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 

[ccp4bb] exclude water from anisotropic refinement in PDB_REDO

[Abhishek Anan]

Hi all,

Is there a way to exclude water from anisotropic refinement in PDB_REDO?

Best regards,

Abhishek

Re: [ccp4bb] AW: Another troublesome dataset (High Rfree after MR)

[Phil Jeffrey]

Rarely do I disagree with the wit and wisdom of James Holton, but R1 is 
not a property that Macromolecular World is unaware of.


R1 is just Rwork.
It's just R1 = Σ | |Fo| – |Fc| | / Σ |Fo|

However e.g. George Sheldrick's SHELXL reports it based on a 4 sig(F) 
cutoff as well as on all data.  Example:


R1 =  0.0421 for 27579 Fo > 4sig(Fo) and  0.0488 for all   30318 data
wR2 = 0.1153, GooF = S = 1.083,  Restrained GooF = 1.083  for all data
(this Small Molecule World structure is not yet finished)

wR2 is a weighted R-factor based on |F|^2

See: http://shelx.uni-ac.gwdg.de/SHELX/shelxl_user_guide.pdf

The CIF file stores the two different R1 values as:
_refine_ls_R_factor_all   0.0488
_refine_ls_R_factor_gt0.0421

So, don't expect that labeling anything "R1" uniquely defines whatever 
sigma cutoff you are actually using.  It's not implicit.  You must 
specify it but preferably don't report it at all, and just use it for 
diagnostic purposes.


Phil Jeffrey
Princeton



On 10/16/17 11:02 AM, James Holton wrote:


If you suspect that weak data (such as all the spot-free hkls beyond 
your anisotropic resoluiton limits) are driving up your Rwork/Rfree, 
then a good sanity check is to compute "R1".  Most macromolecular 
crystallographers don't know what "R1" is, but it is not only 
commonplace but required in small-molecule crystallography.  All you do 

Re: [ccp4bb] AW: Another troublesome dataset (High Rfree after MR)

[Dale Tronrud]

   Discarding weak data was not the way macromolecular refinement was
done prior to 1990.  Discarding data to lower your R-value is a bad
practice now and was a bad practice back then.  It is my recollection
that some people using X-plor adopted this practice, along with
discarding all low resolution data, but outside of that community these
methods were frowned upon.

   I agree that looking at the agreement between model and data for
subsets of your data is a useful tool for identifying pathologies, but
discarding data in refinement simply because they disagree with your
model is deception.  I know that James is not recommending this, but
that is what some people in that bad period in the 1990's were doing.
Most of us were not!

