Re: [ccp4bb] High R/Rfree after MR

[Diana Tomchick]

I sent this on Friday to Gianluca but forgot to also send it to the bulletin 
board, so I’m posting it now, as it relates to Eleanor’s response.

__

It would also be worthwhile to consider the possibility of out-of-register 
errors in the sequence assignment for your model. I have seen cases at a 
similar resolution limit and anisotropic that had high free-R values due to 
this problem. A sequence assignment error in 10-15% of the the total scattering 
mass can be the difference between free R-values of 38% versus 32%, for 
example. With low resolution models, one frequently has one or two (or more) of 
what I like to refer to as “disembodied helixes”—I.e., helixes that have both 
N- and C- termini with insufficient electron density to connect them to the 
rest of the protein. These are great candidates for out-of-register errors.

I find Buccaneer to be a great program for re-building an initial MR model and 
correcting such errors. It won’t fix everything, especially if the local 
electron density is poor, but it does a surprisingly good job with maps in the 
2.8-3.6 Angstrom range. And if you’re reasonably sure of the sequence 
assignment in the majority of the structure solution, you can fix the portion 
that you think is correct and ask it to re-build the questionable part.

It’s my go-to method for these type of problematic structures,

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Oct 14, 2017, at 9:10 AM, Eleanor Dodson 
> wrote:

I would be worried by a structure with these R values, whether or not there is 
anisotropy to consider.

Several times when this has happened to me the spacegroup turns out to be wrong.

You do not give any details of the solution but especially if there is some 
non-crystallographic translation you can assign the space group wrongly - get a 
MR solution which fits some of the amplitudes perfectly ( eg h k 2l, and the 
rest approximately so the maps look reasonable.

Have you run MR in all possible spacegroup for your point group?

Eleanor

On 13 October 2017 at 22:08, Gerard Bricogne 
> wrote:
Dear Randy,

On Fri, Oct 13, 2017 at 04:11:44PM +0100, Randy Read wrote:
> Just to add to this point.  The MR algorithms in Phaser are now able
> to make better use of intensity data, which is particularly
> important when you have any very weak data.  Having weak data can’t
> be avoided when you have serious anisotropy (or tNCS or a
> combination of the two).  Unfortunately, if you use amplitudes that
> have been through the French & Wilson (truncate) algorithm, the real
> variation in intensity is partially masked because the posterior
> amplitude values are computed on the prior assumption that all the
> reflections in a resolution shell have the same underlying intensity
> distribution.

 Your "unfortunately" calsue may be the case with the UCLA server,
but your statement is not true about what the STARANISO server (or
STARANISO as invoked within autoPROC) does: as I already indicated in
a reply to you a few months ago in this BB, the version of TRUNCATE in
STARANISO applies the French & Wilson procedure with a prior Wilson
probability whose expectation value for the intensity is modulated by
the anisotropy of the dataset. This is clearly explained on the server
- see

   http://staraniso.globalphasing.org/staraniso_about.html#step16


 With best wishes,

  Gerard.

--
> The UCLA server actually uses Phaser under the hood — what they add is to 
> turn the anisotropic B-values into suggested resolution limits in the 
> different directions.  However, I don’t think they allow you yet to submit 
> intensities, which would be better.
>
> Best wishes,
>
> Randy Read
>
> -
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical ResearchTel: +44 1223 
> 336500
> Wellcome Trust/MRC Building Fax: +44 1223 
> 336827
> Hills RoadE-mail: 
> rj...@cam.ac.uk
> Cambridge CB2 0XY, U.K.   
> www-structmed.cimr.cam.ac.uk
>
> > On 13 Oct 2017, at 10:29, vincent Chaptal 
> > > wrote:
> >
> > Dear Gia and Paul,
> >
> > about anisotropy, one point to keep in mind is that it is not necessarily 
> > linked to the difference in resolution limits.
> > In fact I am at the moment working 

Re: [ccp4bb] High R/Rfree after MR

[Eleanor Dodson]

I would be worried by a structure with these R values, whether or not there
is anisotropy to consider.

Several times when this has happened to me the spacegroup turns out to be
wrong.

You do not give any details of the solution but especially if there is some
non-crystallographic translation you can assign the space group wrongly -
get a MR solution which fits some of the amplitudes perfectly ( eg h k 2l,
and the rest approximately so the maps look reasonable.

