Re: [ccp4bb] XSCALE!!! ERROR

[Nicolas Foos]

Hello Bashir,

Maybe it's because your dataset present too much weak diffraction spots. 
You can try to change the setting for the strong pixel detection. You 
can Try to decrease a little bit de I/sigma. Have you define the 
resolution range?


I read on your message that you resolution start at 5 A. To my mind it's 
too high, you should have more low resolution. Perhaps your problem com 
from this specificity of you dataset.


Try first  to set your resolution range from inf. to 3.25

Hope to help you.

Nicolas

Le 07/09/11 21:39, Muhammed bashir Khan a écrit :

Dear All;

I am trying to to xsale several data sets together, but it gives an error of

!!! ERROR !!! INSUFFICIENT NUMBER OF COMMON STRONG REFLECTIONS.
PROGRAM IS UNABLE TO PRODUCE A SCALED DATA SET.

resolution of data sets is ranged from 3.25 to 5A

Any suggestion will be highly encouraged.

Thanks in adv.

Bashir

Re: [ccp4bb] XSCALE!!! ERROR

[Christian Roth]

Am Mittwoch 07 September 2011 21:39:07 schrieb Muhammed bashir Khan:
> Dear All;
> 
> I am trying to to xsale several data sets together, but it gives an error
>  of
> 
> !!! ERROR !!! INSUFFICIENT NUMBER OF COMMON STRONG REFLECTIONS.
>PROGRAM IS UNABLE TO PRODUCE A SCALED DATA SET.
> 
> resolution of data sets is ranged from 3.25 to 5A
> 
> Any suggestion will be highly encouraged.
> 
> Thanks in adv.
> 
> Bashir
> 

Hi Bashir,

we had already such an error twice. In one case one of the integration failed 
and the XDS_ASCII.HKL was rubbish. there was the wrong space group and so no 
common reflections. 
In an other case many overloaded reflection led to  too few reflections which 
could be scaled together, because more than 50 % of the reflections were 
rejected in the CORRECT step.  In this case we had to recollect the data.

Christian

Re: [ccp4bb] XSCALE!!! ERROR

[Kay Diederichs]

>
>
>  Original Message 
> Subject: XSCALE!!! ERROR
> Date: Wed, 7 Sep 2011 21:39:07 +0200
> From: Muhammed bashir Khan 
>
> Dear All;
>
> I am trying to to xsale several data sets together, but it gives an
> error of
>
> !!! ERROR !!! INSUFFICIENT NUMBER OF COMMON STRONG REFLECTIONS.
> PROGRAM IS UNABLE TO PRODUCE A SCALED DATA SET.
>
> resolution of data sets is ranged from 3.25 to 5A
>
> Any suggestion will be highly encouraged.
>
> Thanks in adv.
>
> Bashir
>

Bashir,

the problem is what the error message tries to convey: scaling 
_requires_ common reflections, and there are two or more partitions of 
your "several data sets" which do not overlap in this sense. xscale 
prints out the number of common reflections ... inspect the list in 
XSCALE.LP !


Two ways to proceed:
- find out whether there are datasets that have no common reflections 
with any of the others. Remove those, and scale the others.

- add more datasets

HTH,

Kay
--
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email: kay.diederi...@uni-konstanz.deTel +49 7531 88 4049 Fax 3183
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Re: [ccp4bb] How to help crystal grow bigger

[SnowDeer]

To Boaz: I seperated the large crystals and checked its diffraction already.
While the diffraction is quite poor, only several dots could be seen. T_T
I washed the seeds twice with my buffer and seed the drop immediately after
setting it. Thanks for your advices and I will try the additives.

To Charles, Enrico, Bernie & Tiantian: Thanks for your kindly advices, I set
different conditions for the buffers with glycerol and different
protein/reservoir volume ratios already following your instructions. :)

To David: Hmm...my crystals are smaller than the smallest loop T_T. It's
quite hard for me to pick them up (due to my clumsy fingers...lol). Thanks
for your advices and the review.