Dale Tronrud

On 10/16/2017 8:02 AM, James Holton wrote:
> 
> If you suspect that weak data (such as all the spot-free hkls beyond
> your anisotropic resoluiton limits) are driving up your Rwork/Rfree,
> then a good sanity check is to compute "R1".  Most macromolecular
> crystallographers don't know what "R1" is, but it is not only
> commonplace but required in small-molecule crystallography.  All you do
> is throw out all the weak data, say everything with I/sigma < 2 or 3,
> and then re-compute your R factors.  That is, use something like
> "sftools" to select only clearly "observed" reflections, and feed that
> data file back into your refinement program.  In fact, refining only
> against data with I/sigma>3 is the way macromolecular refinement was
> done up until about 1990.  These days, for clarity, you may want to call
> the resulting Rwork/Rfree as R1work and R1free.
> 
> If you do this, and your R1work/R1free are still just as bad as
> Rwork/Rfree, then weak data are not your problem.  You'd be surprised
> how often this is the case.  Next on the list are things like wrong
> symmetry choice, such as twinning masquerading as a symmetry operator,
> or disorder, as in large regions of the molecule that are too fluttery
> to peak above 1 sigma.  The list goes on, but doing the weak-data
> rejection test really helps narrow it down.
> 
> -James Holton
> MAD Scientist
> 
> 
> On 10/16/2017 3:55 AM, herman.schreu...@sanofi.com wrote:
>>
>> Dear Michael,
>>
>>  
>>
>> Did you ask Phaser to check for all possible space groups? There are
>> still I422 and I4 you did not mention. If the space group that came
>> out of Phaser is different from the space group used for processing,
>> subsequent refinement programs may use the wrong space group from the
>> processing. This should be easy to check.
>>
>>  
>>
>> The other suggestion I have is to try a different processing program.
>> Although XDS is excellent, I find that sometimes it has difficulties
>> with ice rings, which reveal themselves not in the processing, but in
>> the subsequent refinement. You may want to try Mosflm or some other
>> processing program.
>>
>>  
>>
>> Best,
>>
>> Herman
>>
>>  
>>
>> *Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag
>> von *Michael Jarva
>> *Gesendet:* Sonntag, 15. Oktober 2017 03:09
>> *An:* CCP4BB@JISCMAIL.AC.UK
>> *Betreff:* [EXTERNAL] [ccp4bb] Another troublesome dataset (High Rfree
>> after MR)
>>
>>  
>>
>> To add to the current anisotropic discussion I recently got a dataset
>> I’m unable to refine and I’m hoping I could get some help on figuring
>> out if there’s anything I can do.
>>
>>  
>>
>> I get a clear cut solution with Phaser using the same protein as
>> search model and got a TFZ of >16, LLG >200, and a packing that makes
>> sense, so I don’t doubt the solution. However, the maps look terrible,
>> more like something I would expect from a 3.65Å dataset rather than
>> the 2.65Å it supposedly is.
>>
>>  
>>
>> The dataset merges well in I4122 to 2.65Å with an overall Rmerge of 5%
>> and a CC1/2 of >0.5 in the outer shell (see the bottom for full
>> summary). There is some minor radiation damage but I could cut out
>> most of it due to the high symmetry.
>>
>>  
>>
>> Xtriage reports no indication of twinning, but does say that the data
>> is moderately anisotropic, so I ran the unmerged data through the
>> StarAniso server, which reported the ellipsoidal resolution limits to
>> be 2.304, 2.893, and 3.039. Refining with the anisotropically
>> truncated data improves the maps somewhat, but I am still unable to
>> get the Rfree below 38%. I tried using both phenix.refine and buster
>> with similar results.
>>
>>  
>>
>> I’ve considered the choice of space group and tried I41, F222, I212121
>> , and C2, but with the same results, and Zanuda tells me the same thing.
>>
>> Lastly, there is some minor ice rings, so my last try was to exclud
>> the ice ring resolutions, but this made little to no difference.
>>
>>  
>>
>> Normally I would just write this off as the data being bad but this
>> time all the statistics tell me this should be doable so I’m curious
>> what has gone wrong. 
>>
>>  
>>
>> Cheers
>>
>> Michael Jarva
>>
>>  
>>
>>  
>>
>> Summary data for 

Re: [ccp4bb] AW: Another troublesome dataset (High Rfree after MR)

[George Sheldrick]

Dear James.

What small molecule programs report often looks like:

R1 =  0.1550 for   17413 Fo > 4sig(Fo)  and  0.2058 for all   23715 data
R1(Free) =  0.2208 for   1938 Fo > 4sig(Fo) and  0.2766 for all   2635 data

from a well-known small molecule program being (mis)used to refine a 
protein. This clearly shows the influence of the weak outer reflections, 
however if properly weighted they still make a useful contribution to 
the refinement. Although for historical reasons this quotes I>4sigma(F), 
it corresponds to I>2sigma(I) and the structure was refined against 
intensities, not Fs. In the above case, the large gap betwen R1 and 
R1(free) also suggests that the structure has been a over-refined by 
using anisotropic temperature factors with too lax restraints. R1 is 
unweighted and based on Fs, not intensities, but in practice it seems to 
be a more useful criterion than a weighted or unweighted R2 (i.e. based 
on intensities).


Best wishes, George



On 10/16/2017 05:02 PM, James Holton wrote:


If you suspect that weak data (such as all the spot-free hkls beyond 
your anisotropic resoluiton limits) are driving up your Rwork/Rfree, 
then a good sanity check is to compute "R1".  Most macromolecular 
crystallographers don't know what "R1" is, but it is not only 
commonplace but required in small-molecule crystallography.  All you 
do is throw out all the weak data, say everything with I/sigma < 2 or 
3, and then re-compute your R factors.  That is, use something like 
"sftools" to select only clearly "observed" reflections, and feed that 
data file back into your refinement program.  In fact, refining only 
against data with I/sigma>3 is the way macromolecular refinement was 
done up until about 1990.  These days, for clarity, you may want to 
call the resulting Rwork/Rfree as R1work and R1free.


