autoBUSTER Example Application: CYS-CYS alternate conformations where one disulfide is bonded and the other broken

  • We will use as an example 1osg.
  • The protein and lead up autobuster runs to this tutorial are described in the basic NCS setup tutorial.
  • After the AutoBusterExample1osgBasicNCS/1osgBasicNCS_003_autoncs_followon (file not found) refinement the strongest difference map features are six strong negative peaks (from -8.2 to -5.6 sigma) centered on each of the disulfide bonds in BAFF protein:


  • The situation can arise because of radiation damage resulting a partial reduction of the disulfide.

Starting Files
SSBOND   1 CYS A  232    CYS A  245
SSBOND   2 CYS B  232    CYS B  245
SSBOND   3 CYS C  232    CYS C  245
SSBOND   4 CYS D  232    CYS D  245
SSBOND   5 CYS E  232    CYS E  245
SSBOND   6 CYS F  232    CYS F  245

Possibility A: remove all restraints across the disulfide
  • Because the input pdb file does not have SSBOND records for the disulfide bond no bond or angle restraints will be used for the link.
  • But because of the lack of the bond the two sulfur atoms across each disulfide would be regarded as having a bad non-bonded contact.
EXCLUDE A|232 A|245
EXCLUDE B|232 B|245
EXCLUDE C|232 C|245
EXCLUDE D|232 D|245
EXCLUDE E|232 E|245
EXCLUDE F|232 F|245
  • can be used to turn off the bad contact term across each disulfide. If this is done then no restraints are used between the CYS residues - neither bonded nor nonbonded contact. Essentially the disulfide bond is allowed to lengthen without any penalty.
  • results: refinement stats
structure Rwork/Rfree MolProbity Ramachandran favored MolProbity score
pdb 0.1843/0.2423 94.52% 2.81
003_autoncs_followon (previous step) 0.1875/0.2234 95.50% 3.01
004_remove_all_restraints 0.1856/0.2213 95.94% 3.04
    • So relaxing the bond restraints on the disulfides results in 0.2% drop in both Rwork and Rfree.
  • results on structure/maps

Possibility B: create alternate conformations and use utility distance restraints to make one disulfide bonded and the other reduced
  • A limitation with the current release is that two alternate structures have to have the same bonding structure.
  • page under construction. In the mean time document 1osg_disulfide_break.pdf provides a preliminary description of what can be done.

Page by Oliver Smart original version 29 May 2008. Address problems, corrections and clarifications