Re: [ccp4bb] Molecular replacement problem

[Nukri Sanishvili]

Hi Robert,
In addition to the great suggestions you already have received, maybe you
should also consider SIMBAD or similar programs? The behavior you are
describing is typical of, albeit not exclusive to, having crystallized a
contaminant protein.
Good luck!
Nukri


On Thu, Jun 18, 2020, 08:01 Robert S Phillips  wrote:

> I've been pulling out my hair with this for a few months now.  I have data
> sets to 2.6 A for a new enzyme in the aminotransferase superfamily.
> Unfortunately, the closest structure is only 25% identity.  MR with PHASER
> using the monomer was a complete failure.  Since the minimum structure of
> enzymes in the family is a dimer (the active site is formed at the
> monomer-monomer interface), I used dimers for MR with PHASER.  Most of the
> results were marginal, but one looks good.  However, it will not refine.
> Everything I have done with this solution has failed, simulated annealing,
> morphing, etc., it will not refine; AUTOBUILD and BUCCANEER with it do not
> give any useable models, since they have poor statistics and low
> completeness.  The output from PHASER is below.
>
> ** SINGLE solution
>
> ** Solution written to PDB file:  DGL_phaser.1.pdb
> ** Solution written to MTZ file:  DGL_phaser.1.mtz
>Solution annotation (history):
>SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1
> PAK=0 LLG=994 TFZ==20.1
>SOLU SPAC P 2 2 21
>SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00
> BFAC -0.12 MULT 2 #TFZ==20.1
>SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89
>
> With LLG = 994 and TFZ = 20.1, isn't this a real solution?
>
> Robert S. Phillips
> Professor of Chemistry and of Biochemistry and Molecular Biology
> University of Georgia
> Athens, GA 30602
> Phone: (706) 542-1996
> Fax: (706) 542-9454
> E-mail: rsphill...@chem.uga.edu
> Web:  http://tryptophan.net
> 
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
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Re: [ccp4bb] Molecular replacement problem

[Fischmann, Thierry]

It may be worth trying rigid body-refinement of the solution, using two rigid 
bodies, one per monomer, if you haven’t tried already. Then perform positional 
refinement.

When refining with the original sequence, first remember that R/Rfree are bound 
to be and stay high with at most 25% homology. Does the map look reasonable? 
Can you see density matching the true sequence, even if refining with the 
molecular replacement model, in places where the side-chains are ordered (not 
facing the solvent) but there is an unmistakable side-chain difference (i.e. 
from a small side chain such as Ala or Val etc. to Phe Tyr Trp, or vice-versa)

If so you can either use an automated approach to fit the new sequence or do it 
“manually” in Coot, then refine again. R’s should drop quickly.

I’ve had good success using the above approach, all refinements performed with 
autoBUSTER.

Thierry

From: CCP4 bulletin board  On Behalf Of Robert S Phillips
Sent: Thursday, June 18, 2020 9:01 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Molecular replacement problem

EXTERNAL EMAIL – Use caution with any links or file attachments.
I've been pulling out my hair with this for a few months now.  I have data sets 
to 2.6 A for a new enzyme in the aminotransferase superfamily.  Unfortunately, 
the closest structure is only 25% identity.  MR with PHASER using the monomer 
was a complete failure.  Since the minimum structure of enzymes in the family 
is a dimer (the active site is formed at the monomer-monomer interface), I used 
dimers for MR with PHASER.  Most of the results were marginal, but one looks 
good.  However, it will not refine.  Everything I have done with this solution 
has failed, simulated annealing, morphing, etc., it will not refine; AUTOBUILD 
and BUCCANEER with it do not give any useable models, since they have poor 
statistics and low completeness.  The output from PHASER is below.

** SINGLE solution

** Solution written to PDB file:  DGL_phaser.1.pdb
** Solution written to MTZ file:  DGL_phaser.1.mtz
   Solution annotation (history):
   SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1 PAK=0 
LLG=994 TFZ==20.1
   SOLU SPAC P 2 2 21
   SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00 BFAC 
-0.12 MULT 2 #TFZ==20.1
   SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89

With LLG = 994 and TFZ = 20.1, isn't this a real solution?

Robert S. Phillips
Professor of Chemistry and of Biochemistry and Molecular Biology
University of Georgia
Athens, GA 30602
Phone: (706) 542-1996
Fax: (706) 542-9454
E-mail: rsphill...@chem.uga.edu<mailto:rsphill...@chem.uga.edu>
Web:  
http://tryptophan.net<https://pod51004.outlook.com/owa/redir.aspx?C=ccbf42ffea5f48b1bf8e9bb950454bab=http%3a%2f%2ftryptophan.net>



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Re: [ccp4bb] Molecular replacement problem

[Eleanor Dodson]

Or try the other orthorhombic SGs..?
Some general ideas for any such problem...

Is there evidence for a dimer in the asymmetric unit, or could your dimer
be generated by the crystal 2-folds?
Checks on data - all easily accessed from CCP4I2 ..
First is the data OK - Wilson plot? twinning? r factors v batch etc..
Second - likely contents of asymmetric unit?
Third , if there is likelty to be more than one molecule per asymm unit
a)Is there non-crystallographic translation
b) is the self rotation any help?

But here your solution is  the identity - ie the model fits your data
without either rotation or translation!

 SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00
BFAC -0.12 MULT 2 #TFZ==20.
Could the model structure be isomorphous with your new one?
Eleanor

On Thu, 18 Jun 2020 at 14:06, David Briggs  wrote:

> Hi Robert,
>
> Have you tried lower symmetry spacegroups? Maybe your crystal is
> 'almost-but-not-quite' orthorhombic and is in fact monoclinic, pretending
> to be orthorhombic.
>
> Zanuda can do this for you.
>
> https://www.ccp4.ac.uk/newsletters/newsletter48/articles/Zanuda/zanuda.html
>
> Good luck,
>
> Dave
>
> --
>
> Dr David C. Briggs
>
> Senior Laboratory Research Scientist
>
> Signalling and Structural Biology Lab
>
> The Francis Crick Institute
>
> London, UK
>
> ==
>
> Diamond User Committee MX representative
>
> ==
>
> about.me/david_briggs
> --
> *From:* CCP4 bulletin board  on behalf of Robert S
> Phillips 
> *Sent:* 18 June 2020 14:00
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* [ccp4bb] Molecular replacement problem
>
> I've been pulling out my hair with this for a few months now.  I have data
> sets to 2.6 A for a new enzyme in the aminotransferase superfamily.
> Unfortunately, the closest structure is only 25% identity.  MR with PHASER
> using the monomer was a complete failure.  Since the minimum structure of
> enzymes in the family is a dimer (the active site is formed at the
> monomer-monomer interface), I used dimers for MR with PHASER.  Most of the
> results were marginal, but one looks good.  However, it will not refine.
> Everything I have done with this solution has failed, simulated annealing,
> morphing, etc., it will not refine; AUTOBUILD and BUCCANEER with it do not
> give any useable models, since they have poor statistics and low
> completeness.  The output from PHASER is below.
>
> ** SINGLE solution
>
> ** Solution written to PDB file:  DGL_phaser.1.pdb
> ** Solution written to MTZ file:  DGL_phaser.1.mtz
>Solution annotation (history):
>SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1
> PAK=0 LLG=994 TFZ==20.1
>SOLU SPAC P 2 2 21
>SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00
> BFAC -0.12 MULT 2 #TFZ==20.1
>SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89
>
> With LLG = 994 and TFZ = 20.1, isn't this a real solution?
>
> Robert S. Phillips
> Professor of Chemistry and of Biochemistry and Molecular Biology
> University of Georgia
> Athens, GA 30602
> Phone: (706) 542-1996
> Fax: (706) 542-9454
> E-mail: rsphill...@chem.uga.edu
> Web:  http://tryptophan.net
> <https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fpod51004.outlook.com%2Fowa%2Fredir.aspx%3FC%3Dccbf42ffea5f48b1bf8e9bb950454bab%26URL%3Dhttp%253a%252f%252ftryptophan.net=02%7C01%7C%7Cd804f2e8aae94225588808d81387ab6d%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637280820702952452=DR11jkYMX2Y0wTZjbuY362yf%2FGNaIN5htG6HQaJ0XcE%3D=0>
>
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Re: [ccp4bb] Molecular replacement problem

[Roger Rowlett]

I managed to solve a structure by MR at 2.4 A with a 27% identity model.
Like you, I had to use a dimer search model to make any headway. To get
usable maps and an initial model, I used Chainsaw to truncate the search
model, Phaser (MR), Parrot (DM) with NCS averaging, then auto building with
Buccaneer. I had the advantage of 8 chains in the ASU, which greatly
improved the NCS averaging. NCS helps as the square root of the chains
averaged.

Before going down this road, all potential MR solutions were carefully
checked by examining crystal lattice packing to ensure the MR solution was
reasonable and space group was likely correct, and that I had the correct
number of chains per ASU. My initial MR attempts, based on Matthew's number
estimates, were 2 chains short, which was obvious from crystal packing.

Cheers, and good luck!

Roger Rowlett
Dorothy & Gordon Kline Professor, Emeritus
Colgate University

On Thu, Jun 18, 2020, 9:01 AM Robert S Phillips  wrote:

> I've been pulling out my hair with this for a few months now.  I have data
> sets to 2.6 A for a new enzyme in the aminotransferase superfamily.
> Unfortunately, the closest structure is only 25% identity.  MR with PHASER
> using the monomer was a complete failure.  Since the minimum structure of
> enzymes in the family is a dimer (the active site is formed at the
> monomer-monomer interface), I used dimers for MR with PHASER.  Most of the
> results were marginal, but one looks good.  However, it will not refine.
> Everything I have done with this solution has failed, simulated annealing,
> morphing, etc., it will not refine; AUTOBUILD and BUCCANEER with it do not
> give any useable models, since they have poor statistics and low
> completeness.  The output from PHASER is below.
>
> ** SINGLE solution
>
> ** Solution written to PDB file:  DGL_phaser.1.pdb
> ** Solution written to MTZ file:  DGL_phaser.1.mtz
>Solution annotation (history):
>SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1
> PAK=0 LLG=994 TFZ==20.1
>SOLU SPAC P 2 2 21
>SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00
> BFAC -0.12 MULT 2 #TFZ==20.1
>SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89
>
> With LLG = 994 and TFZ = 20.1, isn't this a real solution?
>
> Robert S. Phillips
> Professor of Chemistry and of Biochemistry and Molecular Biology
> University of Georgia
> Athens, GA 30602
> Phone: (706) 542-1996
> Fax: (706) 542-9454
> E-mail: rsphill...@chem.uga.edu
> Web:  http://tryptophan.net
> 
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Molecular replacement problem

[David Briggs]

Hi Robert,

Have you tried lower symmetry spacegroups? Maybe your crystal is 
'almost-but-not-quite' orthorhombic and is in fact monoclinic, pretending to be 
orthorhombic.

Zanuda can do this for you.

https://www.ccp4.ac.uk/newsletters/newsletter48/articles/Zanuda/zanuda.html

Good luck,

Dave


--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

Diamond User Committee MX representative

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Robert S 
Phillips 
Sent: 18 June 2020 14:00
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Molecular replacement problem

I've been pulling out my hair with this for a few months now.  I have data sets 
to 2.6 A for a new enzyme in the aminotransferase superfamily.  Unfortunately, 
the closest structure is only 25% identity.  MR with PHASER using the monomer 
was a complete failure.  Since the minimum structure of enzymes in the family 
is a dimer (the active site is formed at the monomer-monomer interface), I used 
dimers for MR with PHASER.  Most of the results were marginal, but one looks 
good.  However, it will not refine.  Everything I have done with this solution 
has failed, simulated annealing, morphing, etc., it will not refine; AUTOBUILD 
and BUCCANEER with it do not give any useable models, since they have poor 
statistics and low completeness.  The output from PHASER is below.