If you do this, and your R1work/R1free are still just as bad as 
Rwork/Rfree, then weak data are not your problem.  You'd be surprised 
how often this is the case.  Next on the list are things like wrong 
symmetry choice, such as twinning masquerading as a symmetry operator, 
or disorder, as in large regions of the molecule that are too fluttery 
to peak above 1 sigma.  The list goes on, but doing the weak-data 
rejection test really helps narrow it down.


-James Holton
MAD Scientist


On 10/16/2017 3:55 AM, herman.schreu...@sanofi.com wrote:


Dear Michael,

Did you ask Phaser to check for all possible space groups? There are 
still I422 and I4 you did not mention. If the space group that came 
out of Phaser is different from the space group used for processing, 
subsequent refinement programs may use the wrong space group from the 
processing. This should be easy to check.


The other suggestion I have is to try a different processing program. 
Although XDS is excellent, I find that sometimes it has difficulties 
with ice rings, which reveal themselves not in the processing, but in 
the subsequent refinement. You may want to try Mosflm or some other 
processing program.


Best,

Herman

*Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag 
von *Michael Jarva

*Gesendet:* Sonntag, 15. Oktober 2017 03:09
*An:* CCP4BB@JISCMAIL.AC.UK
*Betreff:* [EXTERNAL] [ccp4bb] Another troublesome dataset (High 
Rfree after MR)


To add to the current anisotropic discussion I recently got a dataset 
I’m unable to refine and I’m hoping I could get some help on figuring 
out if there’s anything I can do.


I get a clear cut solution with Phaser using the same protein as 
search model and got a TFZ of >16, LLG >200, and a packing that makes 
sense, so I don’t doubt the solution. However, the maps look 
terrible, more like something I would expect from a 3.65Å dataset 
rather than the 2.65Å it supposedly is.


The dataset merges well in I4122 to 2.65Å with an overall Rmerge of 
5% and a CC1/2 of >0.5 in the outer shell (see the bottom for full 
summary). There is some minor radiation damage but I could cut out 
most of it due to the high symmetry.


Xtriage reports no indication of twinning, but does say that the data 
is moderately anisotropic, so I ran the unmerged data through the 
StarAniso server, which reported the ellipsoidal resolution limits to 
be 2.304, 2.893, and 3.039. Refining with the anisotropically 
truncated data improves the maps somewhat, but I am still unable to 
get the Rfree below 38%. I tried using both phenix.refine and buster 
with similar results.


I’ve considered the choice of space group and tried I41, F222, 
I212121 , and C2, but with the same results, and Zanuda tells me the 
same thing.


Lastly, there is some minor ice rings, so my last try was to exclud 
the ice ring resolutions, but this made little to no difference.


Normally I would just write this off as the data being bad but this 
time all the statistics tell me this should be doable so I’m curious 
what has gone wrong.


Cheers

Michael Jarva

Summary data forProject: XDSproject Crystal: XDScrystal Dataset: 

Re: [ccp4bb] AW: Another troublesome dataset (High Rfree after MR)

[James Holton]

If you suspect that weak data (such as all the spot-free hkls beyond 
your anisotropic resoluiton limits) are driving up your Rwork/Rfree, 
then a good sanity check is to compute "R1".  Most macromolecular 
crystallographers don't know what "R1" is, but it is not only 
commonplace but required in small-molecule crystallography.  All you do 
is throw out all the weak data, say everything with I/sigma < 2 or 3, 
and then re-compute your R factors.  That is, use something like 
"sftools" to select only clearly "observed" reflections, and feed that 
data file back into your refinement program.  In fact, refining only 
against data with I/sigma>3 is the way macromolecular refinement was 
done up until about 1990.  These days, for clarity, you may want to call 
the resulting Rwork/Rfree as R1work and R1free.


If you do this, and your R1work/R1free are still just as bad as 
Rwork/Rfree, then weak data are not your problem.  You'd be surprised 
how often this is the case.  Next on the list are things like wrong 
symmetry choice, such as twinning masquerading as a symmetry operator, 
or disorder, as in large regions of the molecule that are too fluttery 
to peak above 1 sigma.  The list goes on, but doing the weak-data 
rejection test really helps narrow it down.


-James Holton
MAD Scientist


On 10/16/2017 3:55 AM, herman.schreu...@sanofi.com wrote:


Dear Michael,

Did you ask Phaser to check for all possible space groups? There are 
still I422 and I4 you did not mention. If the space group that came 
out of Phaser is different from the space group used for processing, 
subsequent refinement programs may use the wrong space group from the 
processing. This should be easy to check.