Have you run MR in all possible spacegroup for your point group?

Eleanor

On 13 October 2017 at 22:08, Gerard Bricogne  wrote:

> Dear Randy,
>
> On Fri, Oct 13, 2017 at 04:11:44PM +0100, Randy Read wrote:
> > Just to add to this point.  The MR algorithms in Phaser are now able
> > to make better use of intensity data, which is particularly
> > important when you have any very weak data.  Having weak data can’t
> > be avoided when you have serious anisotropy (or tNCS or a
> > combination of the two).  Unfortunately, if you use amplitudes that
> > have been through the French & Wilson (truncate) algorithm, the real
> > variation in intensity is partially masked because the posterior
> > amplitude values are computed on the prior assumption that all the
> > reflections in a resolution shell have the same underlying intensity
> > distribution.
>
>  Your "unfortunately" calsue may be the case with the UCLA server,
> but your statement is not true about what the STARANISO server (or
> STARANISO as invoked within autoPROC) does: as I already indicated in
> a reply to you a few months ago in this BB, the version of TRUNCATE in
> STARANISO applies the French & Wilson procedure with a prior Wilson
> probability whose expectation value for the intensity is modulated by
> the anisotropy of the dataset. This is clearly explained on the server
> - see
>
>http://staraniso.globalphasing.org/staraniso_about.html#step16
>
>
>  With best wishes,
>
>   Gerard.
>
> --
> > The UCLA server actually uses Phaser under the hood — what they add is
> to turn the anisotropic B-values into suggested resolution limits in the
> different directions.  However, I don’t think they allow you yet to submit
> intensities, which would be better.
> >
> > Best wishes,
> >
> > Randy Read
> >
> > -
> > Randy J. Read
> > Department of Haematology, University of Cambridge
> > Cambridge Institute for Medical ResearchTel: +44 1223 336500
> > Wellcome Trust/MRC Building Fax: +44 1223 336827
> > Hills Road
> E-mail: rj...@cam.ac.uk
> > Cambridge CB2 0XY, U.K.
> www-structmed.cimr.cam.ac.uk
> >
> > > On 13 Oct 2017, at 10:29, vincent Chaptal 
> wrote:
> > >
> > > Dear Gia and Paul,
> > >
> > > about anisotropy, one point to keep in mind is that it is not
> necessarily linked to the difference in resolution limits.
> > > In fact I am at the moment working on one of these cases, with
> extremely large difference in resolution limits, but relatively low
> anisotropy. Anisotropy is more a deviation from "normal" intensity falloff
> as a function of resolution. There is a thin difference/relationship
> between the two concepts that I think is worth investigating.
> > >
> > > we have performed a statistical analysis of this phenomenon and the
> paper is in revision at the moment, but if you want to know where your
> anisotropy stands in respect to all the other PDBs out there, feel free to
> contact me off list.
> > > You mention MR: Phaser calculates the anisotropy so you can find the
> value in the first lines of the log.
> > >
> > > Staraniso or the UCLA server are good to test if you have anisotropy.
> Staraniso has a newer way of dealing with intensity falloff and accounting
> for it.
> > >
> > > All the best
> > > Vincent
> > >
> > >
> > >
> > >
> > > On 13/10/2017 10:58, Paul Miller wrote:
> > >> I had a similar problem to what you describe. In my case the dataset
> was severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors
> were stuck similar to yours but the map looked good. I was told by someone
> with a much better appreciation of the theory than myself that the
> anisotropy was causing the problem.
> > >>
> > >> It would be interesting to know from an expert in anisotropy e.g. the
> creators of UCLA anisotropy server or Startaniso whether anisotropy can
> cause this problem and whether there is any way around it.
> > >>
> > >> Cheers, Paul
> > >>
> > >> Paul Steven Miller (PhD)
> > >> Postdoctoral Researcher
> > >> University of Oxford
> > >> Wellcome Trust Centre for Human Genetics
> > >> Division of Structural Biology
> > >> Roosevelt Drive
> > >> Oxford
> > >> OX3 7BN
> > >>
> > >>
> > >>  Original message 
> > >>
> > >>> Date: Fri, 13 Oct 2017 10:30:22 +0200
> > >>> From: CCP4 bulletin board
> > >>>  (on behalf of Gianluca Cioci <
> gianluca.ci...@gmail.com>
> > >>> )
> > >>> Subject: [ccp4bb] High R/Rfree after MR
> > >>> To:
> > >>> 