** SINGLE solution

** Solution written to PDB file:  DGL_phaser.1.pdb
** Solution written to MTZ file:  DGL_phaser.1.mtz
   Solution annotation (history):
   SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1 PAK=0 
LLG=994 TFZ==20.1
   SOLU SPAC P 2 2 21
   SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00 BFAC 
-0.12 MULT 2 #TFZ==20.1
   SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89

With LLG = 994 and TFZ = 20.1, isn't this a real solution?

Robert S. Phillips
Professor of Chemistry and of Biochemistry and Molecular Biology
University of Georgia
Athens, GA 30602
Phone: (706) 542-1996
Fax: (706) 542-9454
E-mail: rsphill...@chem.uga.edu
Web:  
http://tryptophan.net<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fpod51004.outlook.com%2Fowa%2Fredir.aspx%3FC%3Dccbf42ffea5f48b1bf8e9bb950454bab%26URL%3Dhttp%253a%252f%252ftryptophan.net=02%7C01%7C%7Cd804f2e8aae94225588808d81387ab6d%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637280820702952452=DR11jkYMX2Y0wTZjbuY362yf%2FGNaIN5htG6HQaJ0XcE%3D=0>



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[ccp4bb] Molecular replacement problem

[Robert S Phillips]

I've been pulling out my hair with this for a few months now.  I have data sets 
to 2.6 A for a new enzyme in the aminotransferase superfamily.  Unfortunately, 
the closest structure is only 25% identity.  MR with PHASER using the monomer 
was a complete failure.  Since the minimum structure of enzymes in the family 
is a dimer (the active site is formed at the monomer-monomer interface), I used 
dimers for MR with PHASER.  Most of the results were marginal, but one looks 
good.  However, it will not refine.  Everything I have done with this solution 
has failed, simulated annealing, morphing, etc., it will not refine; AUTOBUILD 
and BUCCANEER with it do not give any useable models, since they have poor 
statistics and low completeness.  The output from PHASER is below.

** SINGLE solution

** Solution written to PDB file:  DGL_phaser.1.pdb
** Solution written to MTZ file:  DGL_phaser.1.mtz
   Solution annotation (history):
   SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1 PAK=0 
LLG=994 TFZ==20.1
   SOLU SPAC P 2 2 21
   SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00 BFAC 
-0.12 MULT 2 #TFZ==20.1
   SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89

With LLG = 994 and TFZ = 20.1, isn't this a real solution?

Robert S. Phillips
Professor of Chemistry and of Biochemistry and Molecular Biology
University of Georgia
Athens, GA 30602
Phone: (706) 542-1996
Fax: (706) 542-9454
E-mail: rsphill...@chem.uga.edu
Web:  
http://tryptophan.net



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Re: [ccp4bb] molecular replacement problem.

[Eleanor Dodson]

Umm - this is tricky.
First of all you need to reindex the C2221 data into the P21 cell - do you
know the operator?
 then expand that data set to spacegroup P21. There is a cad option to do
this..
Then add that FreeR to the re-processed P21 data.
Eleanor


On 24 March 2013 14:37, Appu kumar appu.kum...@gmail.com wrote:

 Sorry for the misconception. Yes i am expanding the space group from
 merged mtz file.  Actually i have enough number of images collected. when i
 indexed, integrate, and scale the data in either C2221 or P 21, it fetches
 the  overall 98% completeness. But when i am trying to reindex the data
 from C2221 to P21 keeping the Rfree flag of C2221, the completeness of data
 reduced drastically to 40%. This is what i am not getting. I am a beginner
 so i have to read a lot which i am doing also, but i had few  practical
 confusion which i shared  and off course  i am getting good response. Thank
 you all for your kind response  and educating me on the problem i faced.
 Thank you all for your valuable response.

 On 24 March 2013 15:15, vellieux frederic.velli...@ibs.fr wrote:

  Hello,

 Here we deal with symmetry and the unique part of reciprocal space (the
 reciprocal space asymmetric unit so to speak).

 C222(1) has eight asymmetric units (international tables, space group 20);

 P2(1) only has two. Assuming that Friedel's law does apply, then the
 minimum rotation range to collect a non-redundant data set (one observation
 per reflection) is 90 degrees, provided that the crystal is correctly and
 perfectly aligned. Normally with our current data collection methods where
 the crystal is randomly oriented, we would collect more than 90 degrees
 (180 degrees, or 360 degrees with the Pilatus detectors on an intense SR
 beamline where you cannot really check during data collection how well the
 crystal fares during exposure to the X-rays - shoot first, think later.

 The reciprocal space asymmetric unit in C222(1) is smaller.

 I assume that what you are doing is to take the reduced data set file (an
 MTZ file probably) and reduce the symmetry from C222(1) to P2(1). You will
 not cover the monoclinic reciprocal space asymmetric unit by doing so.

 The way to do it is to take the file from processing, before
 (crystallographic symmetry) merging of the equivalents, and perform the
 scaling and merging in the P2(1) space group. Or reprocess the data frames
 in P2(1) if you have lost the unmerged data file.

 Now of course this will still give you a poor completeness if you have
 used a strategy to optimize data collection in the orthorhombic space group
 (you won't have collected enough data then for good completeness in the
 monoclinic space group).

 I hope this is clear !

 HTH,

 Fred.


