AutoBusterExample1osgDisulfideAlternateBroken

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

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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:
• 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

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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

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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

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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.

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Page by Oliver Smart original version 29 May 2008. Address problems, corrections and clarifications buster-develop@globalphasing.com