Re: [ccp4bb] CCP4 mailing list

[Eleanor Dodson]

Well - I seem to get many things twice!  Prob. my fault - and it fills 
up my "Deleted" box..


 Eleanor

Thomas Stout wrote:
 
Has anyone else noticed that they're only getting some of the postings 
on the CCP4 mailing list?
 
Lately I've been noticing that I often see responses to queries, but 
not the query itself.   This was further highlighted for me recently 
when I never received my own posting back from the mailing list.
 
Not that I am asking for MORE e-mail, but it is odd.

-Tom
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Re: [ccp4bb] Merging datasets

[Eleanor Dodson]

There might be many reasons..

Do you have a spacegroup with alternate indexing? If so the two crystals 
may not be using the same convention.

See $CHTML/reindexing.html for more details.

Or is one data set a high resolution pass and one a low resolution pass? 
Sometimes then there are saturated spots which mess up the scaling..


I usually merge both sets independently first - cad them together and 
run SCALEIT to check the degree of agreement.


SCALEIT gives a useful list of outliers and you might be able to 
pinpoint the problem from that.



Kianoush Sadre-Bazzaz wrote:

Dear CCP4,

I have a couple of datasets native and heavy-atom-derivatized. The 
spacegroup and cell dimensions of all are the same (or very 
similar).For some reason I cannot merge any of the datasets 
together. If for example I rescale one native dataset by itself, my 
Rmerge is about 10%, but when I add 50 images (or so) of another, 
supposedly isomorphous crystal, my Rmerge goes up to 20%.


I was wondering if anyone has encountered a similar problem or can 
offer any suggestions. Thanks so much,

Kianoush

Choose the right car based on your needs. Check out Yahoo! Autos new 
Car Finder tool. 
 

Re: [ccp4bb] CCP4 mailing list

[Michele Lunelli]

Maybe some post is rejected because (incorrectly) marked as spam, it happens the 
same to me with yahoo..


Michele

Thomas Stout wrote:
 
Has anyone else noticed that they're only getting some of the postings 
on the CCP4 mailing list?
 

[ccp4bb] AMORE_NSOL

[Xue-Yuan Pei]

Dear ALL:
I am runging AMORE and get an error message:
 AMORE:   NSOL set too small
When I checked the document, I could not find the keyword to reset NSOL.
Any one can give a suggestion?
Regards,
xue

Re: [ccp4bb] AMORE_NSOL

[Eleanor Dodson]

Xue-Yuan Pei wrote:

Dear ALL:
I am runging AMORE and get an error message:
 AMORE:   NSOL set too small
When I checked the document, I could not find the keyword to reset NSOL.
Any one can give a suggestion?
Regards,
xue


  



This is a program parameter - it means you are working with more than 
499 possible solutions to the ROTATION or TRANSLATION search.


That seems more than you should need - you can set PKLIM to something 
higher perhaps?

Eleanor

Re: [ccp4bb] needles vs crystals

[Jens-Christian Navarro Poulsen]

Dear Shivesh

 

I would also go along with Marks suggestions, but also include a second
round of screening in which you seed all of the new screening
experiments with you "needle" seed stock.

 

Best wishes

 

Jens-Christian

 



From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
[EMAIL PROTECTED]
Sent: 7. august 2007 19:18
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] needles vs crystals

 

Shivesh,

In addition to the comments that have been made already, I would try
seeding with the needles (or pieces thereof) - macroscopic, or take a
couple of needles and crush them up into really small pieces and use
various dilutions of these very small pieces for seeding. You could do
that by serial dilution and adding a small amount to each new drop, or
you could use the cat whisker /horse hair approach to distribute some of
the nuclei to new drops.

Good luck.

Mark

 

 

-Original Message-
From: shivesh kumar <[EMAIL PROTECTED]>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tue, 7 Aug 2007 8:45 am
Subject: [ccp4bb] needles vs crystals

Dear all,

I am trying to crystallize a 7kDa protein with MPD as a precipitant.I
got the needles with precipitation in two days at 30% and in 4-5
days,needles appear in all the drops(pH 3.8-4.8).The concentration of
protein is around 20mg/ml and the volume of mother liquor in
500microlt.What should I do to get good crystals,the temp. I am trying
is 16C.Thanx in advance for the suggestions.

Shivesh Kumar

 

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[ccp4bb] mr query

[Kolstoe S.E.]