The other suggestion I have is to try a different processing program. 
Although XDS is excellent, I find that sometimes it has difficulties 
with ice rings, which reveal themselves not in the processing, but in 
the subsequent refinement. You may want to try Mosflm or some other 
processing program.


Best,

Herman

*Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag 
von *Michael Jarva

*Gesendet:* Sonntag, 15. Oktober 2017 03:09
*An:* CCP4BB@JISCMAIL.AC.UK
*Betreff:* [EXTERNAL] [ccp4bb] Another troublesome dataset (High Rfree 
after MR)


To add to the current anisotropic discussion I recently got a dataset 
I’m unable to refine and I’m hoping I could get some help on figuring 
out if there’s anything I can do.


I get a clear cut solution with Phaser using the same protein as 
search model and got a TFZ of >16, LLG >200, and a packing that makes 
sense, so I don’t doubt the solution. However, the maps look terrible, 
more like something I would expect from a 3.65Å dataset rather than 
the 2.65Å it supposedly is.


The dataset merges well in I4122 to 2.65Å with an overall Rmerge of 5% 
and a CC1/2 of >0.5 in the outer shell (see the bottom for full 
summary). There is some minor radiation damage but I could cut out 
most of it due to the high symmetry.


Xtriage reports no indication of twinning, but does say that the data 
is moderately anisotropic, so I ran the unmerged data through the 
StarAniso server, which reported the ellipsoidal resolution limits to 
be 2.304, 2.893, and 3.039. Refining with the anisotropically 
truncated data improves the maps somewhat, but I am still unable to 
get the Rfree below 38%. I tried using both phenix.refine and buster 
with similar results.


I’ve considered the choice of space group and tried I41, F222, I212121 
, and C2, but with the same results, and Zanuda tells me the same thing.


Lastly, there is some minor ice rings, so my last try was to exclud 
the ice ring resolutions, but this made little to no difference.


Normally I would just write this off as the data being bad but this 
time all the statistics tell me this should be doable so I’m curious 
what has gone wrong.


Cheers

Michael Jarva

Summary data for    Project: XDSproject Crystal: XDScrystal 
Dataset: XDSdataset

   Overall  InnerShell  OuterShell
Low resolution limit   34.87 34.87  2.78
High resolution limit   2.65  8.79  2.65
Rmerge  (within I+/I-) 0.052     0.026 1.595
Rmerge  (all I+ and I-)    0.057 0.030 1.805
Rmeas (within I+/I-)   0.062 0.031 1.924
Rmeas (all I+ & I-)    0.063 0.033 1.993
Rpim (within I+/I-)    0.032 0.017 1.042
Rpim (all I+ & I-) 0.025 0.014 0.817
Rmerge in top intensity bin    0.030    - -
Total number of observations   19931   566  2681
Total number unique 3597   114   471
Mean((I)/sd(I)) 11.3  42.3   0.8
Mn(I) half-set correlation CC(1/2) 0.999 0.999 0.575
Completeness

Re: [ccp4bb] TYC not linked to previoys amino-acid

[Garib Murshudov]

But it is not L-peptide. And I would not like to make it L-peptide in the 
standard ccp4 dictionary. 
You can add trans link between GLY and TYC. But it would not be nice. The best 
solution is to use TYR and amilate C terminus.

Garib


On 16 Oct 2017, at 07:40, Eleanor Dodson  wrote:

