Re: [ccp4bb] sudden drop in R/Rfree
No comments on much of your message but I cannot believe you can get better results by cutting anisotropic 2.1A data to 3.3A!! You MUST be throwing away valuable experimental information,, remember crystallography is not about getting low R factors but about getting a model consistent with your experiment. eleanor On Mar 5 2012, Rajesh kumar wrote: Dear All, Thanks for all the suggestion. Data is anisotropic. Since anisotropic correction with the UCLA server didnt give any good Molprobity scores and didnt reduce the R/Rfree at 2.1A, I am using ~3.3A data for further refinement. Using Tom's suggestion of target restrains reduced the gap brtween R/Rfee to with in 5%. I haven't tried Pavel' suggestion but when I do I will post the results. I used a 2.3 A MR model as target to get R/Rfree 0.2209/0.2597. When I used coot real space refine zone to fix few molprobity outliers, most of the favored residues become outliers. Almost all residues changed to become outliers. I am wondering why all the residues are moving from favored to outlier regions after restrained refinement. I thought all the residues would be fixed (fit) to the map and wouldn't change during the coot exercise, and this is what I see in the 2.3 A target/model structure ( with R/Rfree 0.1693/0.1961). I don't know if is this common for a low resolution or I didn't understand this whole thing of refinement correctly. Thanks for all the help, Regards,Raj Date: Sun, 4 Mar 2012 21:02:51 + From: eleanor.dod...@york.ac.uk Subject: Re: [ccp4bb] sudden drop in R/Rfree To: CCP4BB@JISCMAIL.AC.UK Presumably your data is quite anisotropic, and low resolution, so it is quite likely that a TLS model will give much better description of the B factors than more classical refinement. Modelling solvent at that resolotion will be tricky of course. Elesnor On Mar 4 2012, Joseph Cockburn wrote: > Hi Rajesh, If you're seeing a lot of extra density coming up in the > map in regions where you previously added waters, is it possible that > this extra density corresponds to a part of your protein that you > previously thought was disordered and is thus missing from the current > model? At this resolution you wouldn't expect to see many waters. > Also, to the best of my knowledge, the relative weighting of the X-ray > and geometry terms in BUSTER is set by the program so as to produce a > rmsd in bond lengths equal to a target value. The default value of > this is 0.01 Ang (I think) but you can change this using the -r option > on the command line. Using a lower value will reduce the weight on the > X-ray term and may lower the R/R-free gap. Best wishes, Joe > > > >> >> >> 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 >> >> > -- Professor Eleanor Dodson YSNL, Dept of Chemistry University of York Heslington YO10 5YW tel: 00 44 1904 328259 Fax: 00 44 1904 328266 -- Professor Eleanor Dodson YSNL, Dept of Chemistry University of York Heslington YO10 5YW tel: 00 44 1904 328259 Fax: 00 44 1904 328266
Re: [ccp4bb] sudden drop in R/Rfree
Dear All, Thanks for all the suggestion. Data is anisotropic. Since anisotropic correction with the UCLA server didnt give any good Molprobity scores and didnt reduce the R/Rfree at 2.1A, I am using ~3.3A data for further refinement. Using Tom's suggestion of target restrains reduced the gap brtween R/Rfee to with in 5%. I haven't tried Pavel' suggestion but when I do I will post the results. I used a 2.3 A MR model as target to get R/Rfree 0.2209/0.2597. When I used coot real space refine zone to fix few molprobity outliers, most of the favored residues become outliers. Almost all residues changed to become outliers. I am wondering why all the residues are moving from favored to outlier regions after restrained refinement. I thought all the residues would be fixed (fit) to the map and wouldn't change during the coot exercise, and this is what I see in the 2.3 A target/model structure ( with R/Rfree 0.1693/0.1961). I don't know if is this common for a low resolution or I didn't understand this whole thing of refinement correctly. Thanks for all the help, Regards,Raj > Date: Sun, 4 Mar 2012 21:02:51 + > From: eleanor.dod...@york.ac.uk > Subject: Re: [ccp4bb] sudden drop in R/Rfree > To: CCP4BB@JISCMAIL.AC.UK > > Presumably your data is quite anisotropic, and low resolution, so it is > quite likely that a TLS model will give much better description of the B > factors than more classical refinement. > > Modelling solvent at that resolotion will be tricky of course. > Elesnor > > > > On Mar 4 2012, Joseph Cockburn wrote: > > >Hi Rajesh, > >If you're seeing a lot of extra density coming up in the map in regions > >where you previously added waters, is it possible that this extra density > >corresponds to a part of your protein that you previously thought was > >disordered and is thus missing from the current model? At this resolution > >you wouldn't expect to see many waters. > >Also, to the best of my knowledge, the relative weighting of the X-ray and > >geometry terms in BUSTER is set by the program so as to produce a rmsd in > >bond lengths equal to a target value. The default value of this is 0.01 > >Ang (I think) but you can change this using the -r option on the command > >line. Using a lower value will reduce the weight on the X-ray term and may > >lower the R/R-free gap. > >Best wishes, > >Joe > > > > > > > >> > >> > >> 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 > >> > >> > > > > -- > Professor Eleanor Dodson > YSNL, Dept of Chemistry > University of York > Heslington YO10 5YW > tel: 00 44 1904 328259 > Fax: 00 44 1904 328266
