Re: [ccp4bb] No diffraction

2012-02-07 Thread Jesse 
This might be obvious, but make sure you washed the crystals
thoroughly before dissolving them for SDS-PAGE or mass spec

-Jesse

On Thu, Jan 26, 2012 at 10:48 AM, Katherine Sippel
 wrote:
> Might I suggest consulting the CCP4 user community wiki on the topic:
>
> http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Improving_crystal_quality
>
> Good luck,
>
> Katherine
>
>
>
> On Thu, Jan 26, 2012 at 9:33 AM, Theresa H. Hsu 
> wrote:
>>
>> Dear crystallographers
>>
>> I have a protein of 90 kDa forming dimers. Crystals formed with microbatch
>> and vapor diffusion method in 24 hours but no diffraction at home source.
>> Dissolved crystals was confirmed to be the protein with mass spec.
>>
>> Any suggestions to improve diffraction would be welcome.
>>
>> Thanking you in advance.
>>
>> Theresa
>
>

Re: [ccp4bb] No diffraction

2012-01-27 Thread Joe Watts 
1. Try room temperature mounts (as suggested by others)
2. Expose the hell out of the crystal (5 min) on home source or go synchrotron 
3. Run your protein through another column (ion exchange) even if it looks pure
4. Try an additive screen
5. Try limited proteolysis or methylation

6. If none of theat works, clone the same protein from another 
organism/strain/variant, ad try again

Re: [ccp4bb] No diffraction

2012-01-26 Thread Kevin Jin 
Maybe, you can adjust the ion strength of your condition.




On Thu, Jan 26, 2012 at 8:32 AM, Kevin Jin  wrote:
> For crystallization:
>
> Your xtal may come out a little bit fast. If the condition contain
> alcohol,  such as IPA, you may have to modify it.
>
> If you let people know the condition, it may be more helpful.
>
> Also, please check the purity of your protein.
>
> Kevin
>
> On Thu, Jan 26, 2012 at 7:33 AM, Theresa H. Hsu  wrote:
>> Dear crystallographers
>>
>> I have a protein of 90 kDa forming dimers. Crystals formed with microbatch 
>> and vapor diffusion method in 24 hours but no diffraction at home source. 
>> Dissolved crystals was confirmed to be the protein with mass spec.
>>
>> Any suggestions to improve diffraction would be welcome.
>>
>> Thanking you in advance.
>>
>> Theresa

Re: [ccp4bb] No diffraction

2012-01-26 Thread Kevin Jin 
For crystallization:

Your xtal may come out a little bit fast. If the condition contain
alcohol,  such as IPA, you may have to modify it.

If you let people know the condition, it may be more helpful.

Also, please check the purity of your protein.

Kevin

On Thu, Jan 26, 2012 at 7:33 AM, Theresa H. Hsu  wrote:
> Dear crystallographers
>
> I have a protein of 90 kDa forming dimers. Crystals formed with microbatch 
> and vapor diffusion method in 24 hours but no diffraction at home source. 
> Dissolved crystals was confirmed to be the protein with mass spec.
>
> Any suggestions to improve diffraction would be welcome.
>
> Thanking you in advance.
>
> Theresa

Re: [ccp4bb] No diffraction

2012-01-26 Thread Patrick Shaw Stewart 
Theresa

You should also try microseeding into *random screens *by making a seed
stock with the crystals that you have, to use with both microbatch and
vapor diffusion experiments.  You will often pick up new and better
conditions and you're more likely to get well-formed crystals right out of
the screens.

I hope it works

Patrick

_

Refs: Allan D’Arcy, Frederic Villarda, May Marsh. An automated microseed
matrix-screening method for protein crystallization.  Acta
Crystallographica section D 63 (2007), 550–554.

Galina Obmolova,* Thomas J. Malia, Alexey Teplyakov, Raymond Sweet and Gary
L. Gilliland. 'Promoting crystallization of antibody–antigen complexes via
microseed matrix screening.' Acta Crystallographica Section D 66 (2010)
927–933.  Open-access at
http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf

A. G. Villaseñor, A. Wong, A. Shao, A. Garg, A. Kuglstatter and S. F.
Harris. 'Acoustic matrix microseeding: improving protein crystal growth
with minimal chemical bias.' Acta Crystallographica Section D 66 (2010)
568-576.

Gregory Ireton and Barry Stoddard.  'Microseed matrix screening to improve
crystals of yeast cytosine deaminase'.  Acta Crystallographica section D60
(2004) 601–605.

Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E.
Chayen and Peter F.M. Baldock. 'Random Microseeding: A Theoretical and
Practical Exploration of Seed Stability and Seeding Techniques for
Successful Protein Crystallization'. Cryst. Growth Des., 2011, 11 (8), pp
3432–3441. On-line at http://pubs.acs.org/doi/abs/10.1021/cg2001442.  * If
you don't have a subscription to Crystal Growth and Design, click
**here*
* to obtain a free copy (we're limited to 50 downloads in the first year).*



On 26 January 2012 15:33, Theresa H. Hsu  wrote:

> Dear crystallographers
>
> I have a protein of 90 kDa forming dimers. Crystals formed with microbatch
> and vapor diffusion method in 24 hours but no diffraction at home source.
> Dissolved crystals was confirmed to be the protein with mass spec.
>
> Any suggestions to improve diffraction would be welcome.
>
> Thanking you in advance.
>
> Theresa
>



-- 
 patr...@douglas.co.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36

Re: [ccp4bb] No diffraction

2012-01-26 Thread Roger Rowlett 

  
  
As other have/surely will suggest, by all means
  try RT collection to establish if your crystals really diffract
  OK, or if your cryo conditions are killing them. We have lots of
  experience in our lab getting "beamstop" diffraction with certain
  samples when subjected to cryoprotection. Depending on the
intensity of your home source (and its life-cycle age), small
crystals may not be large enough to produce usable or visible
diffraction. With our home source, we really need 0.3 mm crystals
(larger if thin plates) to get usable diffraction.

Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

On 1/26/2012 10:33 AM, Theresa H. Hsu wrote:

  Dear crystallographers

I have a protein of 90 kDa forming dimers. Crystals formed with microbatch and vapor diffusion method in 24 hours but no diffraction at home source. Dissolved crystals was confirmed to be the protein with mass spec.

Any suggestions to improve diffraction would be welcome.

Thanking you in advance.

Theresa

Re: [ccp4bb] No diffraction

2012-01-26 Thread George Sheldrick 
It depends a lot on which home source and which synchrotron, there are 
enormous differences. Goettingen is uniquely well placed because we can 
reach four synchrotrons in a few (3-7) hours by high speed train and in 
theory at least five more with a longer train journey, trains are very 
convenient for transporting crystals. Two of these synchrotrons do not 
give a higher resolution than our home system, but at least they can 
vary the wavelength. However if we think we can see at least two 
reflections at home, of course we take the crystal to a (suitable) 
synchrotron.


George

On 01/26/2012 04:54 PM, Francis E Reyes wrote:

Ditto to Poul's advice.

I've had many many many cases where crystals diffract poorly (or not at all) on 
home sources only to show excellent diffraction at a synchrotron. (Whether or 
not a home source is properly calibrated is probably the biggest issue, but 
that's for another discussion).



On Jan 26, 2012, at 8:33 AM, Theresa H. Hsu wrote:


Dear crystallographers

I have a protein of 90 kDa forming dimers. Crystals formed with microbatch and 
vapor diffusion method in 24 hours but no diffraction at home source. Dissolved 
crystals was confirmed to be the protein with mass spec.

Any suggestions to improve diffraction would be welcome.

Thanking you in advance.

Theresa



-
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder

Re: [ccp4bb] No diffraction

2012-01-26 Thread Francis E Reyes 
Ditto to Poul's advice. 

I've had many many many cases where crystals diffract poorly (or not at all) on 
home sources only to show excellent diffraction at a synchrotron. (Whether or 
not a home source is properly calibrated is probably the biggest issue, but 
that's for another discussion). 



On Jan 26, 2012, at 8:33 AM, Theresa H. Hsu wrote:

> Dear crystallographers
> 
> I have a protein of 90 kDa forming dimers. Crystals formed with microbatch 
> and vapor diffusion method in 24 hours but no diffraction at home source. 
> Dissolved crystals was confirmed to be the protein with mass spec.
> 
> Any suggestions to improve diffraction would be welcome.
> 
> Thanking you in advance.
> 
> Theresa



-
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder

Re: [ccp4bb] No diffraction

2012-01-26 Thread Edward Snell 
And ambient if you were using cryo ...

Edward Snell Ph.D.
Assistant Prof. Department of Structural Biology, SUNY Buffalo,
Senior Scientist, Hauptman-Woodward Medical Research Institute
700 Ellicott Street, Buffalo, NY 14203-1102
Phone: (716) 898 8631 Fax: (716) 898 8660
Skype:  eddie.snell Email: esn...@hwi.buffalo.edu
Telepathy: 42.2 GHz

Heisenberg was probably here!

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Poul 
Nissen
Sent: Thursday, January 26, 2012 10:50 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] No diffraction

But first of all: try to add a synchrotron to the crystals

Poul
On 26/01/2012, at 16.48, Katherine Sippel wrote:


Might I suggest consulting the CCP4 user community wiki on the topic:

http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Improving_crystal_quality

Good luck,

Katherine

On Thu, Jan 26, 2012 at 9:33 AM, Theresa H. Hsu 
mailto:theresah...@live.com>> wrote:
Dear crystallographers

I have a protein of 90 kDa forming dimers. Crystals formed with microbatch and 
vapor diffusion method in 24 hours but no diffraction at home source. Dissolved 
crystals was confirmed to be the protein with mass spec.

Any suggestions to improve diffraction would be welcome.

Thanking you in advance.

Theresa

Re: [ccp4bb] No diffraction

2012-01-26 Thread Poul Nissen 
But first of all: try to add a synchrotron to the crystals

Poul
On 26/01/2012, at 16.48, Katherine Sippel wrote:

> Might I suggest consulting the CCP4 user community wiki on the topic: 
> 
> http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Improving_crystal_quality
> 
> Good luck,
> 
> Katherine
> 
> 
> On Thu, Jan 26, 2012 at 9:33 AM, Theresa H. Hsu  wrote:
> Dear crystallographers
> 
> I have a protein of 90 kDa forming dimers. Crystals formed with microbatch 
> and vapor diffusion method in 24 hours but no diffraction at home source. 
> Dissolved crystals was confirmed to be the protein with mass spec.
> 
> Any suggestions to improve diffraction would be welcome.
> 
> Thanking you in advance.
> 
> Theresa
>

Re: [ccp4bb] No diffraction

2012-01-26 Thread Katherine Sippel 
Might I suggest consulting the CCP4 user community wiki on the topic:

http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Improving_crystal_quality

Good luck,

Katherine


On Thu, Jan 26, 2012 at 9:33 AM, Theresa H. Hsu wrote:

> Dear crystallographers
>
> I have a protein of 90 kDa forming dimers. Crystals formed with microbatch
> and vapor diffusion method in 24 hours but no diffraction at home source.
> Dissolved crystals was confirmed to be the protein with mass spec.
>
> Any suggestions to improve diffraction would be welcome.
>
> Thanking you in advance.
>
> Theresa
>