Dear ccp4bb,
 
In searching for a mr solution I ran both phaser and molrep, with both
programs finding nice solutions in P21212 (cell 81.083  104.435  109.732
90.00  90.00  90.00, resolution 2.65A). For various reasons I refined
the phaser and molrep solutions separately, and both behaved in exactly
the same way giving R=0.24, Rfree=0.28 after adding ligands, mutations
etc. Today I decided to compare the two models (which I figured were
exactly the same) however found that the asymmetric units did not
superimpose. Normally this is just a case of each mr program finding a
different asymmetric unit in the same cell, however this time there is a
distinct clash between the two solutions with about 50 atoms
overlapping. After playing around with various symmetry equivalents I
have found that the difference between the solutions is approximately a
30A translation along the x axis. As there is no signs of any twinning,
and both solutions behave in exactly the same way in refinement, is this
a case of phaser and molrep using different origins or is there
something more insidious going on?
 
Thanks for any advice,
 
Simon
 

Re: [ccp4bb] mr query

[Ian Tickle]

Hi Simon

You didn't say whether you have just 1 mol/a.u. or more than one.  In
the latter case you can get the effect you observe because as well as
considering possible origin shifts you also have to consider the
possibility that say the A and B molecules have been switched, i.e. like
the origin shifts the labels A & B assigned by the MR program are
completely arbitrary.  I had a similar case with 2 mols/a.u. where I got
solutions from different MR programs with identical R factors around 20%
and the molecules were apparently shifted by about 3 Ang relative to
each other.  As soon as I switched the A and B labels in one of the
solutions it was obvious that the solutions were in fact identical (this
happened because in that particular case the A-B translation vector was
almost 1/2 of a lattice translation).

However if you have only 1 mol/a.u. then this doesn't help - in which
case I'm as baffled as you, sorry!

Cheers

-- Ian

> -Original Message-
> From: [EMAIL PROTECTED] 
> [mailto:[EMAIL PROTECTED] On Behalf Of Kolstoe S.E.
> Sent: 08 August 2007 16:51
> To: ccp4bb@jiscmail.ac.uk
> Subject: mr query
> 
> Dear ccp4bb,
>  
> In searching for a mr solution I ran both phaser and molrep, 
> with both programs finding nice solutions in P21212 (cell 
> 81.083  104.435  109.732  90.00  90.00  90.00, resolution 
> 2.65A). For various reasons I refined the phaser and molrep 
> solutions separately, and both behaved in exactly the same 
> way giving R=0.24, Rfree=0.28 after adding ligands, mutations 
> etc. Today I decided to compare the two models (which I 
> figured were exactly the same) however found that the 
> asymmetric units did not superimpose. Normally this is just a 
> case of each mr program finding a different asymmetric unit 
> in the same cell, however this time there is a distinct clash 
> between the two solutions with about 50 atoms overlapping. 
> After playing around with various symmetry equivalents I have 
> found that the difference between the solutions is 
> approximately a 30A translation along the x axis. As there is 
> no signs of any twinning, and both solutions behave in 
> exactly the same way in refinement, is this a case of phaser 
> and molrep using different origins or is there something more 
> insidious going on?
>  
> Thanks for any advice,
>  
> Simon
>  
> 


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Re: [ccp4bb] highly soluble proteins

[Das, Debanu]

Hi Yvonne,
   Were you able to check the polydispersity of your highly soluble protein 
sample by DLS? Non-monodispersity could result in reduced propensity for 
crystallization. 
   Another thing you could then try is something like the "Optimum Solubility 
Screen" which basically involves buffer exchanges into different pH conditions 
to find a condition which is monodisperse. You can refer to the following paper 
for details:

Optimum solubility (OS) screening: an efficient method to optimize buffer 
conditions for homogeneity and crystallization of proteins.
Acta Crystallogr D Biol Crystallogr. 2004 Sep;60(Pt 9):1670-3. Epub 2004 Aug 2. 
Jancarik J, Pufan R, Hong C, Kim SH, Kim R.

More recently, similar approach has been also reported in:

Crystallization Optimum Solubility Screening: using crystallization results to 
identify the optimal buffer for protein crystal formation.
Acta Crystallograph Sect F Struct Biol Cryst Commun. 2005 Dec 1;61(Pt 
12):1035-8. Epub 2005 Nov 5. Collins B, Stevens RC, Page R.

