Disulfide constrained Fabs overcome target size limitation for high-resolution single particle cryoEM

Le résumé fourni par la source

High-resolution structures of proteins and protein complexes are critical to understanding molecular mechanisms of biological processes and in the discovery of therapeutic molecules. CryoEM has revolutionized structure determination of large proteins and their complexes, but a vast majority of proteins, including that underlie human diseases are small (<50 kDa) and usually beyond its reach due to low signal-to- noise images and difficulties in particle alignment. Previously reported solutions (structure chaperones), which directly bind to and increase the overall size of the particle are limited by inherent flexibility and not being bound to their targets in a rigid manner, resulting in the target being poorly resolved compared to the chaperones themselves. Here we present an iterative, structure-guided protein engineering using disulfides, of the Fab (Antibody fragment) scaffold, and present a solution that transforms the typically flexible Fab into a conformationally rigid scaffold with a distinctive shape, and maintains affinity to antigen. The designs are transferable across fabs from different species and chimeras. When bound to proteins even as small as 6–20 kDa, Rigid Fabs allow their structure determination to high resolutions of ∼2.3–2.5 Å for the protein of interest. Since a Fab can be discovered for practically any protein, our Rigid Fab design presents a general approach to overcome the target size limitation of single particle cryoEM.

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