From: Afshan Begum
Date: 18 October 2011 12:45
Best Regards
AFSHAN
----------
From: <elizabeth.duke
Hi
You don't give any details of how you are collecting your data or what the protein is (small, medium, large, solvent content, unit cell etc etc). But at first glance your crystal is huge by "modern day standards"..... Very often large crystals are of poorer overall quality than smaller ones.....
If you are trying to "freeze" a crystal of that size you are likely to run into problems with data quality.
I would suggest trying a smaller crystal...... but without more information it is difficult to provide any real advice.
Good luck!
Liz
Dr. Liz Duke
Principal Beamline Scientist
Diamond Light Source
Harwell Science and Innovation Campus
Chilton
OX11 0DE
UK
----------
From: Charles Allerston
Silver bullet
Additive screens
Methylation
Construct boundaries
in situ proteolysis
Have you got a ligand? Stick in in there!
Do you have an expression tag? Cleave it/leave it on.
What is in your protein buffer? Mess about with that?
Temperature gradients
How is your purity?
How quickly are you getting this in to trays? Can you speed this up? Might be a factor.
Annealing at the beamline?
etc.
cheers,
charlie
----------
From: vandana kukshal
Hello
Vandana kukshal
----------
From: James Holton
An excellent review of this subject was published not long ago:
http://dx.doi.org/10.1107/S0907444905032130
It is even open access!
But, in general the "trick" to improving diffraction is to get your molecules clean, get them to all adopt the same conformation, and then sit still. Adding a column (even if you think your prep is clean) can help, as can fractional recrystallization. Not many people know this, but even the much maligned hen egg white lysozyme doesn't diffract very well if the prep is contaminated with "lysozyme dimers", which is why commercial lysozyme is purified by fractional recrystallization. There is a heat-shock treatment described in the above paper, and of course additive screens. The reason why additive screens work seems to be because if you can get something to stick to the protein, it is more likely to sit still.
-James Holton
MAD Scientist
Date: 18 October 2011 12:45
Dear ccp4 user
I am facing one crucial problem regarding diffraction. Actually the size of my crystal is good enough 0.5mm but it was diffracted only 4 A.
The conditions of crystallization recipes are 4.5% PEG 1000, 0.1M Tris and 25mM Na citrate. I really need your suggestions regarding how can i improve my diffraction quality?
Your support is highly appreciable.
I am facing one crucial problem regarding diffraction. Actually the size of my crystal is good enough 0.5mm but it was diffracted only 4 A.
The conditions of crystallization recipes are 4.5% PEG 1000, 0.1M Tris and 25mM Na citrate. I really need your suggestions regarding how can i improve my diffraction quality?
Your support is highly appreciable.
Best Regards
AFSHAN
----------
From: <elizabeth.duke
Hi
You don't give any details of how you are collecting your data or what the protein is (small, medium, large, solvent content, unit cell etc etc). But at first glance your crystal is huge by "modern day standards"..... Very often large crystals are of poorer overall quality than smaller ones.....
If you are trying to "freeze" a crystal of that size you are likely to run into problems with data quality.
I would suggest trying a smaller crystal...... but without more information it is difficult to provide any real advice.
Good luck!
Liz
Dr. Liz Duke
Principal Beamline Scientist
Diamond Light Source
Harwell Science and Innovation Campus
Chilton
OX11 0DE
UK
----------
From: Charles Allerston
Silver bullet
Additive screens
Methylation
Construct boundaries
in situ proteolysis
Have you got a ligand? Stick in in there!
Do you have an expression tag? Cleave it/leave it on.
What is in your protein buffer? Mess about with that?
Temperature gradients
How is your purity?
How quickly are you getting this in to trays? Can you speed this up? Might be a factor.
Annealing at the beamline?
etc.
cheers,
charlie
----------
From: vandana kukshal
Hello
You can do dehydration of crystal to get better diffraction .......go through this reference............
Dehydration Converts DsbG Crystal Diffraction from Low to High Resolution
Structure, Vol. 11, 139–145, February, 2003,
--
Vandana kukshal
----------
From: James Holton
An excellent review of this subject was published not long ago:
http://dx.doi.org/10.1107/S0907444905032130
It is even open access!
But, in general the "trick" to improving diffraction is to get your molecules clean, get them to all adopt the same conformation, and then sit still. Adding a column (even if you think your prep is clean) can help, as can fractional recrystallization. Not many people know this, but even the much maligned hen egg white lysozyme doesn't diffract very well if the prep is contaminated with "lysozyme dimers", which is why commercial lysozyme is purified by fractional recrystallization. There is a heat-shock treatment described in the above paper, and of course additive screens. The reason why additive screens work seems to be because if you can get something to stick to the protein, it is more likely to sit still.
-James Holton
MAD Scientist
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