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Publication Abstract from PubMed

We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of approximately 120 kDa, called Legobody, does not perturb the nanobody-target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-A overall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-A resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.

Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies).,Wu X, Rapoport TA Proc Natl Acad Sci U S A. 2021 Oct 12;118(41):e2115001118. doi: , 10.1073/pnas.2115001118. PMID:34620716^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.