using protein scaffold vs carrier protein

Nanobodies have several attractive features. Nanobodies, derived from single-chain antibodies of camelids, are also becoming popular as versatile binding partners of target proteins. In addition, the size of the Fabs (∼50 kDa) and the existence of a somewhat flexible hinge region between the two subdomains still make structural analysis challenging. Only a few examples of their application for cryo-EM analysis have been reported ( 9– 11), in part because the selection of appropriate Fabs is not trivial.

Fab fragments can be used as a fiducial marker to facilitate image alignment in cryo-EM images ( 8), but they have been mainly used in X-ray crystallography. However, the intrinsic conformational heterogeneity of DARPins limits their potential to achieve high-resolution structures of small proteins ( 7), and so far only a few DARPins have been selected against membrane proteins. In recent studies, DARPins selected against GFP were grafted onto large scaffolds and used to visualize GFP by cryo-EM ( 6, 7). More promising is the use of a binding partner that can be selected with a screening platform, such as modified ankyrin repeat proteins (DARPins), Fab fragments of antibodies, or nanobodies. It is thus a major goal in the field to expand the use of cryo-EM to the routine analysis of small proteins. However, structure determination of small proteins is of great interest, as most proteins have sizes below 100 kDa and ∼50% are smaller than 50 kDa, including many membrane proteins and proteins of medical importance. Without symmetry, small particles require optimal conditions, such as a highly homogeneous sample, rigid protein conformation, and random particle distribution in thin ice, conditions that are difficult to achieve with most samples ( 2). Small targets often lack recognizable shape features that can facilitate initial image alignment at low resolution. The structural analysis of small particles (∼100 kDa or less) is much more difficult. Large molecules are relatively easy to identify in noisy low-dose images of vitrified samples and have sufficient contrast and features to determine their orientation and position for alignment and averaging. Cryo-EM analysis has several advantages over X-ray crystallography or NMR ( 1), but the method becomes increasingly challenging for smaller proteins. Single-particle electron cryo-microscopy (cryo-EM) has become the method of choice for the determination of protein structures. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis. 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 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 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. We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM).

Using protein scaffold vs carrier protein