Also, other than reductive methylation and surface mutagenesis, you can also 
consider protein truncation based on structure prediction progams and homology 
modeling.

Regards,
Debanu.

--
Debanu Das,
JCSG, SSRL.
-


-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Stephen Graham
Sent: Tuesday, August 07, 2007 11:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] highly soluble proteins

Reductive methylation of amino groups (lysines and the N-terminus) is a fairly 
routine chemical modification that should drop the solubility of your protein.  
An easy-to-use protocol can be found in Walter et al
(2007) "Lysine Methylation as a Routine Rescue Strategy for Protein 
Crystallization."  Structure, 14:1617-1622

Cheers,

Stephen

On 8/7/07, [EMAIL PROTECTED] <[EMAIL PROTECTED]> wrote:
> Yvonne,
>
> Several 'old' proteins have been crystallized from insanely high 
> concentraitons - concanavalin A for instance can be grown from 100-250 
> mg/ml solutions by means of 'salting in' using microdialysis. This is 
> of course highly labor-intensive and also expensive on the protein side.
>
> Look at the distribution of charged residues on the protein 
> (especially helpful if you have a model of some sort). I would 
> recommend mutating some of them to non-charged or even nonpolar 
> residues in order to lower solubility. Alternatively, you could try 
> preparing chemical derivatives of the protein using e.g. heavy atoms or 
> amine-modifying reagents like NHS.
> If you block enough amines, your solubility should go down and your pI 
> will change as well. Likewise you could try modifying exposed acidic 
> residues etc.
>
> Mutagenesis can in the end be easier to do because chemical 
> modification tends to produce complex mixtures of products, unless you 
> do it with a huge excess of the reagent and allow the reaction to 
> proceed to exhaustion.
>
> Artem
>
> > Our lab is trying to crystallize a highly soluble (100+ mg/ml) 
> > protein with a molecular weight of 35 kd.
> >
> > The protein was screened against 1536 conditions at 20 mg/mL. Most 
> > drops were either clear or produced "bubbles" (often oily looking). 
> > The few that had precipitate contained high concentrations of K3PO4, 
> > cobalt, or zinc. We have tried repeating some of the bubble 
> > conditions at 100+ mg/mL and are still getting clear drops or bubbles.
> >
> > Is there something about highly soluble proteins and/or secreted 
> > proteins and/or proteins with unusual portions of their sequence 
> > that needs to be considered in order to successfully crystallize it?
> >
> > I am considering trying "salting out" using dialysis, and also 
> > adding ligands/inhibitors. The protein is in 50 mM NaCl plus 50 mM buffer.
> >
> > I welcome thoughts and suggestions on crystallization ideas, 
> > publications, etc.
> >
> > Thank you
> >
> > Yvonne
> >
>


--
Dr Stephen Graham
Nuffield Medical Fellow
Division of Structural Biology
Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United 
Kingdom
Phone: +44 1865 287 549

Re: [ccp4bb] mr query

[Eleanor Dodson]

Can you combine the PHIC and FOM from each of your solutions, then do 
the Phase comparison in Clipper utilities checking for alternate 
origins? That gives a good indication of what is going on.


Do you  have more than one molecule in the asymm unit? You can finish up 
with A_phaser  matching one symm equiv of B_molrep and B_phaser matching 
a different symm equivalent of A_molrep..


Eleanor

Kolstoe S.E. wrote:

Dear ccp4bb,
 
In searching for a mr solution I ran both phaser and molrep, with both 
programs finding nice solutions in P21212 (cell 81.083  104.435  
109.732  90.00  90.00  90.00, resolution 2.65A). For various reasons I 
refined the phaser and molrep solutions separately, and both behaved 
in exactly the same way giving R=0.24, Rfree=0.28 after adding 
ligands, mutations etc. Today I decided to compare the two models 
(which I figured were exactly the same) however found that the 
asymmetric units did not superimpose. Normally this is just a case of 
each mr program finding a different asymmetric unit in the same cell, 
however this time there is a distinct clash between the two solutions 
with about 50 atoms overlapping. After playing around with various 
symmetry equivalents I have found that the difference between the 
solutions is approximately a 30A translation along the x axis. As 
there is no signs of any twinning, and both solutions behave in 
exactly the same way in refinement, is this a case of phaser and 
molrep using different origins or is there something more insidious 
going on?
 
