Dear All,
Thanks for all the suggestions. Lot to learn when its low resolution.
I have few more details....5th round of refinement gave R/Rfree 0.2016/0.2767
after reducing waters and fixing some outliers and difference has gone up to
7.5%
I have used Buster 2.10.0 for refinement. First round after molecular
replacement I used Refmac for refinement. There after I used Buster with
autoncs_noprune and sim_swap_equiv_plus as it was low resolution in 2nd and 3rd
round. Fourth round I used -autoncs and -TLS options. Here are the details of
NCS and TLS paprameters.
REMARK 3 SIMILARITY.
REMARK 3 NCS.
REMARK 3 NCS REPRESENTATION : RESTRAINT LSSR (-AUTONCS)
REMARK 3 TARGET RESTRAINTS.
REMARK 3 TARGET REPRESENTATION : NONE
REMARK 3 TARGET STRUCTURE : NULL
REMARK 3
REMARK 3 TLS DETAILS.
REMARK 3 NUMBER OF TLS GROUPS : 2
REMARK 3
REMARK 3 TLS GROUP : 1
REMARK 3 SET : { A|* }
REMARK 3 ORIGIN FOR THE GROUP (A): -40.7570 45.8964 0.0654
REMARK 3 T TENSOR
REMARK 3 T11: 0.6834 T22: 0.5325
REMARK 3 T33: -0.6079 T12: -0.0436
REMARK 3 T13: -0.1033 T23: 0.0313
REMARK 3 L TENSOR
REMARK 3 L11: 1.6212 L22: 0.8001
REMARK 3 L33: 6.4728 L12: 0.0460
REMARK 3 L13: 0.0051 L23: 0.7670
REMARK 3 S TENSOR
REMARK 3 S11: -0.2854 S12: 0.5434 S13: 0.0788
REMARK 3 S21: -0.5270 S22: 0.3608 S23: 0.0731
REMARK 3 S31: -0.7141 S32: 0.2459 S33: -0.0754
REMARK 3
REMARK 3 TLS GROUP : 2
REMARK 3 SET : { B|* }
REMARK 3 ORIGIN FOR THE GROUP (A): -29.9645 52.6711 27.6973
REMARK 3 T TENSOR
REMARK 3 T11: 0.5441 T22: 0.5948
REMARK 3 T33: -0.6079 T12: -0.0298
REMARK 3 T13: -0.0992 T23: -0.0084
REMARK 3 L TENSOR
REMARK 3 L11: 1.7807 L22: 1.1381
REMARK 3 L33: 4.5990 L12: 0.7938
REMARK 3 L13: 0.4959 L23: 1.3513
REMARK 3 S TENSOR
REMARK 3 S11: -0.2911 S12: -0.0941 S13: 0.1302
REMARK 3 S21: -0.3180 S22: 0.1719 S23: -0.0989
REMARK 3 S31: -1.2554 S32: 0.5321 S33: 0.1192
I deleted Lot of waters on Ethan's suggestion and now have only 12 of them. I
am refining now and it takes a while for a buster run in our computer and I
will let you know the results.
So, I would appreciate if you could suggest what NCS restrains are needed and
where they were needed and why, if the above method is not a systematic
approach use NCS after obtaining a solution. And I have same question for the
TLS. I guess this would benefit many people like me who are dealing with their
first low resolution data.
Many thanks to Francis for pointing good reference.Let me know if I need to
give more information.I appreciate all the suggestions and your valuable time.
Thanks,RajDate: Fri, 2 Mar 2012 16:45:04 +0000
From: twom...@globalphasing.com
Subject: Re: [ccp4bb] sudden drop in R/Rfree
To: CCP4BB@JISCMAIL.AC.UK
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
From: Rajesh kumar <ccp4...@hotmail.com>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Friday, March 2, 2012 10:54 AM
Subject: [ccp4bb] sudden drop in R/Rfree
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.
ThanksRaj
