Tightening the Cross-Linking Distance Restraints for Better Resolution of Protein Structure and Dynamics

Abstract

Chemical cross-linking coupled with mass spectrometry (CXMS) has been increasingly used in structural biology. CXMS distance restraints are usually applied to Cα or Cβ atoms of the cross-linked residues, with upper bounds typically over 20 A. The incorporation of loose CXMS restraints only marginally improves the resolution of the calculated structures. Here we present a new format of CXMS distance restraints, which works by first modifying the cross-linked residue with a rigid extension derived from the cross-linker. With the flexible side-chain explicitly represented, the reformatted restraint can be applied to the modification group instead, with an upper bound of 6 A or less. The short distance restraint can be represented and back-calculated simply with a straight-line. The use of tighter restraints not only afford better-resolved structures but also uncover protein dynamics. Together, the new approach enables more information extracted from the CXMS data.