National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM052586
United States
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2021 Title: Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies). Authors: Xudong Wu / Tom A Rapoport / Abstract: 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 ...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 ∼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-Å 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-Å resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.
Method to determine structure: MOLECULAR REPLACEMENT Starting model: Fab with similar sequence Resolution: 1.83→28.45 Å / SU ML: 0.17 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 25.15 / Stereochemistry target values: ML
Rfactor
Num. reflection
% reflection
Rfree
0.2106
1997
4.05 %
Rwork
0.1796
47254
-
obs
0.1808
49251
99.03 %
Solvent computation
Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
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