Development of immobilized antibody-based affinity grid strategy and application in on-grid purification of target proteins for high-resolution cryo-EM

Abstract

AbstractIn cryo-electron microscopy (cryo-EM), sample preparation, especially for rare or fragile macromolecular assemblies and those suffering from air-water interface denaturation and particle orientation distribution problems, is a major bottleneck. Here, we developed and characterized an immobilized antibody-based affinity grid (IAAG) strategy based on the high-affinity PA tag/NZ-1 antibody epitope tag system. We used Pyr-NHS as a linker to immobilize NZ-1 Fab on the graphene oxide or carbon covered grid surface. We showed that the IAAG grid can enrich the PA-tagged target proteins, and overcome preferred orientation problems. Furthermore, we demonstrated that our IAAG strategy can be utilized for on-grid purification of low-abundance target complexes from cell lysates and enables atomic resolution cryo-EM. This approach greatly streamlines the purification process, reduces the need for large quantities of biological samples, and addresses common challenges encountered in cryo-EM sample preparation. Collectively, our IAAG strategy provides an efficient and robust means for combined sample purification and vitrification feasible for high-resolution cryo-EM. This approach also has the potential for broader applicability in cryo-EM and cryo-ET.