Re: [ccp4bb] sudden drop in R/Rfree
Presumably your data is quite anisotropic, and low resolution, so it is quite likely that a TLS model will give much better description of the B factors than more classical refinement. Modelling solvent at that resolotion will be tricky of course. Elesnor On Mar 4 2012, Joseph Cockburn wrote: Hi Rajesh, If you're seeing a lot of extra density coming up in the map in regions where you previously added waters, is it possible that this extra density corresponds to a part of your protein that you previously thought was disordered and is thus missing from the current model? At this resolution you wouldn't expect to see many waters. Also, to the best of my knowledge, the relative weighting of the X-ray and geometry terms in BUSTER is set by the program so as to produce a rmsd in bond lengths equal to a target value. The default value of this is 0.01 Ang (I think) but you can change this using the -r option on the command line. Using a lower value will reduce the weight on the X-ray term and may lower the R/R-free gap. Best wishes, Joe 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 -- Professor Eleanor Dodson YSNL, Dept of Chemistry University of York Heslington YO10 5YW tel: 00 44 1904 328259 Fax: 00 44 1904 328266
Re: [ccp4bb] sudden drop in R/Rfree
Hi Rajesh, If you're seeing a lot of extra density coming up in the map in regions where you previously added waters, is it possible that this extra density corresponds to a part of your protein that you previously thought was disordered and is thus missing from the current model? At this resolution you wouldn't expect to see many waters. Also, to the best of my knowledge, the relative weighting of the X-ray and geometry terms in BUSTER is set by the program so as to produce a rmsd in bond lengths equal to a target value. The default value of this is 0.01 Ang (I think) but you can change this using the -r option on the command line. Using a lower value will reduce the weight on the X-ray term and may lower the R/R-free gap. Best wishes, Joe > > > 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 > >
Re: [ccp4bb] sudden drop in R/Rfree
On 2 March 2012 18:01, Jacob Keller wrote: > Can't there be a "group of atoms?" For sure, but doesn't a given parameter either apply to a single atom or to a group of atoms? Cheers -- Ian
Re: [ccp4bb] sudden drop in R/Rfree
Can't there be a "group of atoms?" JPK On Fri, Mar 2, 2012 at 12:00 PM, Ian Tickle wrote: > > I'm aware of this document. Personally I prefer "ADP = Atomic > Displacement > > Parameters" over anything ele, because, given that Atomic Displacement > > Parameters can be parameterized in many different ways, it makes it > easier > > to operate with such terms like: > > > > - isotropic Atomic Displacement Parameters (isotropic ADP); > > - anisotropic Atomic Displacement Parameters (anisotropic ADP); > > - group Atomic Displacement Parameters (group ADP); > > - group isotropic Atomic Displacement Parameters (group isotropic ADP; > > example: when refining one isotropic ADP per set of selected atoms); > > - group anisotropic Atomic Displacement Parameters (group anisotropic > ADP; > > example TLS); > > - ... etc, etc... > > > > (no intention to open another "can of worms" ! -:) ) > > Hi Pavel, but couldn't you simplify it further by omitting the word > "atomic" throughout? In fact isn't "group (an)isotropic Atomic > Displacement Parameter" a contradiction in terms? Surely it can be > either a group parameter or an atomic parameter but not both at the > same time? > > I think the worms are already out and making good their escape :). > > Cheers > > -- Ian > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] sudden drop in R/Rfree
> I'm aware of this document. Personally I prefer "ADP = Atomic Displacement > Parameters" over anything ele, because, given that Atomic Displacement > Parameters can be parameterized in many different ways, it makes it easier > to operate with such terms like: > > - isotropic Atomic Displacement Parameters (isotropic ADP); > - anisotropic Atomic Displacement Parameters (anisotropic ADP); > - group Atomic Displacement Parameters (group ADP); > - group isotropic Atomic Displacement Parameters (group isotropic ADP; > example: when refining one isotropic ADP per set of selected atoms); > - group anisotropic Atomic Displacement Parameters (group anisotropic ADP; > example TLS); > - ... etc, etc... > > (no intention to open another "can of worms" ! -:) ) Hi Pavel, but couldn't you simplify it further by omitting the word "atomic" throughout? In fact isn't "group (an)isotropic Atomic Displacement Parameter" a contradiction in terms? Surely it can be either a group parameter or an atomic parameter but not both at the same time? I think the worms are already out and making good their escape :). Cheers -- Ian
Re: [ccp4bb] sudden drop in R/Rfree
Thanks Ian, I'm aware of this document. Personally I prefer "ADP = Atomic Displacement Parameters" over anything ele, because, given that Atomic Displacement Parameters can be parameterized in many different ways, it makes it easier to operate with such terms like: - isotropic Atomic Displacement Parameters (isotropic ADP); - anisotropic Atomic Displacement Parameters (anisotropic ADP); - group Atomic Displacement Parameters (group ADP); - group isotropic Atomic Displacement Parameters (group isotropic ADP; example: when refining one isotropic ADP per set of selected atoms); - group anisotropic Atomic Displacement Parameters (group anisotropic ADP; example TLS); - ... etc, etc... (no intention to open another "can of worms" ! -:) ) All the best, Pavel On Fri, Mar 2, 2012 at 9:38 AM, Ian Tickle wrote: > > (*) ADP = Atomic Displacement Parameters > > or "anisotropic displacement parameters"? > > See http://ww1.iucr.org/comm/cnom/adp/finrepone/finrepone.html > > Section 1.5 Comments about terminology > > Cheers > > -- Ian >