Re: [ccp4bb] High R/Rfree after MR

[Gerard Bricogne]

Dear Randy,

On Fri, Oct 13, 2017 at 04:11:44PM +0100, Randy Read wrote:
> Just to add to this point.  The MR algorithms in Phaser are now able
> to make better use of intensity data, which is particularly
> important when you have any very weak data.  Having weak data can’t
> be avoided when you have serious anisotropy (or tNCS or a
> combination of the two).  Unfortunately, if you use amplitudes that
> have been through the French & Wilson (truncate) algorithm, the real
> variation in intensity is partially masked because the posterior
> amplitude values are computed on the prior assumption that all the
> reflections in a resolution shell have the same underlying intensity
> distribution. 
 
 Your "unfortunately" calsue may be the case with the UCLA server,
but your statement is not true about what the STARANISO server (or
STARANISO as invoked within autoPROC) does: as I already indicated in
a reply to you a few months ago in this BB, the version of TRUNCATE in
STARANISO applies the French & Wilson procedure with a prior Wilson
probability whose expectation value for the intensity is modulated by
the anisotropy of the dataset. This is clearly explained on the server
- see 

   http://staraniso.globalphasing.org/staraniso_about.html#step16


 With best wishes,
 
  Gerard.

--
> The UCLA server actually uses Phaser under the hood — what they add is to 
> turn the anisotropic B-values into suggested resolution limits in the 
> different directions.  However, I don’t think they allow you yet to submit 
> intensities, which would be better.
> 
> Best wishes,
> 
> Randy Read
> 
> -
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical ResearchTel: +44 1223 336500
> Wellcome Trust/MRC Building Fax: +44 1223 336827
> Hills RoadE-mail: 
> rj...@cam.ac.uk
> Cambridge CB2 0XY, U.K.   
> www-structmed.cimr.cam.ac.uk
> 
> > On 13 Oct 2017, at 10:29, vincent Chaptal  wrote:
> > 
> > Dear Gia and Paul, 
> > 
> > about anisotropy, one point to keep in mind is that it is not necessarily 
> > linked to the difference in resolution limits.
> > In fact I am at the moment working on one of these cases, with extremely 
> > large difference in resolution limits, but relatively low anisotropy. 
> > Anisotropy is more a deviation from "normal" intensity falloff as a 
> > function of resolution. There is a thin difference/relationship between the 
> > two concepts that I think is worth investigating. 
> > 
> > we have performed a statistical analysis of this phenomenon and the paper 
> > is in revision at the moment, but if you want to know where your anisotropy 
> > stands in respect to all the other PDBs out there, feel free to contact me 
> > off list. 
> > You mention MR: Phaser calculates the anisotropy so you can find the value 
> > in the first lines of the log. 
> > 
> > Staraniso or the UCLA server are good to test if you have anisotropy. 
> > Staraniso has a newer way of dealing with intensity falloff and accounting 
> > for it. 
> > 
> > All the best
> > Vincent
> > 
> > 
> > 
> > 
> > On 13/10/2017 10:58, Paul Miller wrote:
> >> I had a similar problem to what you describe. In my case the dataset was 
> >> severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors were 
> >> stuck similar to yours but the map looked good. I was told by someone with 
> >> a much better appreciation of the theory than myself that the anisotropy 
> >> was causing the problem.
> >> 
> >> It would be interesting to know from an expert in anisotropy e.g. the 
> >> creators of UCLA anisotropy server or Startaniso whether anisotropy can 
> >> cause this problem and whether there is any way around it.
> >> 
> >> Cheers, Paul
> >> 
> >> Paul Steven Miller (PhD)
> >> Postdoctoral Researcher
> >> University of Oxford
> >> Wellcome Trust Centre for Human Genetics
> >> Division of Structural Biology
> >> Roosevelt Drive
> >> Oxford
> >> OX3 7BN
> >> 
> >> 
> >>  Original message 
> >> 
> >>> Date: Fri, 13 Oct 2017 10:30:22 +0200
> >>> From: CCP4 bulletin board 
> >>>  (on behalf of Gianluca Cioci 
> >>> 
> >>> )
> >>> Subject: [ccp4bb] High R/Rfree after MR  
> >>> To: 
> >>> CCP4BB@JISCMAIL.AC.UK
> >>> 
> >>> 
> >>>   Dear All,
> >>>   I am trying to refine a structure at 3.3A. Model has
> >>>   60% identity to the target. Maps look OK (for 3.3A)
> >>>   and rebuilding in Coot is relatively
> >>>   straightforward. However, after some rebuilding
> >>>   cycles the R factors are stuck at 0.37/0.39
> >>>   (REFMAC). 
> >>>   XTRIAGE tells me that everything is normal (no twin,
> >>>   98% completeness, R=3.5% in the low resolution bin),
> >>>   perhaps some anisotropy is present. 
> >>>   I have already refined 2 homologous structures at
> >>>   resolutions going from 3.2 

