From: SnowDeer
Date: 5 September 2011 09:06
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
----------
From: Enrico Stura
dear SnowDeer,
The first step to see if bigger crystals can be grown is to use bigger protein drops and vary
the precipitant/protein ratio at the beginning of the vapour diffusion experiment.
You can work out for yourself why this should give you all the informations that you need in subsequent experiments.
Enrico.
--
Enrico A. Stura D.Phil. (Oxon) ,
Room 19, Bat.152,
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
----------
From: ChenTiantian
Agree with Enrico.
Reducing the concentration of your protein sample to make it form less nuclei, then it might grow bigger.
Best R,
tiantian
--
Shanghai Institute of Materia Medica, Chinese Academy of Sciences
Address: Room 101, 646 Songtao Road, Zhangjiang Hi-Tech Park,
Shanghai, 201203
----------
From: David Waterman
Dear SnowDeer,
----------
From: 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
----------
From: 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.
----------
From: Brad Bennett
Hi SnowDeer-
Date: 5 September 2011 09:06
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
----------
From: Enrico Stura
dear SnowDeer,
The first step to see if bigger crystals can be grown is to use bigger protein drops and vary
the precipitant/protein ratio at the beginning of the vapour diffusion experiment.
You can work out for yourself why this should give you all the informations that you need in subsequent experiments.
Enrico.
Enrico A. Stura D.Phil. (Oxon) ,
Room 19, Bat.152,
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
----------
From: ChenTiantian
Agree with Enrico.
Reducing the concentration of your protein sample to make it form less nuclei, then it might grow bigger.
Best R,
tiantian
Shanghai Institute of Materia Medica, Chinese Academy of Sciences
Address: Room 101, 646 Songtao Road, Zhangjiang Hi-Tech Park,
Shanghai, 201203
----------
From: David Waterman
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.
Best wishes
-- David
-- David
----------
From: 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
----------
From: 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.
----------
From: Brad Bennett
Hi SnowDeer-
What's your precipitant? Have you tried lowering that as much as possible? This will likely delay nucleation and crystal growth but the crystals will also likely be larger (and perhaps more well ordered). Of course, you may reach a lower limit where you prohibit nucleation. You'll have to determine this empirically.
How about smaller reservoir volumes while keeping the drop volume the same?
Have you tried setting up and storing trays at lower temperatures, 10 C or less? Sitting drop vs. hanging drop? Different oils in your vapor diffusion experiments, like Al's oil?
Also, I had success with growing large crystals by setting up microbatch drops under oil. If you'd like details, I can provide them off-board.
Cheers-
Brad
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