I have another question: I usually stored my protein samples aliquots at -80
degree and thaw the small aliquots when I need to use. While my senior said
it will harm the protein so she suggested to keep them at 4 degree. So it is
possible that I got the small crystals coz the freezing and thawing alter
the proteins?

Thanks very much.
SnowDeer

On Tue, Sep 6, 2011 at 9:39 PM, David Waterman  wrote:

> Dear SnowDeer,
>
> Just how small is too small? If you have access to a microfocus beamline
> you might find you can collect decent diffraction data from crystals with
> dimensions in the single digit microns. Fishing tiny crystals is difficult,
> but something like the MicroMesh "tennis racquet" mounts can help. Having
> multiple crystals on the same pin is in fact a rather helpful way of
> screening lots of samples. Please don't discount your crystals _only_
> because they are small!
>
> Shameless plug: there is a review on microcrystallography here that you
> might find interesting.
> http://www.tandfonline.com/doi/abs/10.1080/0889311X.2010.527964
>
> Best wishes
>
> -- David
>
>
>
> On 5 September 2011 09:06, SnowDeer  wrote:
>
>> Dear All:
>>
>> Recently I am working on a protein which can already grow nice
>> pyramid-like crystals after the condition was optimized, while the crystals
>> are too small to be picked up. The crystals grew quite fast and densely, so
>> I tried to put 100ul paraffin oil inside the 600ul reservoir solution or put
>> the plate under 16 degree to slow down the evaporation, while the crystals
>> were still the same. I also tried macro or micro seeding with or without the
>> paraffin oil. Macroseeding would give a larger crystal (not very nice) with
>> many small crystals in the drop even I washed the seeds carefully. For
>> microseeding, the same small crystals grew.
>>
>> I don't have many experiences in crystallography, so I have no idea how to
>> make it grow bigger...
>>
>> Any suggestion is most welcome.
>>
>> Thanks.
>> SnowDeer
>>
>
>

Re: [ccp4bb] How to help crystal grow bigger

[Enrico Stura]

SnowDeer,

Additives are indeed a good idea, make sure you do it in an informed  
manner, each additive is different and you will
get a benefit only if you know how to do it right. I do not recommend a  
random approach.


For example, you mention glycerol. There is a lot to know on the subject  
and probably more to discover.
Glycerol will help freezing/thawing cycles and improve protein stability  
if you want to work at higher temperatures than
4°C. You should check increased stability effect out since it is easier to  
work outside a cold room.
I assume you are trying to set up your drops at the same temperature at  
which they will equilibrate. Which is a good thing to do.


Yet, plan your experiments carefully. Also look at previous messages on  
ccp4bb on the subject of glycerol
in particular regarding propanediol. Annie Hassell among others has posted  
some very good comments. This bullettin board
has been very keen on the subject in the past, so you can learn a lot from  
the archives.


Glycerol is also great to reduce nucleation. If you decide to add glycerol  
to the protein solution (for solubility, but in your case it might be for  
stability
reasons), you also need to have a higher (double) glycerol concentration  
in the reservoir else you will risk finding that your drops will get  
biggger and
not smaller. This note of caution applies to vapour diffusion set ups as  
equilibration can be tricky in such context:
Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011)  
Practical Use of Glycerol in Protein Crystallization. Cryst. Growth & Des.  
11 :2755–2762.

http://pubs.acs.org/doi/abs/10.1021/cg101364m

Good luck, but most important work with precision. To go from small to big  
crystals you need to be very
precise on how you set up your experiments and understand the rate at  
which you equilibrate your drops.



Enrico.

On Thu, 08 Sep 2011 09:52:40 +0200, SnowDeer  wrote:

To Boaz: I seperated the large crystals and checked its diffraction  
already.

While the diffraction is quite poor, only several dots could be seen. T_T
I washed the seeds twice with my buffer and seed the drop immediately  
after

setting it. Thanks for your advices and I will try the additives.