Thanks for any advice,
 
Simon
 

[ccp4bb] refmac5 refinement with PEG400

[Craig McElroy]

Hi all,
   I am trying to refine a structure with a PEG400 molecule using 
refmac 5.3. I have created the necessary library file and read it into 
the refinement, but for some reason refmac still quites and complains 
that it has come across a new ligand. I am obviously doing something 
stupid, but could someone please tell me what it is. I have attached the 
refmac log file and the library file.

Thanks
Craig

--
Craig McElroy, Ph.D.
Department of Molecular and Cellular Biochemistry
Ohio State University
483 Hamilton Hall
1645 Neil Ave.
Columbus, OH 43210
(614) 688-8630

global_
_lib_name mon_lib
_lib_version  4.30
_lib_update   02/03/07
# 
#
# ---   LIST OF MONOMERS ---
#
data_comp_list
loop_
_chem_comp.id
_chem_comp.three_letter_code
_chem_comp.name
_chem_comp.group
_chem_comp.number_atoms_all
_chem_comp.number_atoms_nh
_chem_comp.desc_level
1PE  1PE '.   ' .  38  16 .
#
# --- DESCRIPTION OF MONOMERS ---
#
data_comp_1PE
#
loop_
_chem_comp_atom.comp_id
_chem_comp_atom.atom_id
_chem_comp_atom.type_symbol
_chem_comp_atom.type_energy
_chem_comp_atom.partial_charge
_chem_comp_atom.x
_chem_comp_atom.y
_chem_comp_atom.z
 1PE   OH7OOH1   0.000  7.502   80.947   62.566
 1PE   HOH7   HH 0.000  6.928   80.255   62.922
 1PE   C16CCH2   0.000  7.150   81.207   61.200
 1PE   H161   HH 0.000  6.086   81.445   61.142
 1PE   H162   HH 0.000  7.356   80.318   60.601
 1PE   C26CCH2   0.000  7.971   82.389   60.665
 1PE   H261   HH 0.000  7.727   83.290   61.232
 1PE   H262   HH 0.000  7.738   82.549   59.611
 1PE   OH6OO20.000  9.361   82.097   60.808
 1PE   C15CCH2   0.000 10.112   83.217   60.305
 1PE   H151   HH 0.000  9.856   84.115   60.871
 1PE   H152   HH 0.000  9.876   83.373   59.251
 1PE   C25CCH2   0.000 11.614   82.924   60.458
 1PE   H251   HH 0.000 12.133   83.816   60.815
 1PE   H252   HH 0.000 12.034   82.620   59.497
 1PE   OH5OO20.000 11.776   81.863   61.409
 1PE   C14CCH2   0.000 13.161   81.589   61.545
 1PE   H141   HH 0.000 13.685   82.479   61.900
 1PE   H142   HH 0.000 13.578   81.282   60.584
 1PE   C24CCH2   0.000 13.330   80.458   62.559
 1PE   H241   HH 0.000 12.599   80.587   63.360
 1PE   H242   HH 0.000 14.338   80.504   62.978
 1PE   OH4OO20.000 13.130   79.184   61.926
 1PE   C13CCH2   0.000 13.313   78.221   62.961
 1PE   H131   HH 0.000 12.580   78.420   63.745
 1PE   H132   HH 0.000 14.320   78.344   63.367
 1PE   C23CCH2   0.000 13.139   76.788   62.449
 1PE   H231   HH 0.000 13.418   76.714   61.396
 1PE   H232   HH 0.000 12.109   76.451   62.577
 1PE   OH3OO20.000 14.011   75.969   63.233
 1PE   C22CCH2   0.000 13.890   74.609   62.815
 1PE   H221   HH 0.000 14.162   74.514   61.762
 1PE   H222   HH 0.000 12.866   74.260   62.959
 1PE   C12CCH2   0.000 14.847   73.762   63.671
 1PE   H121   HH 0.000 14.816   74.121   64.702
 1PE   H122   HH 0.000 15.861   73.866   63.282
 1PE   OH2OOH1   0.000 14.460   72.395   63.632
 1PE   HOH2   HH 0.000 15.112   71.864   64.108
loop_
_chem_comp_tree.comp_id
_chem_comp_tree.atom_id
_chem_comp_tree.atom_back
_chem_comp_tree.atom_forward
_chem_comp_tree.connect_type
 1PE  OH7n/aC16START
 1PE  HOH7   OH7.  .
 1PE  C16OH7C26.
 1PE  H161   C16.  .
 1PE  H162   C16.  .
 1PE  C26C16OH6.
 1PE  H261   C26.  .
 1PE  H262   C26.  .
 1PE  OH6C26C15.
 1PE  C15OH6C25.
 1PE  H151   C15.  .
 1PE  H152   C15.  .
 1PE  C25C15OH5.
 1PE  H251   C25.  .
 1PE  H252   C25.  .
 1PE  OH5C25C14.
 1PE  C14OH5C24.
 1PE  H141   C14.  .
 1PE  H142   C14.  .
 1PE  C24C14OH4.
 1PE  H241   C24.  .
 1PE  H242   C24.  .
 1PE  OH4C24C13.
 1PE  C13OH4C23.
 1PE  H131   C13.  .
 1PE  H132   C13.  .
 1PE  C2

