Re: [ccp4bb] Phenix refine has a problem with the added H, plz help

[Pavel Afonine]

Dear Mohd,

FYI:
there is specific mailing list for PHENIX specific questions (
http://phenix-online.org/).

Pavel.

On Tue, Apr 26, 2011 at 10:56 AM, Salameh, Mohd A., Ph.D. <
salameh.m...@mayo.edu> wrote:

>  Dear All,
>
> I normally refine my structure without the hydrogens until the final stages
> . But today I’m facing a real trouble with phenix.refine!! It seems
> phenix.refine does not recognize the hydrogen atoms and writes out this
> message: “Fatal problems interpreting PDB file: number of atoms with
> unknown nonbonded energy type symbols: 1420 … “. After comparing this PDB
> and previous PDB files with added H I can see some format changes but I
> doubt it to be the problem!
>
> I wonder if anyone had encountered similar problem and have any suggestion
> that can help overcoming this annoying trouble.
>
> Many Thanks
>
> Mohd
>
>

Re: [ccp4bb] twinned refinement

[Pavel Afonine]

Hi Karolina,

I have also tried phenix (ver.1.7-650) with and without TLS, but only in
> the first macrocycle both R and Rfree go down; in the subsequent cycles
> Rfree increases significantly.
>
> I will appreciate your comments on that.


this shouldn't happen or must have a reasonable explanation. In any case, if
you send me the inputs (data and model files) (please send to my email and
NOT to the whole mailing list) I will do my best to provide the solution as
soon as I have the files.

Pavel.

[ccp4bb] twinned refinement

[Karolina Michalska]

Hello everyone,

I'm having some troubles refining against twinned data. The space group
is I4, twin fraction 35%, 1.5 A resolution. I have selected Rfree set in
Phenix using lattice symmetry, so I believe twin operator was taken into
account.  Twinned refinement in Refmac without TLS gives R/Rfree
0.148/0.173, which seems OK, but I wanted to try TLS refinement and then
the program crashed with something like:

 ***TLS refinement cycle***5

   1
  6.3690789E-02  5.8194529E-02 -0.1404448 -1.0440781E-02
-4.1422732E-03
  1.5739001E-02
  9.1476954E-04 -5.5926095E-04  4.0188796E-04  1.7031586E-04 
2.0264904E-04
 -1.7866226E-04
  7.5498177E-04 -1.2186267E-03  1.7816454E-04  1.3809106E-03 
7.6890434E-03
  1.4953420E-03 -2.4529384E-03 -5.5452576E-04
 -0.1526335
 Problem
 xyz 2420   7.378197  -3.33  0.5405517
-2.3370266E-02
  0.3058620  0.3236508 -2.1583334E-02 -2.1171659E-02
-2.8666053E-03
  2.4862900E-02 -2.3370266E-02  0.2921907  0.3391090
-4.1098855E-03
 -0.8417150 -0.5399062
***
This is from Refmac_5.5.0109 but I have also tried Refmac_5.5.0110 and
different number of TLS groups always with the same result.

I have also tried phenix (ver.1.7-650) with and without TLS, but only in
the first macrocycle both R and Rfree go down; in the subsequent cycles
Rfree increases significantly.

I will appreciate your comments on that.

Karolina

[ccp4bb] Phenix refine has a problem with the added H, plz help

[Salameh, Mohd A., Ph.D.]

Dear All,
I normally refine my structure without the hydrogens until the final
stages. But today I'm facing a real trouble with phenix.refine!! It
seems phenix.refine does not recognize the hydrogen atoms and writes out
this message: "Fatal problems interpreting PDB file: number of atoms
with unknown nonbonded energy type symbols: 1420 ... ". After comparing
this PDB and previous PDB files with added H I can see some format
changes but I doubt it to be the problem!
I wonder if anyone had encountered similar problem and have any
suggestion that can help overcoming this annoying trouble.
Many Thanks
Mohd  

[ccp4bb] In XDS, anomalous correction column has negative values

[Narayanan Ramasubbu]

Dear All:
What does it mean when I get negative values under Anomalous Corr column 
after running XDS? I set the Friedel Law=False even though I suspect 
that my signal is very very weak.

Thanks
Subbu

[ccp4bb] Workshop "Diffraction Data Collection Using Synchrotron Radiation" - Deadline approaching

[Manfred S. Weiss]

Please note that there is only a few more days left to register for the 
workshop

announced below.

