[ccp4bb] How to help crystal grow bigger

2011-09-05 Thread SnowDeer 
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

2011-09-05 Thread 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.


On Mon, 05 Sep 2011 10:06:22 +0200, 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



--
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

Re: [ccp4bb] How to help crystal grow bigger

2011-09-05 Thread ChenTiantian 
>"vary the precipitant/protein ratio"
Agree with Enrico.
Reducing the concentration of your protein sample to make it form less
nuclei, then it might grow bigger.

Best R,

tiantian

On Mon, Sep 5, 2011 at 7:48 PM, Enrico Stura  wrote:

> 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.
>
>
>
> On Mon, 05 Sep 2011 10:06:22 +0200, 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
>>
>
>
> --
> 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
>



-- 
Shanghai Institute of Materia Medica, Chinese Academy of Sciences
Address: Room 101, 646 Songtao Road, Zhangjiang Hi-Tech Park,
Shanghai, 201203

Re: [ccp4bb] How to help crystal grow bigger

2011-09-06 Thread 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.
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

2011-09-08 Thread 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

2011-09-08 Thread 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

Re: [ccp4bb] How to help crystal grow bigger

2011-09-09 Thread 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

On Thu, Sep 8, 2011 at 3:52 AM, 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
>
> 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
>>>
>>
>>
>