Re: [ccp4bb] refmac5 refinement with PEG400

[Kumar, Abhinav]

Trying using "Make check none" in the script file.

Thanks 
Abhinav 

Stanford Synchrotron Radiation Laboratory 
Joint Center for Structural Genomics 
Mail Stop 99 
Phone: (650) 926-2992 
Fax: (650) 926-3292 

 

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Craig McElroy
Sent: Wednesday, August 08, 2007 1:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] refmac5 refinement with PEG400

Hi all,
I am trying to refine a structure with a PEG400 molecule using 
refmac 5.3. I have created the necessary library file and read it into 
the refinement, but for some reason refmac still quites and complains 
that it has come across a new ligand. I am obviously doing something 
stupid, but could someone please tell me what it is. I have attached the 
refmac log file and the library file.
Thanks
Craig

-- 
Craig McElroy, Ph.D.
Department of Molecular and Cellular Biochemistry
Ohio State University
483 Hamilton Hall
1645 Neil Ave.
Columbus, OH 43210
(614) 688-8630

Re: [ccp4bb] refmac5 refinement with PEG400

[Craig McElroy]

Just a quick clarification... I'm already using "make check none" in the 
script file, and the PEG400 pdb from which I made the library file is 
from the HICCUP server and was called 1PE.

Thanks
Craig

Craig McElroy, Ph.D.
Department of Molecular and Cellular Biochemistry
Ohio State University
483 Hamilton Hall
1645 Neil Ave.
Columbus, OH 43210
(614) 688-8630



Kumar, Abhinav wrote:

Trying using "Make check none" in the script file.

Thanks 
Abhinav 

Stanford Synchrotron Radiation Laboratory 
Joint Center for Structural Genomics 
Mail Stop 99 
Phone: (650) 926-2992 
Fax: (650) 926-3292 

 


-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Craig McElroy
Sent: Wednesday, August 08, 2007 1:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] refmac5 refinement with PEG400

Hi all,
I am trying to refine a structure with a PEG400 molecule using 
refmac 5.3. I have created the necessary library file and read it into 
the refinement, but for some reason refmac still quites and complains 
that it has come across a new ligand. I am obviously doing something 
stupid, but could someone please tell me what it is. I have attached the 
refmac log file and the library file.

Thanks
Craig

  

Re: [ccp4bb] refmac5 refinement with PEG400

[Manish C Pathak]

Dear Craig,

It seems Refmac is not reading the user supplied dictionary. I expected a line
near Makecif parameters like this.

"User supplied dictionary entries: /home/pathak/junk.cif"

regards
Manish


Craig McElroy <[EMAIL PROTECTED]> wrote: Hi all,
I am trying to refine a structure with a PEG400 molecule using 
refmac 5.3. I have created the necessary library file and read it into 
the refinement, but for some reason refmac still quites and complains 
that it has come across a new ligand. I am obviously doing something 
stupid, but could someone please tell me what it is. I have attached the 
refmac log file and the library file.
Thanks
Craig

-- 
Craig McElroy, Ph.D.
Department of Molecular and Cellular Biochemistry
Ohio State University
483 Hamilton Hall
1645 Neil Ave.
Columbus, OH 43210
(614) 688-8630




   
-
Shape Yahoo! in your own image.  Join our Network Research Panel today!

Re: [ccp4bb] refmac5 refinement with PEG400

[Kumar, Abhinav]

Could the length of the path in the file names be an issue?
Your dir names are very long.