Manfred



After the successful first two workshops on "Diffraction Data Collection
Using Synchrotron Radiation", which took place in 2007 and 2009, a third
edition of the workshop will be held from July 07-09, 2011 at the Helmholtz
Zentrum Berlin fuer Materialien und Energie (HZB) in Berlin.

The workshop is sponsored by the German Society for Crystallography
(DGK) and organised by the DGK Working Group 1 (Biological Structures)
in cooperation with Dr. Manfred Weiss and Dr. Uwe Mueller.

The workshop comprises a series of basic lectures on the topic and two
extended practical sessions. It is aimed at PhD students in Biological
Crystallography with little or no experience in diffraction data collection
at a synchrotron. The practical sessions will take place at the MX
beamlines
located at the electron storage ring BESSY II.

The workshop fee is 50 EUR for DGK-members and 60 EUR for non-members.
This fee covers all conference material as well as board and lodging for
two
nights on the campus in Berlin-Adlershof.

For more information please visit the web page
http://www.helmholtz-berlin.de/bessy-mx-workshop/

Registration is now open. Since the number of students will have to be
limited
to 20 in order to ensure that the practical sessions run efficiently, we
expect that
the workshop will fill up quickly. Participants are also invited to
present a poster.
As last time, the best poster will receive an attractive prize.
##

Manfred Weiss&  Uwe Mueller


--
Dr. Manfred. S. Weiss
Helmholtz-Zentrum Berlin für Materialien und Energie
Macromolecular Crystallography (HZB-MX)
Albert-Einstein-Str. 15
D-12489 Berlin
GERMANY
Fon:   +49-30-806213149
Fax:   +49-30-806214975
Web:   http://www.helmholtz-berlin.de/bessy-mx
Email: mswe...@helmholtz-berlin.de

Helmholtz-Zentrum Berlin für Materialien und Energie GmbH
Hahn-Meitner-Platz 1, 14109 Berlin
Vorsitzender des Aufsichtsrats: Prof. Dr. Dr. h.c. mult. Joachim Treusch
Stellvertretende Vorsitzende: Dr. Beatrix Vierkorn-Rudolph
Geschäftsführer: Prof. Dr. Anke Rita Kaysser-Pyzalla, Prof. Dr. Dr. h.c.
Wolfgang Eberhardt, Dr. Ulrich Breuer
Sitz der Gesellschaft: Berlin
Handelsregister: AG Charlottenburg, 89 HRB 5583

Re: [ccp4bb] Mock Dataset

[Andrew T. Torelli]

Dear Peter,

Perhaps your PI is referring to the collection of datasets and 
tutorials being compiled at the following link.  Raw data (diffraction images) 
are available for various datasets exhibiting different challenges and 
tutorials, put together by the authors of various data processing and structure 
phasing/refinement programs, will provide expert advice on how to tackle these 
"tough, but workable" examples.

Some of the author/speakers involved are still working very 
hard to get their tutorials ready so stay tuned!  You can access all the raw 
data now, and there are links to all the software/documentation.  Please take a 
look at the acknowledgements to recognize the considerable effort put into this 
venture by the author/speakers, corporate sponsors and contributors of the raw 
data.

The website will accompany the "Data Processing With The Pros"  
and "Phasing And Refinement For Dummies: No Book Required" Sessions at the 
upcoming ACA meeting in New Orleans, but the website will be made available as 
a permanent learning resource.  These sessions are geared especially for novice 
to intermediate-advanced crystallographers, but should appeal to anyone.

http://bl831.als.lbl.gov/example_data_sets/ACA2011/index.html

-Andy Torelli

P.S.  It is of course possible that my bias and excitement led me to guess 
incorrectly about the datasets to which your PI is referring ;)

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Peter 
Randolph
Sent: Tuesday, April 26, 2011 8:48 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Mock Dataset

My PI has mentioned a Mock Dataset made available by the ACA for various 
programs (HKL etc) to practice with, but I haven't been able to find it and was 
wondering if anyone knew of any,
Thanks,
Peter Randolph

Re: [ccp4bb] Mock Dataset

[Ed Pozharski]

On Tue, 2011-04-26 at 08:48 -0400, Peter Randolph wrote:
> My PI has mentioned a Mock Dataset made available by the ACA for
> various programs (HKL etc) to practice with, but I haven't been able
> to find it and was wondering if anyone knew of any, 
> Thanks, 
> Peter Randolph