 On 24/03/13 11:20, Appu kumar wrote:

 I run the phenix.xtriage to evaluate the twining but it suggest no
 twining. When i reindex from C2221 to P21, the completeness of data reduced
 from 95 % to 35% whereas the map is very good and Rwork and Rfree are 26/31
 for 2.2 resolution. I do not understand why the completeness of data
 reduced so much on reindexing. please Can anyone explain this phenomenon.
 Thank you

 On 24 March 2013 13:30, Matthias Zebisch 
 matthias.zebi...@bbz.uni-leipzig.de wrote:

  the p21 c2221 ambivalence can mean severe twinning (i had a similar
 case just now - try several crystals from the same condition) !
 What do the twinning statistics suggest?

 cheers, Matthias

 -
 Dr. Matthias Zebisch
 Division of Structural Biology,
 Wellcome Trust Centre for Human Genetics,
 University of Oxford,
 Roosevelt Drive,
 Oxford OX3 7BN, UK

 Phone (+44) 1865 287549;
 Fax (+44) 1865 287547
 Email matth...@strubi.ox.ac.uk
 Website http://www.strubi.ox.ac.uk
 -

   On 3/24/2013 7:46 AM, Appu kumar wrote:

 Thank you for the quick reply. After molecular replacement , i have done
 only few cycle of refinement in refmac. I have not done any solvent
 modification or NCS averaging. I have initially indexed the data in C2221
 but Rfree was not decreasing so i reindexed the data in  data in P121 space
 group keeping the Rfree flag of C2221. While analysing the symmetry mates ,
 i found large space but no density. structure of Ligand binding domain is
 almost identical with 90% identity in sequence. I am stuck with this
 problem and don't know how to process further.
 Please give me your valuable suggestion. I will appreciate your effort.
 Thank you
 Appu

 On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote:

 Dear Appu,

  I am not sure that I have a complete sense of the issue at hand since
 some of the information needed to think your issue through is missing in
 your email. For example, to what high resolution cut-off were the data
 measured? What resolution limits were used for the MR search? How do the
 unit cell dimensions and space group in the two cases compare?

  I am guessing the ligand binding 

Re: [ccp4bb] molecular replacement problem.

[Appu kumar]

Thank you for the quick reply. After molecular replacement , i have done
only few cycle of refinement in refmac. I have not done any solvent
modification or NCS averaging. I have initially indexed the data in C2221
but Rfree was not decreasing so i reindexed the data in  data in P121 space
group keeping the Rfree flag of C2221. While analysing the symmetry mates ,
i found large space but no density. structure of Ligand binding domain is
almost identical with 90% identity in sequence. I am stuck with this
problem and don't know how to process further.
Please give me your valuable suggestion. I will appreciate your effort.
Thank you
Appu

On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote:

 Dear Appu,

 I am not sure that I have a complete sense of the issue at hand since some
 of the information needed to think your issue through is missing in your
 email. For example, to what high resolution cut-off were the data measured?
 What resolution limits were used for the MR search? How do the unit cell
 dimensions and space group in the two cases compare?

 I am guessing the ligand binding domain in your protein has the identical
 sequence to that of the published ligand binding domain that you use as a
 template in your MR search. In any case, here are a couple of my thoughts:

 (1) It might be worth setting up different runs of MR with different
 numbers for expected copies (not just two copies but also one copy and
 three copies just in case you have one of the extreme cases of solvent
 content)?

 (2) If the MR solution is correct and there is physical room for a DNA
 binding domain in your lattice (check by displaying symmetry mates),
 perhaps the DNA binding domain is disordered. In that case (and if all
 attempts with current data fail), you may have to crystallize the protein
 in presence of DNA.


 Good luck!
 Raji




 On Sat, Mar 23, 2013 at 2:26 PM, Appu kumar appu.kum...@gmail.com wrote:

 Dear members,

   I am doing a molecular replacement of a
 transcription factor whose ligand binding structure(24000 Da) is available
 in PDB but not for the DNA binding(13000 Da). When i am searching for the
 two copies from ligand binding domain as a template model, i am getting
 very good solution but i am not getting any density for the DNA binding
 domain to build up in density. The space gorup is P 1 21 1 (4) and unit
 cell parameters are Unit Cell:   57.43   69.36  105.99   90.00   90.00
 90.00. Please guide me how to get the complete model structure. Table below
 show the matthews statistics

  For estimated molecular weight   37000.
 Nmol/asym  Matthews Coeff  %solvent   P(2.20) P(tot)
 _
   1 5.7178.46 0.00 0.01
   2 2.8556.91 0.62 0.70
   3 1.9035.37 0.37 0.29
   4 1.4313.82 0.00 0.00
 _


 The phaser molecular replacement gives the following table.
 istogram of relative frequencies of VM values
--
Frequency of most common VM value normalized to 1
VM values plotted in increments of 1/VM (0.02)

 --- relative frequency ---
 0.0  0.1  0.2  0.3  0.4  0.5  0.6  0.7  0.8  0.9  1.0
 |||||||||||
10.00 -
 8.33 -
 7.14 -
 6.25 -
 5.56 -
 5.00 -
 4.55 -
 4.17 -
 3.85 --
 3.57 ---
 3.33 --
 3.12 --
 2.94  (COMPOSITION*1)
 2.78 ---
 2.63 
 2.50 -
 2.38 
 2.27 --
 2.17 ---
 2.08 --
 2.00 --
 1.92 ---
 1.85 ---
 1.79 ---
 1.72 -
 1.67 -
 1.61 -
 1.56 -
 1.52 -
 1.47 * (COMPOSITION*2)
 1.43 -
 1.39 -
 1.35 -
 1.32 -
 1.28 -
 1.25 -

 $TABLE : Cell Content Analysis:
 $SCATTER
 :N*Composition vs Probability:0|3x0|1:1,2:
 $$
 N*Composition Probability
 $$ loggraph $$
 1 0.306066
 2 0.00141804
 $$

Most probable VM for resolution = 2.27817
Most probable MW of protein in asu for resolution = 92664.2

 Thank a lot in advance





 --
 Raji Edayathumangalam
 Instructor in Neurology, Harvard Medical School
 Research Associate, Brigham and Women's Hospital
 Visiting Research Scholar, Brandeis University

Re: [ccp4bb] molecular replacement problem.