You also have this message in the log file:
==> WARNING:  HKLIN has not been defined

Maybe using file names without path may help when you have all the files in the 
working directory.

Thanks 
Abhinav 

Stanford Synchrotron Radiation Laboratory 
Joint Center for Structural Genomics 
Mail Stop 99 
Phone: (650) 926-2992 
Fax: (650) 926-3292 

 

-Original Message-
From: Craig McElroy [mailto:[EMAIL PROTECTED] 
Sent: Wednesday, August 08, 2007 1:27 PM
To: Kumar, Abhinav
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refmac5 refinement with PEG400

Just a quick clarification... I'm already using "make check none" in the 
script file, and the PEG400 pdb from which I made the library file is 
from the HICCUP server and was called 1PE.
Thanks
Craig

Craig McElroy, Ph.D.
Department of Molecular and Cellular Biochemistry
Ohio State University
483 Hamilton Hall
1645 Neil Ave.
Columbus, OH 43210
(614) 688-8630



Kumar, Abhinav wrote:
> Trying using "Make check none" in the script file.
>
> Thanks 
> Abhinav 
>
> Stanford Synchrotron Radiation Laboratory 
> Joint Center for Structural Genomics 
> Mail Stop 99 
> Phone: (650) 926-2992 
> Fax: (650) 926-3292 
>
>  
>
> -Original Message-
> From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Craig McElroy
> Sent: Wednesday, August 08, 2007 1:11 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] refmac5 refinement with PEG400
>
> Hi all,
> I am trying to refine a structure with a PEG400 molecule using 
> refmac 5.3. I have created the necessary library file and read it into 
> the refinement, but for some reason refmac still quites and complains 
> that it has come across a new ligand. I am obviously doing something 
> stupid, but could someone please tell me what it is. I have attached the 
> refmac log file and the library file.
> Thanks
> Craig
>
>   

Re: [ccp4bb] refmac5 refinement with PEG400

[Craig McElroy]

Indeed, the library file was not being read due to the long names. 
Thanks all who responded.

Craig

Craig McElroy, Ph.D.
Department of Molecular and Cellular Biochemistry
Ohio State University
483 Hamilton Hall
1645 Neil Ave.
Columbus, OH 43210
(614) 688-8630



Kumar, Abhinav wrote:

Could the length of the path in the file names be an issue?
Your dir names are very long.

You also have this message in the log file:
==> WARNING:  HKLIN has not been defined

Maybe using file names without path may help when you have all the files in the 
working directory.

Thanks 
Abhinav 

Stanford Synchrotron Radiation Laboratory 
Joint Center for Structural Genomics 
Mail Stop 99 
Phone: (650) 926-2992 
Fax: (650) 926-3292 

 


-Original Message-
From: Craig McElroy [mailto:[EMAIL PROTECTED] 
Sent: Wednesday, August 08, 2007 1:27 PM

To: Kumar, Abhinav
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refmac5 refinement with PEG400

Just a quick clarification... I'm already using "make check none" in the 
script file, and the PEG400 pdb from which I made the library file is 
from the HICCUP server and was called 1PE.

Thanks
Craig

Craig McElroy, Ph.D.
Department of Molecular and Cellular Biochemistry
Ohio State University
483 Hamilton Hall
1645 Neil Ave.
Columbus, OH 43210
(614) 688-8630



Kumar, Abhinav wrote:
  

Trying using "Make check none" in the script file.

Thanks 
Abhinav 

Stanford Synchrotron Radiation Laboratory 
Joint Center for Structural Genomics 
Mail Stop 99 
Phone: (650) 926-2992 
Fax: (650) 926-3292 

 


-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Craig McElroy
Sent: Wednesday, August 08, 2007 1:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] refmac5 refinement with PEG400

Hi all,
I am trying to refine a structure with a PEG400 molecule using 
refmac 5.3. I have created the necessary library file and read it into 
the refinement, but for some reason refmac still quites and complains 
that it has come across a new ligand. I am obviously doing something 
stupid, but could someone please tell me what it is. I have attached the 
refmac log file and the library file.

Thanks
Craig

  



  

[ccp4bb] SUMMARY: highly soluble proteins

[Yvonne Leduc]

Thank you to all who responded. I find it amazing how generously helpful 
the crystallography community is.

Lots of good ideas.

Yvonne
_

-Original Message-
Sent: Tuesday, August 07, 2007 12:17 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] highly soluble proteins

Our lab is trying to crystallize a highly soluble (100+ mg/ml) protein
with a molecular weight of 35 kd.

