autoBUSTER Example Application: CYS-CYS alternate conformations where one disulfide is bonded and the other broken
- Background
- Starting files
- Possibility A: remove all restraints across the disulfide
- Possibility B: create alternate conformations and use utility distance restraints to make one disulfide bonded and the other reduced
Background
- 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.
- How can this be represented in an autoBUSTER refinement? There are two useful approaches:
Starting Files
- AutoBusterExample1osgBasicNCS/1osg.mtz the structure factors as supplied by the pdb.
- 1osgBasicNCS_003_autoncs_followon_removeSSbond.pdb pdb file resulting from AutoBusterExample1osgBasicNCS/1osgBasicNCS_003_autoncs_followon (file not found) refinement job. The six SSBOND records for the disulfide bonds have been edited out of the pdb file:
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.
- The TNT EXCLUDE cards:
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.
- 1osgDisulfideBreak_004_remove_all_restraints is a shell script that runs the refinement. The script writes out a .Gelly file with the exclude cards and this is used by the autoBUSTER refine command line argument -Gelly. (explanation of shell scripts used here).
- 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 buster-develop@globalphasing.com