Cryo-EM study of a small protein using bispecific Diabody

Abstract

Recent advances in Cryo-EM technology have enabled the determination of protein structures at close to atomic resolution. However, application of this method is still difficult for proteins below ~100 kDa due to lower contrast. Additionally, the structural flexibility of the proteins is a major obstacle to successful EM analysis. In this research, we aim to develop novel "bispecific Diabodies" as tools for structural studies of small and/or flexible proteins. Diabodies are bivalent and bispecific antibody fragments, which consist of two heavy chain variable domains (VHs) and two light chain variable domains (VLs) in a single polypeptide, and so can simultaneously bind two different proteins. For EM applications, Diabodies can serve as rigid linkers connecting small targets and large helper proteins as fiducial markers, allowing easy detection by improving contrast because of the increased size of the whole protein complex. We confirmed the stable formation of "target-Diabody-helper protein" complexes by both size-exclusion chromatography and negative staining EM. Finally, We collect Cryo-EM data for Diabody complex. As result of processing this data, We confirmed that Diabody complex was formed. We plan to expand the use of this technique to eukaryotic membrane proteins, one of the most challenging types in structural biology.