Re: [ccp4bb] High R/Rfree after MR

[Gianluca Cioci]

Thank to all for the replies !

I have tried the UCLA server using the unmerged intensities from XDS
 and the verdict is strong anisotropy
The Rfactors (after Phaser+Refmac) are only slightly lower for the
Corrected.
Non Corrected:Corrected

R factor  0.3778  R factor  0.3732

R free  0.3871 R free  0.3816

I will make a few cycles of rebuilding and see if it makes any difference...


Best regards,


GIA



Best regards,

GIA


On Fri, Oct 13, 2017 at 5:11 PM, Randy Read  wrote:

> Just to add to this point.  The MR algorithms in Phaser are now able to
> make better use of intensity data, which is particularly important when you
> have any very weak data.  Having weak data can’t be avoided when you have
> serious anisotropy (or tNCS or a combination of the two).  Unfortunately,
> if you use amplitudes that have been through the French & Wilson (truncate)
> algorithm, the real variation in intensity is partially masked because the
> posterior amplitude values are computed on the prior assumption that all
> the reflections in a resolution shell have the same underlying intensity
> distribution.
>
> The UCLA server actually uses Phaser under the hood — what they add is to
> turn the anisotropic B-values into suggested resolution limits in the
> different directions.  However, I don’t think they allow you yet to submit
> intensities, which would be better.
>
> Best wishes,
>
> Randy Read
>
> -
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical ResearchTel: +44 1223 336500
> Wellcome Trust/MRC Building Fax: +44 1223 336827
> Hills Road
> E-mail: rj...@cam.ac.uk
> Cambridge CB2 0XY, U.K.
> www-structmed.cimr.cam.ac.uk
>
> > On 13 Oct 2017, at 10:29, vincent Chaptal 
> wrote:
> >
> > Dear Gia and Paul,
> >
> > about anisotropy, one point to keep in mind is that it is not
> necessarily linked to the difference in resolution limits.
> > In fact I am at the moment working on one of these cases, with extremely
> large difference in resolution limits, but relatively low anisotropy.
> Anisotropy is more a deviation from "normal" intensity falloff as a
> function of resolution. There is a thin difference/relationship between the
> two concepts that I think is worth investigating.
> >
> > we have performed a statistical analysis of this phenomenon and the
> paper is in revision at the moment, but if you want to know where your
> anisotropy stands in respect to all the other PDBs out there, feel free to
> contact me off list.
> > You mention MR: Phaser calculates the anisotropy so you can find the
> value in the first lines of the log.
> >
> > Staraniso or the UCLA server are good to test if you have anisotropy.
> Staraniso has a newer way of dealing with intensity falloff and accounting
> for it.
> >
> > All the best
> > Vincent
> >
> >
> >
> >
> > On 13/10/2017 10:58, Paul Miller wrote:
> >> I had a similar problem to what you describe. In my case the dataset
> was severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors
> were stuck similar to yours but the map looked good. I was told by someone
> with a much better appreciation of the theory than myself that the
> anisotropy was causing the problem.
> >>
> >> It would be interesting to know from an expert in anisotropy e.g. the
> creators of UCLA anisotropy server or Startaniso whether anisotropy can
> cause this problem and whether there is any way around it.
> >>
> >> Cheers, Paul
> >>
> >> Paul Steven Miller (PhD)
> >> Postdoctoral Researcher
> >> University of Oxford
> >> Wellcome Trust Centre for Human Genetics
> >> Division of Structural Biology
> >> Roosevelt Drive
> >> Oxford
> >> OX3 7BN
> >>
> >>
> >>  Original message 
> >>
> >>> Date: Fri, 13 Oct 2017 10:30:22 +0200
> >>> From: CCP4 bulletin board
> >>>  (on behalf of Gianluca Cioci <
> gianluca.ci...@gmail.com>
> >>> )
> >>> Subject: [ccp4bb] High R/Rfree after MR
> >>> To:
> >>> CCP4BB@JISCMAIL.AC.UK
> >>>
> >>>
> >>>   Dear All,
> >>>   I am trying to refine a structure at 3.3A. Model has
> >>>   60% identity to the target. Maps look OK (for 3.3A)
> >>>   and rebuilding in Coot is relatively
> >>>   straightforward. However, after some rebuilding
> >>>   cycles the R factors are stuck at 0.37/0.39
> >>>   (REFMAC).
> >>>   XTRIAGE tells me that everything is normal (no twin,
> >>>   98% completeness, R=3.5% in the low resolution bin),
> >>>   perhaps some anisotropy is present.
> >>>   I have already refined 2 homologous structures at
> >>>   resolutions going from 3.2 to 3.8 and there were no
> >>>   problems (final R ~ 0.21/0.24).
> >>>   Any advice ?
> >>>   Thanks,
> >>>   GIA
> >>>
> >
> > --
> > Vincent Chaptal, PhD
> > Institut de Biologie et Chimie des Protéines
> > Drug Resistance and Membrane Proteins Laboratory
> > 7 passage du Vercors
> > 69007 LYON
> > FRANCE
> > +33 4 37 65 29 