> Thank you Garib..
> 
> 
> So you have to copy the $CLIBD/monomers/t/TYC.cif to your own directory and 
> change the string "non-polymer" to "L-peptide" 
> Then use that as a dictionary entry and indeed that behaves as expected - ie 
> forms the peptide link GLY-TYC
> 
> Eleanor 
> 
> On 16 October 2017 at 12:32, Garib Murshudov  wrote:
> These are default values.
> 
> Decision about standard residue types are made using the info from the 
> dictionary.  In the current version it is like:
> 
> TYC  TYC 'L-TYROSINAMIDE  ' non-polymer25  13 
> .
> 
> I.e. it is not a peptide. Strictly speaking it is not a peptide. It would 
> form standard peptide link, at least on the carboxyl side. 
> These cases should be dealt with different types of links.
> 
> How this residue incorporated into the protein? Is it something like 
> C-terminus amilation? If it is the case then better approach is to use TYR 
> and C-terminus amilation modification. I do not remember how it works, but if 
> it is the case then i can find out.
> 
> 
> Regards
> Garib
> 
> 
> On 16 Oct 2017, at 07:06, Eleanor Dodson  wrote:
> 
>> Very puzzled by this - but now I think I understand .
>> 
>> Can you comment Garib please? 
>> 
>> In refmac there is a list of amino acids to be linked 
>> 
>>   SUBROUTINE GET_STANDARD_RES_TYPE(MDOC,LINE,MONN,ITYPE,IERR)
>>  ...
>> C -
>> C ITYPE = 3 RES_LTYPE( 3) =  'peptide '
>> C
>>   DATA RNAME /'CYS','SER','THR','PRO','ALA','GLY','ASN','ASP'
>>  *   ,'GLU','GLN','HIS','ARG','LYS','MET','ILE','LEU'
>>  *   ,'VAL','PHE','TYR','TRP','TRY','HYP','PCA','SAR'
>>  *   ,'ORN','CSS','CSH','DAR','DPR','NLE','DAS','INI'
>>  *   ,'DGL','DGN','DHI','DIL','DIV','DLI','DSN','DSP'
>>  *   ,'ABA','BMT','MLE','MVA','DVA','MSE','PTR','DAL'
>>  *   ,'DPN','DTR','DTH','TYS','CGU','DCY','ILG','OCS'
>>  *   ,'KCX','SAH','SAM','SEP','LLP','5HP','CSO'  /
>> 
>> 
>> 
>> As you can see TYC is not in this list: 
>> 
>> When I change your TYC to TYS the link is formed properly,  ( I have to 
>> delete the NXT but that shouldnt matter..) 
>> 
>> So either TYC and all other possible peptides should be added to the RNAME 
>> list, or better if the dictionary defines a residue as an L-peptide then 
>> that is how it should be handled.
>> 
>> 
>> But for the present you will have to use that work-around of creating a LINK.
>> 
>> Eleanor
>> C
>> 
>> 
>> 
> 
> Dr Garib N Murshudov
> MRC-LMB
> Francis Crick Avenue
> Cambridge 
> CB2 0QH UK
> Web http://www.mrc-lmb.cam.ac.uk, 
> http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
> 
> 
> 
> 

Dr Garib N Murshudov
MRC-LMB
Francis Crick Avenue
Cambridge 
CB2 0QH UK
Web http://www.mrc-lmb.cam.ac.uk, 
http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/

[ccp4bb] AW: Re: [ccp4bb] TYC not linked to previoys amino-acid

[Herman . Schreuder]

Dear Eleanor,
The method I proposed works for any amino acid, not just tyrosine. Having an 
amide as a separate residue may sound strange, but is it exactly how our 
chemists treat it as well.
Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Eleanor 
Dodson
Gesendet: Montag, 16. Oktober 2017 13:40
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] TYC not linked to previoys amino-acid

Thank you Garib..


So you have to copy the $CLIBD/monomers/t/TYC.cif to your own directory and 
change the string "non-polymer" to "L-peptide"
Then use that as a dictionary entry and indeed that behaves as expected - ie 
forms the peptide link GLY-TYC
Eleanor

On 16 October 2017 at 12:32, Garib Murshudov 
> wrote:
These are default values.

Decision about standard residue types are made using the info from the 
dictionary.  In the current version it is like:

TYC  TYC 'L-TYROSINAMIDE  ' non-polymer25  13 .

I.e. it is not a peptide. Strictly speaking it is not a peptide. It would form 
standard peptide link, at least on the carboxyl side.
These cases should be dealt with different types of links.

How this residue incorporated into the protein? Is it something like C-terminus 
amilation? If it is the case then better approach is to use TYR and C-terminus 
amilation modification. I do not remember how it works, but if it is the case 
then i can find out.