The protein was screened against 1536 conditions at 20 mg/mL. Most drops
were either clear or produced "bubbles" (often oily looking). The few
that had precipitate contained high concentrations of K3PO4, cobalt, or
zinc. We have tried repeating some of the bubble conditions at 100+
mg/mL and are still getting clear drops or bubbles.

Is there something about highly soluble proteins and/or secreted
proteins and/or proteins with unusual portions of their sequence that
needs to be considered in order to successfully crystallize it?

I am considering trying "salting out" using dialysis, and also adding
ligands/inhibitors. The protein is in 50 mM NaCl plus 50 mM buffer.

I welcome thoughts and suggestions on crystallization ideas,
publications, etc.

Thank you

Yvonne



Try adjusting the pH close to the pI to reduce solubility.
__

For what it is worth: I've worked with a few highly soluble
proteins(150-200+ mg/mL) I'd start by raising your protein
concentration to at least half of the solubility limit. I hope your
protein is also highly expressing, since the high protein concentrations
will eat through supplies rather quickly! You can also get some
additional advantage by altering the precipitant to protein ratio: so,
rather than setting up 1:1 drops, set them up as 1:3 with the 3X being
precipitant. Finally, a desperation tactic can be to spike the wells of
your drops with highly concentrated salt: this will act as a desiccant
and draw water out of your protein drops -- effectively serving to
concentrate it. I would do this after your screens have equilibrated
for a few days--week(s) if you are not seeing any precipitation
occurring.
_

Effects of Zn and Co may be non-specific, aggregative kind of binding and
ppt formation, and may not be very helpful. Phase separation (bubbles) 
seems

normal at such high []'s

I would try (a) making SeMet protein since most Se-labelled proteins are
less soluble than their native conterparts, but this depends on Met content
and (2) reductive methylation of Lys, as this should also decrease the 
polar

character and therefore solubility. Again, depends on your Lys content.
__

I would suggest the technique of surface entropy reduction. In this 
server you can find all the references and informations about this 
methodology: http://nihserver.mbi.ucla.edu/SER/intro.php



Something you might want to try is reductive methylation of lysine 
residues.

This procedure reduces the solubility of proteins,
by typically 30-60% and - in many cases -
people have managed to crystallize proteins
which were otherwise intractable.

Reference:
Walter et al. (2006) "Lysine methylation as a routine rescue strategy 
for protein crystallization"
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17098187 


In this study we took 10 proteins which
did not crystallize and applied reductive methylation:
In the end, three of them gave high-resolution structures.
_

Hi,

How homogeneous is your sample?

Are your proteins glycosylated?
If so, you may try to deglycosylate them (PNGase F, EndoH, etc...).

You can also try to use detergents (for example beta-OG as an initial 
additive to the drop).


Try changing the pH.

In which buffer is your protein sample?
You can try to change it to for example to something like 10 mM Tris-HCl 
pH 7.5, and then repeat screening with and without detergents.


Try different temperatures (4, 12, 20, 30 °C).

___

We had a similar situation. This might not be applicable to you, but
cutting off our his tag reduced the protein's solubility, and we then
got some excellent crystals.


Reductive methylation of amino groups (lysines and the N-terminus) is
a fairly routine chemical modification that should drop the solubility
of your protein. An easy-to-use protocol can be found in Walter et al
(2007) "Lysine Methylation as a Routine Rescue Strategy for Protein
Crystallization." Structure, 14:1617-1622

___

May I ask if you have tried using The Solubility Tool Kit? I have 
attached a

PDF of the datasheet that accompanies the kit.

Protein exists in solution as a salt and different protein salts have
different sol

[ccp4bb] More than 70 TLS groups

[Craig Morton]

I'm working on a system where I need to use more than the default limit 
of 70 TLS groups. A quick scan of the refmac5 code indicates that the 
variable I need to change before recompiling is in tls.fh and called 
MAXTLSGRP.


Before I go through the recompile and test, only to find there's another 
variable out there that I have to change as well, has someone else used 
more than 70 TLS groups? If so, was there anything else that had to be 
changed in the source code to get refmac (or other subsequent steps like 
tlsanl) to work?


Cheers,

Craig.

