve a previously unknown structure, but also prove to be an accessible means for membrane protein structure determination.