The preparation of diffraction quality crystals remains the major bottleneck in macromolecular X-ray crystallography. A crystallization chaperone is an auxiliary protein, like an antibody fragment, that binds to and increases the crystallization probability of a target molecule of interest. Such chaperones reduce conformational heterogeneity, mask counterproductive surfaces while extending surfaces predisposed to forming crystal contacts, and provide phasing information. Over a decade ago, fragments of monoclonal antibodies were used as crystallization chaperones. This approach has been particularly effective in the determination of high-impact structures of membrane proteins. 

In 1993, scientists from the Vrije Universiteit Brussel discovered the occurence of bona fide antibodies devoid of light chains in Camelidae. The small and rigid recombinant antigen binding fragments (15kD) of these heavy chain only antibodies -known as Nanobodies^TM- proved to be unique research tools in structural biology. By rigidifying flexible regions and obscuring aggregative surfaces, Nanobody complexes warrant conformationally uniform samples that are key to protein structure determination by X-ray crystallography. The elucidation of the first GPCR structure in its active state using conformationally selective Nanobodies demonstrates the power of the Xaperone platform to speed up and reduce the cost of generating diffracting quality crystals of challenging targets.

Xaperones are antigen binding fragments from heavy chain only antibodies that:

bind cryptic epitopes and lock proteins in unique native conformations increase the stability of soluble proteins and solubilized membrane proteins reduce the complexity in conformationally rich proteins and protein complexes increase the polar surface enabling the growth of diffracting crystals allow to affinity-trap active protein

Concerns are often raised about the authenticity of structures determined through chaperone-assisted crystallography. One might think that chaperones can distort a protein into a nonnative structure. Such a view however makes little thermodynamic sense. In order for a chaperone to bind to a particular conformation with reasonably high affinity, that conformation must exist at a detectable concentration. An antibody would pay a huge energetic penalty if it first binds to a low energy state and then distorts the structure into a state so high-energy that it does not appreciably exist in the absence of the bound antibody. Therefore, it is much more likely that immunization and library selection identify a high-affinity antibody that binds to and stabilizes one of the lower energy states. Methods were developed to clone the nanobody repertoire of an immunized dromedary or llama in phage display vectors, and to select the antigen-specific VHHs from these 'immune' nanobody libraries.