If you are looking for some images to practice with, you can find plenty
here:

http://tardis.edu.au/

-- 
"Hurry up before we all come back to our senses!"
   Julian, King of Lemurs

[ccp4bb] Mock Dataset

[Peter Randolph]

My PI has mentioned a Mock Dataset made available by the ACA for various
programs (HKL etc) to practice with, but I haven't been able to find it and
was wondering if anyone knew of any,
Thanks,
Peter Randolph

Re: [ccp4bb] problem with pdb & its symmetry molecule

[Ramanuj Banerjee]

Dear All,
   Thank you for your kind reply. After reading the suggestions, I 
too think that this is the solution. Thank you once again.


  


  Ramanuj Banerjee

Re: [ccp4bb] problem with pdb & its symmetry molecule

[Boaz Shaanan]

This intertwined conformation is very possible and is reminiscent of Trp 
repressor (at least in the view you provided).

 Boaz

Boaz Shaanan, Ph.D.

Dept. of Life Sciences

Ben-Gurion University of the Negev

Beer-Sheva 84105

Israel

Phone: 972-8-647-2220 ; Fax: 646-1710

Skype: boaz.shaanan


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ramanuj 
Banerjee
Sent: Tuesday, April 26, 2011 10:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] problem with pdb & its symmetry molecule

Dear All, I have solved a protein structure (experimentally phased) with 1 
molecule in the asymmetric unit at 2.22 A (high resolution). The present R 
factor is .22 and R free .27 with Ramachandran favoured >98% and R and R free 
are decreasing with refinement.The problem is: when the pdb is opened in pymol 
and symmetry mates generated, the upper part of the molecule shows to be 
intertwined with the symmetry molecule (attached .jpg), but there are no 
clashes in between the two.The electron density is so very fine that no 
alternative choices of chain flow are available.All the processes starting from 
phasing and refinement have been done in Phenix.Is such a thing possible ?

Re: [ccp4bb] problem with pdb & its symmetry molecule

[Michael Thompson]

Ramanuj,

This is definitely possible. Cases of "intertwined homodimers" are rare, but 
there are several known structures that demonstrate this phenomenon, and they 
are very interesting (especially with respect to studying knotted proteins - 
see reference below). Examples are pdb IDs: 2ouf, 1myk, 2rh3.

A cool paper regarding intertwined homodimers and knotted proteins:

Proc Natl Acad Sci U S A. 2010 Nov 30;107(48):20732-7. Epub 2010 Nov 10.
Structure and folding of a designed knotted protein.
King NP, Jacobitz AW, Sawaya MR, Goldschmidt L, Yeates TO.

Your structure is very interesting.

Mike


- Original Message -
From: "Ramanuj Banerjee" 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, April 26, 2011 12:37:29 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] problem with pdb & its symmetry molecule


Dear All, I have solved a protein structure (experimentally phased) with 1 
molecule in the asymmetric unit at 2.22 A (high resolution). The present R 
factor is .22 and R free .27 with Ramachandran favoured >98% and R and R free 
are decreasing with refinement.The problem is: when the pdb is opened in pymol 
and symmetry mates generated, the upper part of the molecule shows to be 
intertwined with the symmetry molecule (attached .jpg), but there are no 
clashes in between the two.The electron density is so very fine that no 
alternative choices of chain flow are available.All the processes starting from 
phasing and refinement have been done in Phenix.Is such a thing possible ? 

[image/jpeg:a copy.jpg]

-- 
Michael C. Thompson

Graduate Student

Biochemistry & Molecular Biology Division

Department of Chemistry & Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu

Re: [ccp4bb] problem with pdb & its symmetry molecule

[Michael Thompson]

Ramanuj,

This is definitely possible. Cases of "intertwined homodimers" are rare, but 
there are several known structures that demonstrate this phenomenon, and they 
are very interesting (especially with respect to studying knotted proteins - 
see reference below). Examples are pdb IDs: 2ouf, 1myk, 2rh3.

A cool paper regarding intertwined homodimers and knotted proteins:

Proc Natl Acad Sci U S A. 2010 Nov 30;107(48):20732-7. Epub 2010 Nov 10.
Structure and folding of a designed knotted protein.
King NP, Jacobitz AW, Sawaya MR, Goldschmidt L, Yeates TO.

Your structure is very interesting.

