On 2 Mar 2012, at 16:02, Regina Kettering wrote:
Rajesh;
I am not sure that you have a high enough data:refinement parameters ratio to refine TLS. It just adds more parameters to refine that can lead to over-refinement of your model, especially at the 3.3 A.
TLS only adds twenty parameters per chain; so it's a really parsimonious thing to do at low resolution.
I'd say that adding lots of waters at 3.3A (at four parameters per added water) was much more likely to be the cause of a very wide R/Rfree gap.
I'm a bit worried that a user working at low resolution on a protein with more than one chain per ASU is not using NCS from the very beginning; that's another good way of adding more restraints and effectively getting the parametersto-data ratio down (because the 'parameters' in that ratio is really 'parameters minus K * number of restraints'; there is scope for a lot of debate as to the right value of K, it clearly depends on the strength of the restraints)
If he's using the Global Phasing refinement software, I would strongly suggest that Rajesh use targetting to the initial molecular replacement result throughout the refinement, as yet a third way of adding more restraints.
Tom Womack (Global Phasing)
HTH,
Regina
Dear All,
I have a 3.3 A data for a protein whose SG is P6522. Model used was wild type structure of same protein at 2.3 A.
After molecular replacement, first three rounds of refinement the R/Rf was 26/32.8, 27.1/31.72 % and 7.35/30.88 % respectively.
In the fourth round I refined with TLS and NCS abd added water and the R/Rf dropped to 19.34/26.46. It has almost 7% difference. I also see lot of unanswerable density in the map where lot of waters were placed. Model fits to the map like a low resolution data with most of side chains don't have best density.
I was not expecting such a sudden drop in the R/Rfree and a difference is 7.2%.
I am wondering if I am in right direction. I am not sure if this usual for 3.3A data or in general any data if we consider the difference.
I appreciate your valuable suggestions.
Thanks
Raj
