Rigid enlargement of sybodies with antibody fragments for cryo-EM analyses of small membrane proteins

Single particle cryo-electron microscopy (cryo-EM) has become the method of choice to determine experimental structures of integral membrane proteins. However, high-resolution structure determination by cryo-EM remains a challenge for membrane proteins that are too small or lack distinctive structural elements for particle alignment. To address this problem, single-domain antibodies called nanobodies and their synthetic variants called sybodies are widely used tools to trap membrane transporters in defined conformations, to enlarge particle sizes and to act as fiducial markers enabling reliable particle alignment. Recently, antibody fragments (Fabs) enlarging nanobodies at their backside in a rigid fashion, called Legobody and NabFab, have been developed. Here, we investigated how Legobodies and NabFabs can be harmonized with sybodies. We show that any sybody can be adapted to the Legobody approach with minimal effort, while only a subset of sybodies belonging to the loop library can be converted into a format recognized by the NabFab without complementarity-determining region-grafting. This technical note will facilitate the usage of Legobodies and NabFabs in the context of sybodies targeting membrane proteins and other small proteins for high-resolution structure determination by cryo-EM.