Regards
Garib


On 16 Oct 2017, at 07:06, Eleanor Dodson 
> wrote:


Very puzzled by this - but now I think I understand .
Can you comment Garib please?
In refmac there is a list of amino acids to be linked

  SUBROUTINE GET_STANDARD_RES_TYPE(MDOC,LINE,MONN,ITYPE,IERR)
 ...
C -
C ITYPE = 3 RES_LTYPE( 3) =  'peptide '
C
  DATA RNAME /'CYS','SER','THR','PRO','ALA','GLY','ASN','ASP'
 *   ,'GLU','GLN','HIS','ARG','LYS','MET','ILE','LEU'
 *   ,'VAL','PHE','TYR','TRP','TRY','HYP','PCA','SAR'
 *   ,'ORN','CSS','CSH','DAR','DPR','NLE','DAS','INI'
 *   ,'DGL','DGN','DHI','DIL','DIV','DLI','DSN','DSP'
 *   ,'ABA','BMT','MLE','MVA','DVA','MSE','PTR','DAL'
 *   ,'DPN','DTR','DTH','TYS','CGU','DCY','ILG','OCS'
 *   ,'KCX','SAH','SAM','SEP','LLP','5HP','CSO'  /



As you can see TYC is not in this list:

When I change your TYC to TYS the link is formed properly,  ( I have to delete 
the NXT but that shouldnt matter..)

So either TYC and all other possible peptides should be added to the RNAME 
list, or better if the dictionary defines a residue as an L-peptide then that 
is how it should be handled.


But for the present you will have to use that work-around of creating a LINK.

Eleanor
C


Dr Garib N Murshudov
MRC-LMB
Francis Crick Avenue
Cambridge
CB2 0QH UK
Web 
http://www.mrc-lmb.cam.ac.uk,
http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/

Re: [ccp4bb] TYC not linked to previoys amino-acid

[Eleanor Dodson]

Thank you Garib..


So you have to copy the $CLIBD/monomers/t/TYC.cif to your own directory and
change the string "non-polymer" to "L-peptide"
Then use that as a dictionary entry and indeed that behaves as expected -
ie forms the peptide link GLY-TYC

Eleanor

On 16 October 2017 at 12:32, Garib Murshudov 
wrote:

> These are default values.
>
> Decision about standard residue types are made using the info from the
> dictionary.  In the current version it is like:
>
> TYC  TYC 'L-TYROSINAMIDE  ' non-polymer25
>  13 .
>
> I.e. it is not a peptide. Strictly speaking it is not a peptide. It would
> form standard peptide link, at least on the carboxyl side.
> These cases should be dealt with different types of links.
>
> How this residue incorporated into the protein? Is it something like
> C-terminus amilation? If it is the case then better approach is to use TYR
> and C-terminus amilation modification. I do not remember how it works, but
> if it is the case then i can find out.
>
>
> Regards
> Garib
>
>
> On 16 Oct 2017, at 07:06, Eleanor Dodson 
> wrote:
>
> Very puzzled by this - but now I think I understand .
>
> Can you comment Garib please?
>
> In refmac there is a list of amino acids to be linked
>
>   SUBROUTINE GET_STANDARD_RES_TYPE(MDOC,LINE,MONN,ITYPE,IERR)
>  ...
> C -
> C ITYPE = 3 RES_LTYPE( 3) =  'peptide '
> C
>   DATA RNAME /'CYS','SER','THR','PRO','ALA','GLY','ASN','ASP'
>  *   ,'GLU','GLN','HIS','ARG','LYS','MET','ILE','LEU'
>  *   ,'VAL','PHE','TYR','TRP','TRY','HYP','PCA','SAR'
>  *   ,'ORN','CSS','CSH','DAR','DPR','NLE','DAS','INI'
>  *   ,'DGL','DGN','DHI','DIL','DIV','DLI','DSN','DSP'
>  *   ,'ABA','BMT','MLE','MVA','DVA','MSE','PTR','DAL'
>  *   ,'DPN','DTR','DTH','TYS','CGU','DCY','ILG','OCS'
>  *   ,'KCX','SAH','SAM','SEP','LLP','5HP','CSO'  /
>
>
>
> As you can see TYC is not in this list:
>
> When I change your TYC to TYS the link is formed properly,  ( I have to
> delete the NXT but that shouldnt matter..)
>
> So either TYC and all other possible peptides should be added to the RNAME
> list, or better if the dictionary defines a residue as an L-peptide then
> that is how it should be handled.
>
>
> But for the present you will have to use that work-around of creating a
> LINK.
>
> Eleanor
> C
>
>
>
>
> Dr Garib N Murshudov
> MRC-LMB
> Francis Crick Avenue
> Cambridge
> CB2 0QH UK
> Web http://www.mrc-lmb.cam.ac.uk,
> http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
>
>
>
>

[ccp4bb] Experiences with Simplex

[Bernhard Rupp]

Hi Fellows,

 

we are dabbling with membrane proteins and thus are wondering if

any of you have experiences with this wunder-method:

 

Making water-soluble integral
 membrane proteins in vivo
using an amphipathic protein fusion strategy. 

