Xudong Wu in the Rapoport lab revolutionizes cryo-EM by breaking its size limit

Target protein and resulting structure

Structure determination by cryo-EM is difficult or impossible for proteins smaller than ∼100 kDa, excluding many membrane proteins and proteins of pharmaceutical interest from the analysis. In this paper, Wu and Rapoport report on a general method that allows structure determination of small proteins for which a tightly binding nanobody is available. The nanobody is rigidly attached to two scaffolds: a Fab fragment of an antibody directed against the nanobody and a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble is called Legobody. The method is demonstrated for two small proteins that have sizes of ∼22 kDa. The Legobody approach can easily be applied to any target protein and should greatly expand the use of cryo-EM single-particle analysis.