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

Abstract

A genetically engineered humanized C H 2-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 P2 1 2 1 2 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.

Atomic force microscopy as an innovative tool for nanoanalysis of native stratum corneum

“…Therefore, the AFM is suitable to complement the structural information of proteins obtained, e.g. by X‐ray diffraction analysis (18). Moreover, the imaging of complex biological structures like the surface of single living cells in nanometer resolution is also reported (19).…”

Initial crystallographic studies of a small heat-shock protein fromXylella fastidiosa

“…In fact, it has been shown that one of the most harmful impurities which limits crystal quality consists of molecules exhibiting structural variability, including clusters or aggregates (McPherson et al, 2004). In this sense, it is noteworthy that an interesting case studied by AFM has been reported in which competition between different oligomeric forms might be responsible for the disorder and low resolution limit of the diffraction patterns (Larson et al, 2005). In the present study, the information obtained from the AFM analysis, although limited, helps us to understand the most probable reason for the relatively poor diffraction properties of the HSP17.9 crystals.…”

The Structure of an Antitumor CH2-domain-deleted Humanized Antibody

“…Details of the crystallization of HuCC49DCH2 form B have been described elsewhere, along with an atomic force microscopy investigation of the crystals. 9 The crystals used for diffraction analysis were grown by vapor-diffusion at 17 8C from 4 M sodium formate (pH 7.2) in the presence of 1.5 mM Triton X-100. Detergent was essential to crystallization, and many detergents were effective in yielding crystals, but, so far, only Triton X-100 produced ordered crystals that diffract to adequate resolution.…”

“…There is evidence from AFM studies of HuCC49DCH2 and its crystals that, under the crystallization conditions noted above, tetrameric assemblies are present in solution, and that crystal growth proceeds through the incorporation of tetramers into the crystal lattice. 9 In contrast to the ring-to-ring interface discussed above, there are only six contacts (interatomic distances !4 Å ) between tetramers along the a axis and a total of only 463 Å 2 of buried surface area per ring assembly.…”

“…9 Those analyses indicated that the crystallization unit was composed of four HuCC49DCH2 antibodies arranged in a toroidal structure. They further showed that the tetrameric aggregates existed independent of the crystals and were present in solution.…”