Re: [ccp4bb] High R/Rfree after MR

[Randy Read]

Just to add to this point.  The MR algorithms in Phaser are now able to make 
better use of intensity data, which is particularly important when you have any 
very weak data.  Having weak data can’t be avoided when you have serious 
anisotropy (or tNCS or a combination of the two).  Unfortunately, if you use 
amplitudes that have been through the French & Wilson (truncate) algorithm, the 
real variation in intensity is partially masked because the posterior amplitude 
values are computed on the prior assumption that all the reflections in a 
resolution shell have the same underlying intensity distribution.  

The UCLA server actually uses Phaser under the hood — what they add is to turn 
the anisotropic B-values into suggested resolution limits in the different 
directions.  However, I don’t think they allow you yet to submit intensities, 
which would be better.

Best wishes,

Randy Read

-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical ResearchTel: +44 1223 336500
Wellcome Trust/MRC Building Fax: +44 1223 336827
Hills RoadE-mail: 
rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   
www-structmed.cimr.cam.ac.uk

> On 13 Oct 2017, at 10:29, vincent Chaptal  wrote:
> 
> Dear Gia and Paul, 
> 
> about anisotropy, one point to keep in mind is that it is not necessarily 
> linked to the difference in resolution limits.
> In fact I am at the moment working on one of these cases, with extremely 
> large difference in resolution limits, but relatively low anisotropy. 
> Anisotropy is more a deviation from "normal" intensity falloff as a function 
> of resolution. There is a thin difference/relationship between the two 
> concepts that I think is worth investigating. 
> 
> we have performed a statistical analysis of this phenomenon and the paper is 
> in revision at the moment, but if you want to know where your anisotropy 
> stands in respect to all the other PDBs out there, feel free to contact me 
> off list. 
> You mention MR: Phaser calculates the anisotropy so you can find the value in 
> the first lines of the log. 
> 
> Staraniso or the UCLA server are good to test if you have anisotropy. 
> Staraniso has a newer way of dealing with intensity falloff and accounting 
> for it. 
> 
> All the best
> Vincent
> 
> 
> 
> 
> On 13/10/2017 10:58, Paul Miller wrote:
>> I had a similar problem to what you describe. In my case the dataset was 
>> severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors were 
>> stuck similar to yours but the map looked good. I was told by someone with a 
>> much better appreciation of the theory than myself that the anisotropy was 
>> causing the problem.
>> 
>> It would be interesting to know from an expert in anisotropy e.g. the 
>> creators of UCLA anisotropy server or Startaniso whether anisotropy can 
>> cause this problem and whether there is any way around it.
>> 
>> Cheers, Paul
>> 
>> Paul Steven Miller (PhD)
>> Postdoctoral Researcher
>> University of Oxford
>> Wellcome Trust Centre for Human Genetics
>> Division of Structural Biology
>> Roosevelt Drive
>> Oxford
>> OX3 7BN
>> 
>> 
>>  Original message 
>> 
>>> Date: Fri, 13 Oct 2017 10:30:22 +0200
>>> From: CCP4 bulletin board 
>>>  (on behalf of Gianluca Cioci 
>>> 
>>> )
>>> Subject: [ccp4bb] High R/Rfree after MR  
>>> To: 
>>> CCP4BB@JISCMAIL.AC.UK
>>> 
>>> 
>>>   Dear All,
>>>   I am trying to refine a structure at 3.3A. Model has
>>>   60% identity to the target. Maps look OK (for 3.3A)
>>>   and rebuilding in Coot is relatively
>>>   straightforward. However, after some rebuilding
>>>   cycles the R factors are stuck at 0.37/0.39