[Appu kumar]

I run the phenix.xtriage to evaluate the twining but it suggest no twining.
When i reindex from C2221 to P21, the completeness of data reduced from 95
% to 35% whereas the map is very good and Rwork and Rfree are 26/31 for 2.2
resolution. I do not understand why the completeness of data reduced so
much on reindexing. please Can anyone explain this phenomenon.
Thank you

On 24 March 2013 13:30, Matthias Zebisch 
matthias.zebi...@bbz.uni-leipzig.de wrote:

  the p21 c2221 ambivalence can mean severe twinning (i had a similar case
 just now - try several crystals from the same condition) !
 What do the twinning statistics suggest?

 cheers, Matthias

 -
 Dr. Matthias Zebisch
 Division of Structural Biology,
 Wellcome Trust Centre for Human Genetics,
 University of Oxford,
 Roosevelt Drive,
 Oxford OX3 7BN, UK

 Phone (+44) 1865 287549;
 Fax (+44) 1865 287547
 Email matth...@strubi.ox.ac.uk
 Website http://www.strubi.ox.ac.uk
 -

 On 3/24/2013 7:46 AM, Appu kumar wrote:

 Thank you for the quick reply. After molecular replacement , i have done
 only few cycle of refinement in refmac. I have not done any solvent
 modification or NCS averaging. I have initially indexed the data in C2221
 but Rfree was not decreasing so i reindexed the data in  data in P121 space
 group keeping the Rfree flag of C2221. While analysing the symmetry mates ,
 i found large space but no density. structure of Ligand binding domain is
 almost identical with 90% identity in sequence. I am stuck with this
 problem and don't know how to process further.
 Please give me your valuable suggestion. I will appreciate your effort.
 Thank you
 Appu

 On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote:

 Dear Appu,

  I am not sure that I have a complete sense of the issue at hand since
 some of the information needed to think your issue through is missing in
 your email. For example, to what high resolution cut-off were the data
 measured? What resolution limits were used for the MR search? How do the
 unit cell dimensions and space group in the two cases compare?

  I am guessing the ligand binding domain in your protein has the
 identical sequence to that of the published ligand binding domain that you
 use as a template in your MR search. In any case, here are a couple of my
 thoughts:

  (1) It might be worth setting up different runs of MR with different
 numbers for expected copies (not just two copies but also one copy and
 three copies just in case you have one of the extreme cases of solvent
 content)?

  (2) If the MR solution is correct and there is physical room for a DNA
 binding domain in your lattice (check by displaying symmetry mates),
 perhaps the DNA binding domain is disordered. In that case (and if all
 attempts with current data fail), you may have to crystallize the protein
 in presence of DNA.


  Good luck!
 Raji




 On Sat, Mar 23, 2013 at 2:26 PM, Appu kumar appu.kum...@gmail.comwrote:

 Dear members,

   I am doing a molecular replacement of a
 transcription factor whose ligand binding structure(24000 Da) is available
 in PDB but not for the DNA binding(13000 Da). When i am searching for the
 two copies from ligand binding domain as a template model, i am getting
 very good solution but i am not getting any density for the DNA binding
 domain to build up in density. The space gorup is P 1 21 1 (4) and unit
 cell parameters are Unit Cell:   57.43   69.36  105.99   90.00   90.00
 90.00. Please guide me how to get the complete model structure. Table below
 show the matthews statistics

  For estimated molecular weight   37000.
 Nmol/asym  Matthews Coeff  %solvent   P(2.20) P(tot)
 _
   1 5.7178.46 0.00 0.01
   2 2.8556.91 0.62 0.70
   3 1.9035.37 0.37 0.29
   4 1.4313.82 0.00 0.00
 _


 The phaser molecular replacement gives the following table.
 istogram of relative frequencies of VM values
--
Frequency of most common VM value normalized to 1
VM values plotted in increments of 1/VM (0.02)

 --- relative frequency ---
 0.0  0.1  0.2  0.3  0.4  0.5  0.6  0.7  0.8  0.9  1.0
 |||||||||||
10.00 -
 8.33 -
 7.14 -
 6.25 -
 5.56 -
 5.00 -
 4.55 -
 4.17 -
 3.85 --
 3.57 ---
 3.33 --
 3.12 --
 2.94  (COMPOSITION*1)
 2.78 ---
 2.63 
 2.50 -
 2.38 
 2.27 

Re: [ccp4bb] molecular replacement problem.

[vellieux]

Hello,

Here we deal with symmetry and the unique part of reciprocal space (the 
reciprocal space asymmetric unit so to speak).


C222(1) has eight asymmetric units (international tables, space group 20);

P2(1) only has two. Assuming that Friedel's law does apply, then the 
minimum rotation range to collect a non-redundant data set (one 
observation per reflection) is 90 degrees, provided that the crystal is 
correctly and perfectly aligned. Normally with our current data 
collection methods where the crystal is randomly oriented, we would 
collect more than 90 degrees (180 degrees, or 360 degrees with the 
Pilatus detectors on an intense SR beamline where you cannot really 
check during data collection how well the crystal fares during exposure 
to the X-rays - shoot first, think later.


The reciprocal space asymmetric unit in C222(1) is smaller.

I assume that what you are doing is to take the reduced data set file 
(an MTZ file probably) and reduce the symmetry from C222(1) to P2(1). 
You will not cover the monoclinic reciprocal space asymmetric unit by 
doing so.


The way to do it is to take the file from processing, before 
(crystallographic symmetry) merging of the equivalents, and perform the 
scaling and merging in the P2(1) space group. Or reprocess the data 
frames in P2(1) if you have lost the unmerged data file.


Now of course this will still give you a poor completeness if you have 
used a strategy to optimize data collection in the orthorhombic space 
group (you won't have collected enough data then for good completeness 
in the monoclinic space group).