Mizrachi D, Chen Y, Liu J, Peng HM, Ke A, Pollack L, Turner RJ, Auchus RJ,
DeLisa MP. Nat Commun. 2015 Apr 8;6:6826. doi: 10.1038/ncomms7826.
PMID:25851941

Any experience report, also negative, would be highly appreciated.

 

Best regards, BR

 

--

Bernhard Rupp

  http://www.hofkristallamt.org/

  b...@hofkristallamt.org

+1 925 209 7429

+43 676 571 0536

--

Many plausible ideas vanish 

at the presence of thought

--

 

Re: [ccp4bb] Problem with *.ref format (from Rigaku)

[Kay Diederichs]

Dear Gottfried,

I think you should take this up with Rigaku. They might have a re-formatting 
program for .ref files, and/or they may be able to tell you how to process the 
Saturn92 data with XDS (I can hardly imagine that they changed the format in a 
XDS-incompatible way).

But you paid for the machine, and that should entitle you to their service.

good luck,
Kay

On Sun, 15 Oct 2017 13:35:34 +0200, Gottfried Palm  
wrote:

>Dear all,
>
>   this is a question about scaling data integrated in CrystalClear 
>(Rigaku data processing gui based on d*trek) in ccp4.
>Since scaling dtprofit.ref files from different scans is sometimes poor 
>or even failing within CrystalClear (i.e. with dtscaleaverage after 
>merging them), I used to try scaling them with scala. I am facing this 
>problem mainly with high resolution / small molecule data collection, 
>where I need up to 20 scans, each 90-180 degrees, for complete low and 
>high resolution.
>
>The procedure, that worked, was using
>
>dtrek2scala for each scan (with scan1.ref and output_scan1.head, then a 
>second run of dtrek2scala for scan2.ref and output_scan2.head, etc.) to 
>create scan1.mtz, scan2.mtz, etc.
>sortmtz with scan1.mtz, scan2.mtz, ... to create a multibatch mtz file
>scala with the multibatch.mtz file to create the final scaled mtz file
>
>Some time ago Rigaku changed the format of the .ref files, so 
>dtrek2scala is not working any more.
>Is there a possibility to change the new .ref format to the old one? Or 
>can I read the new .ref files in scala or better aimless directly?
>
>The alternative to process the images in xds hits a similar problem: The 
>format of the images has also changed and the new .img files (from the 
>Saturn92 detector) are not read anymore (whereas they used to be 
>processable in xds before).
>
>Greetings
>   Gottfried
>
>
>Dr. Gottfried Palm
>Ernst-Moritz-Arndt-Universität
>Inst. für Biochemie (MNF)
>Abt. Biochemie I
>Felix-Hausdorff-Straße 4
>17489 Greifswald

[ccp4bb] AW: Another troublesome dataset (High Rfree after MR)

[Herman . Schreuder]

Dear Michael,

Did you ask Phaser to check for all possible space groups? There are still I422 
and I4 you did not mention. If the space group that came out of Phaser is 
different from the space group used for processing, subsequent refinement 
programs may use the wrong space group from the processing. This should be easy 
to check.

The other suggestion I have is to try a different processing program. Although 
XDS is excellent, I find that sometimes it has difficulties with ice rings, 
which reveal themselves not in the processing, but in the subsequent 
refinement. You may want to try Mosflm or some other processing program.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Michael 
Jarva
Gesendet: Sonntag, 15. Oktober 2017 03:09
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Another troublesome dataset (High Rfree after MR)

To add to the current anisotropic discussion I recently got a dataset I'm 
unable to refine and I'm hoping I could get some help on figuring out if 
there's anything I can do.

I get a clear cut solution with Phaser using the same protein as search model 
and got a TFZ of >16, LLG >200, and a packing that makes sense, so I don't 
doubt the solution. However, the maps look terrible, more like something I 
would expect from a 3.65Å dataset rather than the 2.65Å it supposedly is.

The dataset merges well in I4122 to 2.65Å with an overall Rmerge of 5% and a 
CC1/2 of >0.5 in the outer shell (see the bottom for full summary). There is 
some minor radiation damage but I could cut out most of it due to the high 
symmetry.