>>>   (REFMAC). 
>>>   XTRIAGE tells me that everything is normal (no twin,
>>>   98% completeness, R=3.5% in the low resolution bin),
>>>   perhaps some anisotropy is present. 
>>>   I have already refined 2 homologous structures at
>>>   resolutions going from 3.2 to 3.8 and there were no
>>>   problems (final R ~ 0.21/0.24). 
>>>   Any advice ?
>>>   Thanks,
>>>   GIA
>>> 
> 
> -- 
> Vincent Chaptal, PhD
> Institut de Biologie et Chimie des Protéines
> Drug Resistance and Membrane Proteins Laboratory
> 7 passage du Vercors 
> 69007 LYON
> FRANCE
> +33 4 37 65 29 01
> http://www.ibcp.fr
> 
> 

Re: [ccp4bb] High R/Rfree after MR

[vincent Chaptal]

Dear Gia and Paul,

about anisotropy, one point to keep in mind is that it is not 
necessarily linked to the difference in resolution limits.
In fact I am at the moment working on one of these cases, with extremely 
large difference in resolution limits, but relatively low anisotropy. 
Anisotropy is more a deviation from "normal" intensity falloff as a 
function of resolution. There is a thin difference/relationship between 
the two concepts that I think is worth investigating.


we have performed a statistical analysis of this phenomenon and the 
paper is in revision at the moment, but if you want to know where your 
anisotropy stands in respect to all the other PDBs out there, feel free 
to contact me off list.
You mention MR: Phaser calculates the anisotropy so you can find the 
value in the first lines of the log.


Staraniso or the UCLA server are good to test if you have anisotropy. 
Staraniso has a newer way of dealing with intensity falloff and 
accounting for it.


All the best
Vincent




On 13/10/2017 10:58, Paul Miller wrote:

I had a similar problem to what you describe. In my case the dataset was 
severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors were stuck 
similar to yours but the map looked good. I was told by someone with a much 
better appreciation of the theory than myself that the anisotropy was causing 
the problem.

It would be interesting to know from an expert in anisotropy e.g. the creators 
of UCLA anisotropy server or Startaniso whether anisotropy can cause this 
problem and whether there is any way around it.

Cheers, Paul

Paul Steven Miller (PhD)
Postdoctoral Researcher
University of Oxford
Wellcome Trust Centre for Human Genetics
Division of Structural Biology
Roosevelt Drive
Oxford
OX3 7BN


 Original message 

Date: Fri, 13 Oct 2017 10:30:22 +0200
From: CCP4 bulletin board  (on behalf of Gianluca Cioci 
)
Subject: [ccp4bb] High R/Rfree after MR
To: CCP4BB@JISCMAIL.AC.UK

   Dear All,
   I am trying to refine a structure at 3.3A. Model has
   60% identity to the target. Maps look OK (for 3.3A)
   and rebuilding in Coot is relatively
   straightforward. However, after some rebuilding
   cycles the R factors are stuck at 0.37/0.39
   (REFMAC).
   XTRIAGE tells me that everything is normal (no twin,
   98% completeness, R=3.5% in the low resolution bin),
   perhaps some anisotropy is present.
   I have already refined 2 homologous structures at
   resolutions going from 3.2 to 3.8 and there were no
   problems (final R ~ 0.21/0.24).
   Any advice ?
   Thanks,
   GIA


--

Vincent Chaptal, PhD

Institut de Biologie et Chimie des Protéines

Drug Resistance and Membrane Proteins Laboratory

7 passage du Vercors

69007 LYON

FRANCE

+33 4 37 65 29 01

http://www.ibcp.fr

Re: [ccp4bb] High R/Rfree after MR

[Kay Diederichs]

Gianluca,

you could become an expert yourself, by trying 
- the UCLA anisotropy server
- STARANISO
with these data, and report here what the outcome in each case is - 
Rwork/Rfree, map appearance, ...

