Re: [ccp4bb]: exploding atoms during final refinement

[Garib Murshudov <garib@ysbl.york.ac.uk>]

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Jan Abendroth wrote:

> Dear all,
> some strange things happen during the last cycles of my refinement ...
>
> The situation:
> I am refining quite a large structure consisting of six 50kDa Monomers
> (6*458=2748 residues + some 3800 water + some buffer) with a 1.3 AA
> data-set and refmac5. I always use a script like the one attached
> incuding contributions of hydrogen (without refining them). After
> introduction of anisotropic B-factors and some few multiple
> conformations the refinement is at is final stage with R-values around
> R-work=14.5% and R-free 17.5%. When I changed the restrains on the shape
> the displacement ellipsoids (SPHERE) from default 2 to 5 or higher both
> R-values dropped by 1% but some severe problems occurred and the
> refinement diverged:
>
> The problems:
> >From one cycle to the next within refmac the B-factors of one atom in
> one of the last residues of the sixth monomer suddenly grew a lot (>100
> and much more in following cycles) and became very oblate. The R-values
> inceased by between 1 and more than 5% -  the refinement diverged rather
> than converged. Once a monomer was affected more and more atoms in the
> same monomer showed these strange B-factors in the following cycles.
> This comes along with an increasing number of "Eigenvalue"-,
> sphericity-outlier- and rigid-bond-outlier-warnings
>
> Trials how to fix it:
> I have tried the following things in order to make the refinement
> convergent again:
> - inclusion of ncs: code 4 did not help, code 6 caused a core dump
> - fixing Babinets Solvent B to 200: didn't help
> - restraining B-shifts (HOLD BFAC from def=3 to 10): didn't help
> - newer refmac-5-version (5.0.28): things got much worse!
>
> - starting with isotropic B-factors: helps for a while (5 big cycles)
> - resetting SPHERE from 5 to 2: B-factors remain stable, but both
> R-factors get 1% higher again
>
> Questions:
> - Why does this strange behaviour start in a clearly defined region of
> the structure?
> - Why does this stay in one monomer, the others have the same
> sequece/structure?
> - Why are there severe differences between the refmac-versions?
> - Does refmac have some problems with large structures?
> - Does it make sense to return to tighter SPHERE restraints in spite of
> 1% larger R-values?
> - Does anyone have some similar expriences or some experiences with the
> SPHERE restraints?
>
> Thanks a lot for any hints and suggestions!
> Best regards
>
> Jan
>

Hi Jan,

First: You don't need HGEN in refmac5. It generates hydorgens itself.
(MAKE HYDRogens ALL it is default, alternatives
 NO do not use hydrogens,
 Yes use hydrogens if they are in the input file)
Second
TRY
DAMP 0.5 0.5 to reduce shifts.

Garib


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Garib N. Murshudov, Chemistry Department, University of York, U.K.
Tel: Home +44 (1904) 42 62 87, work:  +44 (1904) 43 25 65
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