No diffraction

From: Theresa H. Hsu
Date: 26 January 2012 15:33


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

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From: 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

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From: Poul Nissen

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

Poul

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From: Edward Snell

And ambient if you were using cryo …

 

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From: 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).

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From: 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

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From: 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,

__

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From: 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).

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From: 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

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From: Kevin Jin


Maybe, you can adjust the ion strength of your condition.

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From: 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

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From: Jesse


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

-Jesse