I hope this is clear !

HTH,

Fred.

On 24/03/13 11:20, Appu kumar wrote:
I run the phenix.xtriage to evaluate the twining but it suggest no 
twining. When i reindex from C2221 to P21, the completeness of data 
reduced from 95 % to 35% whereas the map is very good and Rwork and 
Rfree are 26/31 for 2.2 resolution. I do not understand why the 
completeness of data reduced so much on reindexing. please Can anyone 
explain this phenomenon.

Thank you

On 24 March 2013 13:30, Matthias Zebisch 
matthias.zebi...@bbz.uni-leipzig.de 
mailto:matthias.zebi...@bbz.uni-leipzig.de wrote:


the p21 c2221 ambivalence can mean severe twinning (i had a
similar case just now - try several crystals from the same
condition) !
What do the twinning statistics suggest?

cheers, Matthias

-
Dr. Matthias Zebisch
Division of Structural Biology,
Wellcome Trust Centre for Human Genetics,
University of Oxford,
Roosevelt Drive,
Oxford OX3 7BN, UK

Phone (+44) 1865 287549;
Fax (+44) 1865 287547
emailmatth...@strubi.ox.ac.uk  mailto:matth...@strubi.ox.ac.uk
Websitehttp://www.strubi.ox.ac.uk
-

On 3/24/2013 7:46 AM, Appu kumar wrote:

Thank you for the quick reply. After molecular replacement , i
have done only few cycle of refinement in refmac. I have not done
any solvent modification or NCS averaging. I have initially
indexed the data in C2221 but Rfree was not decreasing so i
reindexed the data in  data in P121 space group keeping the Rfree
flag of C2221. While analysing the symmetry mates , i found large
space but no density. structure of Ligand binding domain is
almost identical with 90% identity in sequence. I am stuck with
this problem and don't know how to process further.
Please give me your valuable suggestion. I will appreciate your
effort.
Thank you
Appu

On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu
mailto:r...@brandeis.edu wrote:

Dear Appu,

I am not sure that I have a complete sense of the issue at
hand since some of the information needed to think your issue
through is missing in your email. For example, to what high
resolution cut-off were the data measured? What resolution
limits were used for the MR search? How do the unit cell
dimensions and space group in the two cases compare?

I am guessing the ligand binding domain in your protein has
the identical sequence to that of the published ligand
binding domain that you use as a template in your MR search.
In any case, here are a couple of my thoughts:

(1) It might be worth setting up different runs of MR with
different numbers for expected copies (not just two copies
but also one copy and three copies just in case you have one
of the extreme cases of solvent content)?

(2) If the MR solution is correct and there is physical room
for a DNA binding domain in your lattice (check by displaying
symmetry mates), perhaps the DNA binding domain is
disordered. In that case (and if all attempts with current
data fail), you may have to crystallize the protein in
presence of DNA.


Good luck!
Raji

Re: [ccp4bb] molecular replacement problem.

[Raji Edayathumangalam]

Dear Appu,

You want to be sure you have good reason to drop the space group from
C222(1) to P2(1). There may be many reasons why your Rfree may not drop
following refinement, especially if you only have one domain in your
protein located and just in case there are more molecules to locate in the
MR search.

For C222(1) data, did Xtriage suggest any alternate space groups? Also,
what program did you use for MR? If you used Phaser with your C222(1) data,
did you ask to search for alternate space groups? Did you check for
translational NCS?

If you want me to take a look at your data, I'd be happy to look at your
scaled data and MR results and try to help you out. If so, please email me
off the bulletin board.

Good luck!
Raji




On Sun, Mar 24, 2013 at 6:45 AM, vellieux frederic.velli...@ibs.fr wrote:

  Hello,

 Here we deal with symmetry and the unique part of reciprocal space (the
 reciprocal space asymmetric unit so to speak).

 C222(1) has eight asymmetric units (international tables, space group 20);

 P2(1) only has two. Assuming that Friedel's law does apply, then the
 minimum rotation range to collect a non-redundant data set (one observation
 per reflection) is 90 degrees, provided that the crystal is correctly and
 perfectly aligned. Normally with our current data collection methods where
 the crystal is randomly oriented, we would collect more than 90 degrees
 (180 degrees, or 360 degrees with the Pilatus detectors on an intense SR
 beamline where you cannot really check during data collection how well the
 crystal fares during exposure to the X-rays - shoot first, think later.

 The reciprocal space asymmetric unit in C222(1) is smaller.

 I assume that what you are doing is to take the reduced data set file (an
 MTZ file probably) and reduce the symmetry from C222(1) to P2(1). You will
 not cover the monoclinic reciprocal space asymmetric unit by doing so.

 The way to do it is to take the file from processing, before
 (crystallographic symmetry) merging of the equivalents, and perform the
 scaling and merging in the P2(1) space group. Or reprocess the data frames
 in P2(1) if you have lost the unmerged data file.

 Now of course this will still give you a poor completeness if you have
 used a strategy to optimize data collection in the orthorhombic space group
 (you won't have collected enough data then for good completeness in the
 monoclinic space group).

 I hope this is clear !

 HTH,

 Fred.