Xtriage reports no indication of twinning, but does say that the data is 
moderately anisotropic, so I ran the unmerged data through the StarAniso 
server, which reported the ellipsoidal resolution limits to be 2.304, 2.893, 
and 3.039. Refining with the anisotropically truncated data improves the maps 
somewhat, but I am still unable to get the Rfree below 38%. I tried using both 
phenix.refine and buster with similar results.

I've considered the choice of space group and tried I41, F222, I212121 , and 
C2, but with the same results, and Zanuda tells me the same thing.
Lastly, there is some minor ice rings, so my last try was to exclud the ice 
ring resolutions, but this made little to no difference.

Normally I would just write this off as the data being bad but this time all 
the statistics tell me this should be doable so I'm curious what has gone wrong.

Cheers
Michael Jarva



Summary data forProject: XDSproject Crystal: XDScrystal Dataset: 
XDSdataset



   Overall  InnerShell  OuterShell

Low resolution limit   34.87 34.87  2.78

High resolution limit   2.65  8.79  2.65



Rmerge  (within I+/I-) 0.052 0.026 1.595

Rmerge  (all I+ and I-)0.057 0.030 1.805

Rmeas (within I+/I-)   0.062 0.031 1.924

Rmeas (all I+ & I-)0.063 0.033 1.993

Rpim (within I+/I-)0.032 0.017 1.042

Rpim (all I+ & I-) 0.025 0.014 0.817

Rmerge in top intensity bin0.030- -

Total number of observations   19931   566  2681

Total number unique 3597   114   471

Mean((I)/sd(I)) 11.3  42.3   0.8

Mn(I) half-set correlation CC(1/2) 0.999 0.999 0.575

Completeness97.9  93.1  99.6

Multiplicity 5.5   5.0   5.7



Anomalous completeness  92.4  92.1  96.8

Anomalous multiplicity   3.0   3.0   3.0

DelAnom correlation between half-sets  0.176 0.258 0.051

Mid-Slope of Anom Normal Probability   1.078   - -



Average unit cell:   82.39   82.39   69.73   90.00   90.00   90.00

Space group: I 41 2 2

Average mosaicity:   0.10

[ccp4bb] Beam-line Scientist and Engineer Positions Elettra, Italian Synchrotron Source, Trieste, Italy

[sushant kumar]

Dear all,
Two beam-lines dedicated to high pressure X-ray diffraction and
macromolecular crystallographic studies have been commissioned at the
Elettra Synchrotron Source in Trieste, Italy, as a part of an Indo-Italian
collaboration funded by the Department of Science and Technology on the
Indian side. There are three vacancies for beam-line scientists/engineers
in this project. Applications from suitable candidates (Indian nationals)
are invited. The applicants should have backgrounds in X-ray diffraction
and structural studies. Expertise in macromolecular crystallography
(including experimental aspects of high-throughput screening and remote
data collection) and/or high pressure crystallographic studies including a
working knowledge of diamond anvil cells is desirable. Prior experience of
working at a synchrotron facility as well as expertise in instrumentation
and data analysis pertaining to diffraction studies will be considered as
advantages. Familiarity with computer programing and interest and knowledge
in instrumentation and software packages like MATLAB, LABVIEW, PYTHON, etc.
would be added assets. Selected candidates will be expected to provide
support to users at the beam lines and assist in the enhancement of
capabilities of the beam-lines as well as carry out other duties assigned
to them. The compensation package includes a monthly all-inclusive pay in
the range of Euro 1600–2100, depending on the experience of the successful
candidate. There is also the possibility of having synchrotron beam time to
run one’s own research programme. Candidates below the age of 35 years
would be preferred, though the age limit may be relaxed in the case of
highly deserving candidates. For the post of beam-line scientist, the
candidate should have a doctoral degree in physics, chemistry, earth
sciences, any branches of life sciences or any other relevant or related
field. For the post of beam-line engineer, the candidate with either
B.Tech./M.Sc. physics or M.Sc. chemistry may apply. Both the posts would
prefer a work experience at synchrotron beam-lines and/or high-pressure
laboratories and an outstanding R record. Interested candidates should
apply within three weeks of the publication of this advertisement, sending
a detailed CV, along with a list of publications, and the names and
addresses of three references to the following address: Prof. D. D. Sarma,
Solid State and Structural Chemistry Unit, Indian Institute of Science,
Bengaluru 560 012; e-mail: elettrabeaml...@gmail.com with a cc to
ddsoffic...@gmail.com.