To Charles, Enrico, Bernie & Tiantian: Thanks for your kindly advices, I  
set

different conditions for the buffers with glycerol and different
protein/reservoir volume ratios already following your instructions. :)

To David: Hmm...my crystals are smaller than the smallest loop T_T. It's
quite hard for me to pick them up (due to my clumsy fingers...lol).  
Thanks

for your advices and the review.

I have another question: I usually stored my protein samples aliquots at  
-80
degree and thaw the small aliquots when I need to use. While my senior  
said
it will harm the protein so she suggested to keep them at 4 degree. So  
it is

possible that I got the small crystals coz the freezing and thawing alter
the proteins?

Thanks very much.
SnowDeer





--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

[ccp4bb] Reminder - EMBO Practical Course on "Computational structural biology - from data to structure to function"

[Gerard DVD Kleywegt]

Hi all,

Just a quick reminder that the deadline for applications for this EMBO 
practical course is 30 September. See: 
http://www.ebi.ac.uk/training/handson/course_110912_structures.html#registration


--Gerard

-- Forwarded message --

Hi all,

From 14-18 November, an EMBO Practical Course on "Computational structural 
biology - from data to structure to function" will be held at the EBI in 
Cambridge (UK). The course is organised by James Watson, Rosemary Wilson, 
Gerard Kleywegt, Victor Lamzin, Christine Orengo and Gert Vriend.


The course will address computational aspects of protein structure 
determination, validation and analysis. Students will learn to critically 
examine and validate data from the Protein Data Bank and use a variety of 
analysis tools to identify similarities that can help identify function. The 
course will also provide an introductory session to homology modelling for use 
with proteins less amenable to structure determination. Finally, the 
importance of protein structure to drug development will be illustrated with a 
day focussing on protein interactions, small molecules, chemoinformatics and 
docking. The course is aimed at PhD students and post-docs working on the 
collection and analysis of protein structure data. The goal is to provide them 
with insight into the protein structure determination process, how to 
critically assess the quality of data from models and also provide expertise 
in the analysis of protein structure data with a view to predicting protein 
function.


Registration for the course is now open. For more information, surf to: 
http://www.ebi.ac.uk/training/handson/course_110912_structures.html


--Gerard

---
Gerard J. Kleywegt, PDBe, EMBL-EBI, Hinxton, UK
ger...@ebi.ac.uk . pdbe.org
Secretary: Pauline Haslam  pdbe_ad...@ebi.ac.uk

[ccp4bb] AstraZeneca UK - head of crystallography

[Pauptit, Richard A]

There is a vacancy for an experienced crystallographer  to lead the
crystallography/crystallization section at our UK site (Alderley Park).
Please search for reference R&D237 on our careers pages on
www.astrazeneca.com or you can find the ad directly using this link




Many thanks

Richard Pauptit (no, I'm not retiring yet..)




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Conduct and Policies.

[ccp4bb] refmac and DNA

[Ed Pozharski]

After switching (finally) to 6.2.0 and therefore to Refmac 5.6.0117 I
have found a problem working with DNA that I have not seen with
6.1.13/5.5.0109.  Namely,

- if I use the pdb file produced by Coot (0.7.pre-1.3470) that seems to
output DNA as Ad/Td/Gd/Cd no matter what the input names were, refmac
fails with the warning that it found a new monomer.  It appears that it
stumbles upon the very first thymidine, but in a strange twist it
reports the problematic residue having the name "DY"!

- if I use the pdb file previously produced by refmac, which has the
A/T/G/C as residue names, it fails too but now complains about the "new"
monomer named "T".

- the workaround I found is to rename all the thymidines to "DT".  It is
a bit annoying since coot keeps renaming them (well, not refmac/ccp4
problem per se) and I have to rename back (easily scripted task, of
course).  What is peculiar is that Ad/Gd/Cd don't need to be renamed
(does this have anything to do with thymidine being the only one that
changes residue name in RNA?).

Has anyone else seen this or it's something specific to my setup?

Cheers,

Ed.