 On 24/03/13 11:20, Appu kumar wrote:

 I run the phenix.xtriage to evaluate the twining but it suggest no
 twining. When i reindex from C2221 to P21, the completeness of data reduced
 from 95 % to 35% whereas the map is very good and Rwork and Rfree are 26/31
 for 2.2 resolution. I do not understand why the completeness of data
 reduced so much on reindexing. please Can anyone explain this phenomenon.
 Thank you

 On 24 March 2013 13:30, Matthias Zebisch 
 matthias.zebi...@bbz.uni-leipzig.de wrote:

  the p21 c2221 ambivalence can mean severe twinning (i had a similar
 case just now - try several crystals from the same condition) !
 What do the twinning statistics suggest?

 cheers, Matthias

 -
 Dr. Matthias Zebisch
 Division of Structural Biology,
 Wellcome Trust Centre for Human Genetics,
 University of Oxford,
 Roosevelt Drive,
 Oxford OX3 7BN, UK

 Phone (+44) 1865 287549;
 Fax (+44) 1865 287547
 Email matth...@strubi.ox.ac.uk
 Website http://www.strubi.ox.ac.uk
 -

   On 3/24/2013 7:46 AM, Appu kumar wrote:

 Thank you for the quick reply. After molecular replacement , i have done
 only few cycle of refinement in refmac. I have not done any solvent
 modification or NCS averaging. I have initially indexed the data in C2221
 but Rfree was not decreasing so i reindexed the data in  data in P121 space
 group keeping the Rfree flag of C2221. While analysing the symmetry mates ,
 i found large space but no density. structure of Ligand binding domain is
 almost identical with 90% identity in sequence. I am stuck with this
 problem and don't know how to process further.
 Please give me your valuable suggestion. I will appreciate your effort.
 Thank you
 Appu

 On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote:

 Dear Appu,

  I am not sure that I have a complete sense of the issue at hand since
 some of the information needed to think your issue through is missing in
 your email. For example, to what high resolution cut-off were the data
 measured? What resolution limits were used for the MR search? How do the
 unit cell dimensions and space group in the two cases compare?

  I am guessing the ligand binding domain in your protein has the
 identical sequence to that of the published ligand binding domain that you
 use as a template in your MR search. In any case, here are a couple of my
 thoughts:

  (1) It might be worth setting up different runs of MR with different
 numbers for expected copies 

Re: [ccp4bb] molecular replacement problem.

[Appu kumar]

Sorry for the misconception. Yes i am expanding the space group from merged
mtz file.  Actually i have enough number of images collected. when i
indexed, integrate, and scale the data in either C2221 or P 21, it fetches
the  overall 98% completeness. But when i am trying to reindex the data
from C2221 to P21 keeping the Rfree flag of C2221, the completeness of data
reduced drastically to 40%. This is what i am not getting. I am a beginner
so i have to read a lot which i am doing also, but i had few  practical
confusion which i shared  and off course  i am getting good response. Thank
you all for your kind response  and educating me on the problem i faced.
Thank you all for your valuable response.

On 24 March 2013 15:15, vellieux frederic.velli...@ibs.fr wrote:

  Hello,

 Here we deal with symmetry and the unique part of reciprocal space (the
 reciprocal space asymmetric unit so to speak).

 C222(1) has eight asymmetric units (international tables, space group 20);

 P2(1) only has two. Assuming that Friedel's law does apply, then the
 minimum rotation range to collect a non-redundant data set (one observation
 per reflection) is 90 degrees, provided that the crystal is correctly and
 perfectly aligned. Normally with our current data collection methods where
 the crystal is randomly oriented, we would collect more than 90 degrees
 (180 degrees, or 360 degrees with the Pilatus detectors on an intense SR
 beamline where you cannot really check during data collection how well the
 crystal fares during exposure to the X-rays - shoot first, think later.

 The reciprocal space asymmetric unit in C222(1) is smaller.

 I assume that what you are doing is to take the reduced data set file (an
 MTZ file probably) and reduce the symmetry from C222(1) to P2(1). You will
 not cover the monoclinic reciprocal space asymmetric unit by doing so.

 The way to do it is to take the file from processing, before
 (crystallographic symmetry) merging of the equivalents, and perform the
 scaling and merging in the P2(1) space group. Or reprocess the data frames
 in P2(1) if you have lost the unmerged data file.

 Now of course this will still give you a poor completeness if you have
 used a strategy to optimize data collection in the orthorhombic space group
 (you won't have collected enough data then for good completeness in the
 monoclinic space group).

 I hope this is clear !

 HTH,

 Fred.


 On 24/03/13 11:20, Appu kumar wrote:

 I run the phenix.xtriage to evaluate the twining but it suggest no
 twining. When i reindex from C2221 to P21, the completeness of data reduced
 from 95 % to 35% whereas the map is very good and Rwork and Rfree are 26/31
 for 2.2 resolution. I do not understand why the completeness of data
 reduced so much on reindexing. please Can anyone explain this phenomenon.
 Thank you

 On 24 March 2013 13:30, Matthias Zebisch 
 matthias.zebi...@bbz.uni-leipzig.de wrote:

  the p21 c2221 ambivalence can mean severe twinning (i had a similar
 case just now - try several crystals from the same condition) !
 What do the twinning statistics suggest?

 cheers, Matthias

 -
 Dr. Matthias Zebisch
 Division of Structural Biology,
 Wellcome Trust Centre for Human Genetics,
 University of Oxford,
 Roosevelt Drive,
 Oxford OX3 7BN, UK

 Phone (+44) 1865 287549;
 Fax (+44) 1865 287547
 Email matth...@strubi.ox.ac.uk
 Website http://www.strubi.ox.ac.uk
 -

   On 3/24/2013 7:46 AM, Appu kumar wrote:

 Thank you for the quick reply. After molecular replacement , i have done
 only few cycle of refinement in refmac. I have not done any solvent
 modification or NCS averaging. I have initially indexed the data in C2221
 but Rfree was not decreasing so i reindexed the data in  data in P121 space
 group keeping the Rfree flag of C2221. While analysing the symmetry mates ,
 i found large space but no density. structure of Ligand binding domain is
 almost identical with 90% identity in sequence. I am stuck with this
 problem and don't know how to process further.
 Please give me your valuable suggestion. I will appreciate your effort.
 Thank you
 Appu

 On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote:

 Dear Appu,

  I am not sure that I have a complete sense of the issue at hand since
 some of the information needed to think your issue through is missing in
 your email. For example, to what high resolution cut-off were the data
 measured? What resolution limits were used for the MR search? How do the
 unit cell dimensions and space group in the two cases compare?