Re: [ccp4bb] sudden drop in R/Rfree
> (*) ADP = Atomic Displacement Parameters But aren't *isotropic* b-factors subsumed under this TLA (three-letter acronym?) JPK On Fri, Mar 2, 2012 at 11:38 AM, Ian Tickle wrote: > > (*) ADP = Atomic Displacement Parameters > > or "anisotropic displacement parameters"? > > See http://ww1.iucr.org/comm/cnom/adp/finrepone/finrepone.html > > Section 1.5 Comments about terminology > > Cheers > > -- Ian > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] sudden drop in R/Rfree
> (*) ADP = Atomic Displacement Parameters or "anisotropic displacement parameters"? See http://ww1.iucr.org/comm/cnom/adp/finrepone/finrepone.html Section 1.5 Comments about terminology Cheers -- Ian
Re: [ccp4bb] sudden drop in R/Rfree
Hi, At 3.3A I would recommend trying a TLS model _instead_ of refining > individual B factors. may be it is implementation/software/mindset dependent, but in phenix.refine refining TLS+individual ADP or simply individual ADP(*) is a better option most of the time at low resolution. For details see pages 30 and 31 here: http://phenix-online.org/newsletter/CCN_2010_07.pdf Pavel (*) ADP = Atomic Displacement Parameters
Re: [ccp4bb] sudden drop in R/Rfree
Dear All,
Thanks for all the suggestions. Lot to learn when its low resolution.
I have few more details5th 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 3NCS.
REMARK 3 NCS REPRESENTATION : RESTRAINT LSSR (-AUTONCS)
REMARK 3TARGET 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 3SET : { A|* }
REMARK 3ORIGIN FOR THE GROUP (A): -40.7570 45.89640.0654
REMARK 3T 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 3L 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 3S 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 3SET : { B|* }
REMARK 3ORIGIN FOR THE GROUP (A): -29.9645 52.6711 27.6973
REMARK 3T 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 3L 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 3S 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 +
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.
Re: [ccp4bb] sudden drop in R/Rfree
I've found the following article to be useful in defining suitable refinement strategies at a particular resolution. 1. Mueller, M., Jenni, S. & Ban, N. Strategies for crystallization and structure determination of very large macromolecular assemblies. Curr Opin Struct Biol 17, 572–579 (2007). It can be used as a rough guide line at the start of your refinement (incomplete model, fresh off of molecular replacement) with slow increase of refinement parameters the better your model becomes (and as Rfree permits). F On Mar 2, 2012, at 9:39 AM, Ethan Merritt wrote: > On Friday, 02 March 2012, 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. > > I'm afraid you've got this completely backwards. > TLS uses very few parameters, and is especially useful at low resolution. > At 3.3A I would recommend trying a TLS model _instead_ of refining > individual B factors. > > NCS restraints also help a lot at low resolution. > > So the drop is believable, but... > > You should first worry about "lots of waters were placed". > It's there that many extra parameters have been added, perhaps leading > to over-fitting. I would not expect 3.3A data to justify placement of > more than a handful of waters at most. > > If you're parameter counting, you might note that 5 water molecules add > more parameters than 1 TLS model. But the TLS model may improve the model > everywhere, whereas the waters will only suppress a few local difference > density peaks. > > cheers, > > Ethan > > >> >> HTH, >> >> Regina >> >> >> >> >> From: Rajesh kumar >> 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. >> >> Thanks >> Raj - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] sudden drop in R/Rfree
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 > 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. > > Thanks > Raj > > > >
Re: [ccp4bb] sudden drop in R/Rfree
On Friday, 02 March 2012, 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. I'm afraid you've got this completely backwards. TLS uses very few parameters, and is especially useful at low resolution. At 3.3A I would recommend trying a TLS model _instead_ of refining individual B factors. NCS restraints also help a lot at low resolution. So the drop is believable, but... You should first worry about "lots of waters were placed". It's there that many extra parameters have been added, perhaps leading to over-fitting. I would not expect 3.3A data to justify placement of more than a handful of waters at most. If you're parameter counting, you might note that 5 water molecules add more parameters than 1 TLS model. But the TLS model may improve the model everywhere, whereas the waters will only suppress a few local difference density peaks. cheers, Ethan > > HTH, > > Regina > > > > > From: Rajesh kumar > 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. > > Thanks > Raj
Re: [ccp4bb] sudden drop in R/Rfree
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. HTH, Regina From: Rajesh kumar 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. Thanks Raj
[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
