Combined use of AFM and X-ray diffraction to analyze crystals of an engineered, domain-deleted antibody

A genetically engineered humanized CH2‐domain‐deleted monoclonal antibody lacking any interchain‐hinge disulfide bonds has been crystallized in the presence of detergent in a form suitable for X‐ray diffraction analysis. The crystals were grown from 4 M formate along with Triton X‐100 and had P21212 space‐group symmetry, with unit‐cell parameters a = 83, b = 224, c = 167 Å. The crystals diffract to beyond 2.8 Å resolution. A disordered crystal form of larger size and more attractive habit was also grown from 4 M formate, but in the presence of the Anapoe series of detergents. Preliminary X‐­ray data, in conjunction with atomic force microscopy images, are consistent with asymmetric units consisting of two intact antibodies forming a circular dimeric ring. The crystallizing unit, which must contain a twofold axis, is a toroidal assembly of four antibodies (two dimeric rings). Competition between dimers and tetramers to enter the lattice, along with a unique kind of planar defect of packing, may be responsible for the unusually high defect density and the disorder of the X‐ray diffraction pattern exhibited by the second crystal form. An approach to crystallizing proteins showing phase separation, particularly intact antibodies, that uses a preliminary detergent test set is described.