  I am guessing the ligand binding domain in your protein has the
 identical sequence to that of the published ligand binding domain that you
 use as a template in your MR search. In any case, here are a couple of my
 thoughts:

  (1) It might be worth setting up different runs of MR with different
 numbers for expected copies (not just two copies but also one copy and
 

[ccp4bb] molecular replacement problem.

[Appu kumar]

Dear members,

  I am doing a molecular replacement of a
transcription factor whose ligand binding structure(24000 Da) is available
in PDB but not for the DNA binding(13000 Da). When i am searching for the
two copies from ligand binding domain as a template model, i am getting
very good solution but i am not getting any density for the DNA binding
domain to build up in density. The space gorup is P 1 21 1 (4) and unit
cell parameters are Unit Cell:   57.43   69.36  105.99   90.00   90.00
90.00. Please guide me how to get the complete model structure. Table below
show the matthews statistics

 For estimated molecular weight   37000.
Nmol/asym  Matthews Coeff  %solvent   P(2.20) P(tot)
_
  1 5.7178.46 0.00 0.01
  2 2.8556.91 0.62 0.70
  3 1.9035.37 0.37 0.29
  4 1.4313.82 0.00 0.00
_


The phaser molecular replacement gives the following table.
istogram of relative frequencies of VM values
   --
   Frequency of most common VM value normalized to 1
   VM values plotted in increments of 1/VM (0.02)

--- relative frequency ---
0.0  0.1  0.2  0.3  0.4  0.5  0.6  0.7  0.8  0.9  1.0
|||||||||||
   10.00 -
8.33 -
7.14 -
6.25 -
5.56 -
5.00 -
4.55 -
4.17 -
3.85 --
3.57 ---
3.33 --
3.12 --
2.94  (COMPOSITION*1)
2.78 ---
2.63 
2.50 -
2.38 
2.27 --
2.17 ---
2.08 --
2.00 --
1.92 ---
1.85 ---
1.79 ---
1.72 -
1.67 -
1.61 -
1.56 -
1.52 -
1.47 * (COMPOSITION*2)
1.43 -
1.39 -
1.35 -
1.32 -
1.28 -
1.25 -

$TABLE : Cell Content Analysis:
$SCATTER
:N*Composition vs Probability:0|3x0|1:1,2:
$$
N*Composition Probability
$$ loggraph $$
1 0.306066
2 0.00141804
$$

   Most probable VM for resolution = 2.27817
   Most probable MW of protein in asu for resolution = 92664.2

Thank a lot in advance

Re: [ccp4bb] molecular replacement problem.

[Raji Edayathumangalam]

Dear Appu,

I am not sure that I have a complete sense of the issue at hand since some
of the information needed to think your issue through is missing in your
email. For example, to what high resolution cut-off were the data measured?
What resolution limits were used for the MR search? How do the unit cell
dimensions and space group in the two cases compare?

I am guessing the ligand binding domain in your protein has the identical
sequence to that of the published ligand binding domain that you use as a
template in your MR search. In any case, here are a couple of my thoughts:

(1) It might be worth setting up different runs of MR with different
numbers for expected copies (not just two copies but also one copy and
three copies just in case you have one of the extreme cases of solvent
content)?

(2) If the MR solution is correct and there is physical room for a DNA
binding domain in your lattice (check by displaying symmetry mates),
perhaps the DNA binding domain is disordered. In that case (and if all
attempts with current data fail), you may have to crystallize the protein
in presence of DNA.


Good luck!
Raji




On Sat, Mar 23, 2013 at 2:26 PM, Appu kumar appu.kum...@gmail.com wrote:

 Dear members,

   I am doing a molecular replacement of a
 transcription factor whose ligand binding structure(24000 Da) is available
 in PDB but not for the DNA binding(13000 Da). When i am searching for the
 two copies from ligand binding domain as a template model, i am getting
 very good solution but i am not getting any density for the DNA binding
 domain to build up in density. The space gorup is P 1 21 1 (4) and unit
 cell parameters are Unit Cell:   57.43   69.36  105.99   90.00   90.00
 90.00. Please guide me how to get the complete model structure. Table below
 show the matthews statistics

  For estimated molecular weight   37000.
 Nmol/asym  Matthews Coeff  %solvent   P(2.20) P(tot)
 _
   1 5.7178.46 0.00 0.01
   2 2.8556.91 0.62 0.70
   3 1.9035.37 0.37 0.29
   4 1.4313.82 0.00 0.00
 _


 The phaser molecular replacement gives the following table.
 istogram of relative frequencies of VM values
--
Frequency of most common VM value normalized to 1
VM values plotted in increments of 1/VM (0.02)

 --- relative frequency ---
 0.0  0.1  0.2  0.3  0.4  0.5  0.6  0.7  0.8  0.9  1.0
 |||||||||||
10.00 -
 8.33 -
 7.14 -
 6.25 -
 5.56 -
 5.00 -
 4.55 -
 4.17 -
 3.85 --
 3.57 ---
 3.33 --
 3.12 --
 2.94  (COMPOSITION*1)
 2.78 ---
 2.63 
 2.50 -
 2.38 
 2.27 --
 2.17 ---
 2.08 --
 2.00 --
 1.92 ---
 1.85 ---
 1.79 ---
 1.72 -
 1.67 -
 1.61 -
 1.56 -
 1.52 -
 1.47 * (COMPOSITION*2)
 1.43 -
 1.39 -
 1.35 -
 1.32 -
 1.28 -
 1.25 -

 $TABLE : Cell Content Analysis:
 $SCATTER
 :N*Composition vs Probability:0|3x0|1:1,2:
 $$
 N*Composition Probability
 $$ loggraph $$
 1 0.306066
 2 0.00141804
 $$

Most probable VM for resolution = 2.27817
Most probable MW of protein in asu for resolution = 92664.2

 Thank a lot in advance





-- 
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University