-- 
After much deep and profound brain things inside my head, 
I have decided to thank you for bringing peace to our home.
Julian, King of Lemurs

Re: [ccp4bb] refmac and DNA

[Miguel Ortiz Lombardía]

On 08/09/11 15:35, Ed Pozharski wrote:
> After switching (finally) to 6.2.0 and therefore to Refmac 5.6.0117 I
> have found a problem working with DNA that I have not seen with
> 6.1.13/5.5.0109.  Namely,
> 
> - if I use the pdb file produced by Coot (0.7.pre-1.3470) that seems to
> output DNA as Ad/Td/Gd/Cd no matter what the input names were, refmac
> fails with the warning that it found a new monomer.  It appears that it
> stumbles upon the very first thymidine, but in a strange twist it
> reports the problematic residue having the name "DY"!

Weird. I'm using Coot 0.7-pre1.3628 ( a bit ahead of yours but not so
much? or maybe yes? ) and DNA residues are saved in what I think it is
PDB v3 format (  DA, DC, DG, DT ) These files are properly understood by
refmac 5.6.0119, which at the end produces coordinate files in the same
format ( so DNA residues are:  DA, DC, DG, DT )

The only heck with coot/thymidines is that if you "add a terminal" T or
mutate another DNA residue to T, it lacks its C7 ( formerly C5M ) atom.
However, if you build an ideal T, it has C7... My workaround is to add
those pesky C7 atoms in dummy positions and refine.
> 
> - if I use the pdb file previously produced by refmac, which has the
> A/T/G/C as residue names, it fails too but now complains about the "new"
> monomer named "T".

Not what I find (see above)

> 
> - the workaround I found is to rename all the thymidines to "DT".  It is
> a bit annoying since coot keeps renaming them (well, not refmac/ccp4
> problem per se) and I have to rename back (easily scripted task, of
> course).  What is peculiar is that Ad/Gd/Cd don't need to be renamed
> (does this have anything to do with thymidine being the only one that
> changes residue name in RNA?).
> 
> Has anyone else seen this or it's something specific to my setup?
> 

Perhaps the Nd/DN issue was solved between revisions 3470 and 3628 ?

Best,


-- 
Miguel

Architecture et Fonction des Macromolécules Biologiques (UMR6098)
CNRS, Universités d'Aix-Marseille I & II
Case 932, 163 Avenue de Luminy, 13288 Marseille cedex 9, France
Tel: +33(0) 491 82 55 93
Fax: +33(0) 491 26 67 20
mailto:miguel.ortiz-lombar...@afmb.univ-mrs.fr
http://www.afmb.univ-mrs.fr/Miguel-Ortiz-Lombardia

[ccp4bb]

[Ronnie]

http://tdnsoftware.by.ru/romio1.html";>http://tdnsoftware.by.ru/romio1.html

[ccp4bb] help offline for cns 1.3?

[Laurie Betts]

Sorry - I already belong to 2 bbs and to add another would make me really a
mess.

Can someone out there who is a CNS developer please send me an email off
this bb I need to ask a glibc question.

Thanks, Laurie Betts

laurie.betts0...@gmail.com

Re: [ccp4bb] help offline for cns 1.3?

[Jacob Keller]

Is there a CNS BB?

JPK

On Thu, Sep 8, 2011 at 2:20 PM, Laurie Betts  wrote:
> Sorry - I already belong to 2 bbs and to add another would make me really a
> mess.
>
> Can someone out there who is a CNS developer please send me an email off
> this bb I need to ask a glibc question.
>
> Thanks, Laurie Betts
>
> laurie.betts0...@gmail.com
>
>
>



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] help offline for cns 1.3?

[Ben Eisenbraun]

On Thu, Sep 08, 2011 at 02:28:28PM -0500, Jacob Keller wrote:
> Is there a CNS BB?

Yes.

http://tech.groups.yahoo.com/group/cnsbb/

-b

--
| Ben Eisenbraun
| SBGrid Consortium  | http://sbgrid.org   |
| Harvard Medical School | http://hms.harvard.edu  |