Tightening the Crosslinking Distance Restraints for Better Resolution of Protein Structure and Dynamics

Abstract

Chemical crosslinking 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 crosslinked residues, with upper bounds typically over 20Å. The incorporation of loose CXMS restraints only marginally improves the resolution of the calculated structures. Here, we present a revised format of CXMS distance restraints, which works by first modifying the crosslinked residue with a rigid extension derived from the crosslinker. With the flexible side chain explicitly represented, the reformatted restraint can be applied to the modification group instead, with an upper bound of 6Å 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, our approach enables more information extracted from the CXMS data.