--
Dr Craig Morton Ph: +61 3 9288 2480
Senior AssociateFx: +61 3 9416 2676
St Vincent's Institute  www.svi.edu.au

Re: [ccp4bb] PDB format survey?

[Ralf W. Grosse-Kunstleve]

Hi George,

> It seems to me that column 21 of an ATOM or HETATM instruction
> is always blank, and column 22 is the chain ID. So if we put a
> two-character chain ID right justified in columns 21 and 22, for the
> vast majority of structures there would be no change, and it would
> be relatively easy to change MMDB, Coot etc.  to accomodate the
> increase in the possible number of chains from 26 to (say) 36^2 =
> 1296 (if digits are allowed too).

I really like this idea and I will make phenix/cctbx work this way.

> The next problem is of course the 5-digit atom serial number in columns
> 7 to 11, which limits the total number of atoms in the structure to a
> paltry 9. Many programs ignore this number, but it is used by the
> CONECT, SSBOND and CISPEP records in the PDB. However column 12 also
> appears to be blank, I think that using it (e.g. with A for atoms
> 10 to 19, B for atoms 20 to 29 etc.) would enable the
> sequence numbers to be recycled and would again require no change for
> the vast majority of PDB files. I personally think that this is a much
> better solution than what the PDB currently does for more than 9
> atoms (they spread the structure over several PDB files with different
> PDB-IDs!).

The solution to this problem is to simply treat the serial numbers and
residue numbers as strings. X-PLOR/CNS has been doing this forever,
maybe other programs, too.
Implementations to generate intuitive, maximally backward compatible
numbers can be found here:

  http://cci.lbl.gov/hybrid_36/

This includes a Fortran-77 implementation without any external
dependencies, heavily tested with a large variety of compilers.

I think these simple tricks will be sufficient until we are all
retired!

Cheers,
Ralf




   

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Yahoo! Answers - Check it out.
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Re: [ccp4bb] PDB format survey?

[Ethan A Merritt]

On Wednesday 08 August 2007 20:47, Ralf W. Grosse-Kunstleve wrote:
> 
> The solution to this problem is to simply treat the serial numbers and
> residue numbers as strings. X-PLOR/CNS has been doing this forever,
> maybe other programs, too.
> Implementations to generate intuitive, maximally backward compatible
> numbers can be found here:
> 
>   http://cci.lbl.gov/hybrid_36/

From that URL:

ATOM  8  SD  MET L  48.231 -64.383  -9.257  1.00 11.54   S
ATOM  9  CE  MET L  49.398 -63.242 -10.211  1.00 14.60   C
ATOM  A  N   VAL LA000  52.228 -67.689 -12.196  1.00  8.76   N
ATOM  A0001  CA  VAL LA000  53.657 -67.774 -12.458  1.00  3.40   C

Could you please clarify this example? 

Is that "A" a hexidecimal number, or is it a decimal number
that just happens to have an "A" in front of it?
[A-Z][0-] gives a larger range of values than 5 bytes of hexadecimal,
so I'm guessing it's the former.  But the example is not clear.

(yes I could download and inspect the source, but I'm lazy tonight)

-- 
Ethan A Merritt
Biomolecular Structure Center
University of Washington, Seattle 98195-7742

Re: [ccp4bb] PDB format survey?

[Edward A. Berry]

Ethan A Merritt wrote:

On Wednesday 08 August 2007 20:47, Ralf W. Grosse-Kunstleve wrote:

Implementations to generate intuitive, maximally backward compatible
numbers can be found here:

  http://cci.lbl.gov/hybrid_36/


From that URL:

ATOM  8  SD  MET L  48.231 -64.383  -9.257  1.00 11.54   S
ATOM  9  CE  MET L  49.398 -63.242 -10.211  1.00 14.60   C
ATOM  A  N   VAL LA000  52.228 -67.689 -12.196  1.00  8.76   N
ATOM  A0001  CA  VAL LA000  53.657 -67.774 -12.458  1.00  3.40   C

Could you please clarify this example? 


Is that "A" a hexidecimal number, or is it a decimal number
that just happens to have an "A" in front of it?
[A-Z][0-] gives a larger range of values than 5 bytes of hexadecimal,
so I'm guessing it's the former.  But the example is not clear.


I'm guessing the former also. A 5-digit hex number would not be
backwards compatible. With this system legacy programs can still
read the files with 9 atoms or less, and anything more than
that they couldn't have handled anyway. Very nice!

Ed