Mike



- Original Message -
From: "Ramanuj Banerjee" 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, April 26, 2011 12:37:29 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] problem with pdb & its symmetry molecule


Dear All, I have solved a protein structure (experimentally phased) with 1 
molecule in the asymmetric unit at 2.22 A (high resolution). The present R 
factor is .22 and R free .27 with Ramachandran favoured >98% and R and R free 
are decreasing with refinement.The problem is: when the pdb is opened in pymol 
and symmetry mates generated, the upper part of the molecule shows to be 
intertwined with the symmetry molecule (attached .jpg), but there are no 
clashes in between the two.The electron density is so very fine that no 
alternative choices of chain flow are available.All the processes starting from 
phasing and refinement have been done in Phenix.Is such a thing possible ? 

[image/jpeg:a copy.jpg]

-- 
Michael C. Thompson

Graduate Student

Biochemistry & Molecular Biology Division

Department of Chemistry & Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu

Re: [ccp4bb] Using chaperones to boost expression in E. coli

[Olga Kolaj]

Michael,

We have put together a review about use of folding modulators to improve
expression in E. coli some time ago which might be useful to you (Kolaj et
al Microb Cell Fact. 2009 Jan 27;8:9). Here is the link:
http://www.ncbi.nlm.nih.gov/pubmed/19173718

Regards

On Fri, Apr 22, 2011 at 4:28 PM, Kothe, Michael
wrote:

>  Dear ccp4bb,
>
>
>
> I am curious to hear of examples where the expression of well-behaved
> protein was achieved by the coexpression of chaperones in E. coli. I know
> the appropriate strains and vectors exist, but I can’t remember hearing of a
> successful case. I have heard anecdotally of several cases where it was
> tried without success (including one attempt I made myself). I also heard of
> concerns that the chaperones might copurify with the (now soluble) protein
> of interest and are difficult to remove.
>
>
>
> Thanks,
>
> Michael
>
>
>

Re: [ccp4bb] problem with pdb & its symmetry molecule

[Anastassis Perrakis]

Yes, its surely possible. If there are no clashes start thinking if this dimer 
is the physiological molecule in solution. 

Check probability that this dimer is stable in solution in Pisa - ebiFold

If so, check first with size exclusion columns if it behaves like dimer in 
solution, preferably use a MALLS setup if available. Ssaxs can confirm it's 
shape. AUC is handy for kD. 

And if its a dimer then of course mutate the interface and assay ... Have fun!

Tassos


Sent from my iPhone

On 26 Apr 2011, at 09:37, Ramanuj Banerjee  wrote:

> Dear All, I have solved a protein structure (experimentally phased) with 1 
> molecule in the asymmetric unit at 2.22 A (high resolution). The present R 
> factor is .22 and R free .27 with Ramachandran favoured >98% and R and R free 
> are decreasing with refinement.The problem is: when the pdb is opened in 
> pymol and symmetry mates generated, the upper part of the molecule shows to 
> be intertwined with the symmetry molecule (attached .jpg), but there are no 
> clashes in between the two.The electron density is so very fine that no 
> alternative choices of chain flow are available.All the processes starting 
> from phasing and refinement have been done in Phenix.Is such a thing possible 
> ?
> 

Re: [ccp4bb] problem with pdb & its symmetry molecule

[Tim Gruene]

Dear Ramanuj Banerjee,

from the attached picture it is difficult to make out N- and C-terminus of your
protein and it looks like two closed molecules intertwined- which would indeed
be very surprising.

However, if you think there is space for the molecules to form such a homodimer
(either in solution or upon formation of the crystal), why should it not be
possible? It seems like a beautiful conformation.

Cheers, Tim


On Tue, Apr 26, 2011 at 08:37:29AM +0100, Ramanuj Banerjee wrote:
> Dear All,
> I have solved a protein structure (experimentally phased) with 1 
> molecule in
> the asymmetric unit at 2.22 A (high resolution). The present R factor is .22
> and R free .27 with Ramachandran favoured >98% and R and R free are
> decreasing with refinement.The problem is: when the pdb is opened in pymol
> and symmetry mates generated, the upper part of the molecule shows to be
> intertwined with the symmetry molecule (attached .jpg), but there are no
> clashes in between the two.The electron density is so very fine that no
> alternative choices of chain flow are available.All the processes starting
> from phasing and refinement have been done in Phenix.Is such a thing
> possible ? 
> 




-- 
--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

phone: +49 (0)551 39 22149

GPG Key ID = A46BEE1A



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