My personal experience is that both refmac and phenix.refine deal well with 
moderate anisotropy (but there is room for improvement, e.g. current 
phenix.refine does not take the sigmas into account). In cases of severe 
anisotropy I had good results with STARANISO - better phasing, better maps, 
better refinement.

best,

Kay

On Fri, 13 Oct 2017 09:58:39 +0100, Paul Miller  wrote:

>I had a similar problem to what you describe. In my case the dataset was 
>severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors were stuck 
>similar to yours but the map looked good. I was told by someone with a much 
>better appreciation of the theory than myself that the anisotropy was causing 
>the problem.
>
>It would be interesting to know from an expert in anisotropy e.g. the creators 
>of UCLA anisotropy server or Startaniso whether anisotropy can cause this 
>problem and whether there is any way around it.
>
>Cheers, Paul
>
>Paul Steven Miller (PhD)
>Postdoctoral Researcher
>University of Oxford
>Wellcome Trust Centre for Human Genetics
>Division of Structural Biology
>Roosevelt Drive
>Oxford
>OX3 7BN
>
>
> Original message 
>>Date: Fri, 13 Oct 2017 10:30:22 +0200
>>From: CCP4 bulletin board  (on behalf of Gianluca 
>>Cioci )
>>Subject: [ccp4bb] High R/Rfree after MR  
>>To: CCP4BB@JISCMAIL.AC.UK
>>
>>   Dear All,
>>   I am trying to refine a structure at 3.3A. Model has
>>   60% identity to the target. Maps look OK (for 3.3A)
>>   and rebuilding in Coot is relatively
>>   straightforward. However, after some rebuilding
>>   cycles the R factors are stuck at 0.37/0.39
>>   (REFMAC).�
>>   XTRIAGE tells me that everything is normal (no twin,
>>   98% completeness, R=3.5% in the low resolution bin),
>>   perhaps some anisotropy is present.�
>>   I have already refined 2 homologous structures at
>>   resolutions going from 3.2 to 3.8 and there were no
>>   problems (final R ~ 0.21/0.24).�
>>   Any advice ?
>>   Thanks,
>>   GIA

Re: [ccp4bb] High R/Rfree after MR

[Paul Miller]

I had a similar problem to what you describe. In my case the dataset was 
severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors were stuck 
similar to yours but the map looked good. I was told by someone with a much 
better appreciation of the theory than myself that the anisotropy was causing 
the problem.

It would be interesting to know from an expert in anisotropy e.g. the creators 
of UCLA anisotropy server or Startaniso whether anisotropy can cause this 
problem and whether there is any way around it.

Cheers, Paul

Paul Steven Miller (PhD)
Postdoctoral Researcher
University of Oxford
Wellcome Trust Centre for Human Genetics
Division of Structural Biology
Roosevelt Drive
Oxford
OX3 7BN


 Original message 
>Date: Fri, 13 Oct 2017 10:30:22 +0200
>From: CCP4 bulletin board  (on behalf of Gianluca Cioci 
>)
>Subject: [ccp4bb] High R/Rfree after MR  
>To: CCP4BB@JISCMAIL.AC.UK
>
>   Dear All,
>   I am trying to refine a structure at 3.3A. Model has
>   60% identity to the target. Maps look OK (for 3.3A)
>   and rebuilding in Coot is relatively
>   straightforward. However, after some rebuilding
>   cycles the R factors are stuck at 0.37/0.39
>   (REFMAC). 
>   XTRIAGE tells me that everything is normal (no twin,
>   98% completeness, R=3.5% in the low resolution bin),
>   perhaps some anisotropy is present. 
>   I have already refined 2 homologous structures at
>   resolutions going from 3.2 to 3.8 and there were no
>   problems (final R ~ 0.21/0.24). 
>   Any advice ?
>   Thanks,
>   GIA