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

  • Author(s): Larson, SB
  • Kuznetsov, YG
  • Day, J
  • Zhou, J
  • Glaser, S
  • Braslawsky, G
  • McPherson, A
  • et al.
Abstract

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 P2 1212 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. © 2005 International Union of Crystallography - all rights reserved.

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