in my opinion one should always first check RT diffraction and we use
the Mitegen plastic cap over the loop. We find the solutions slowly
dry out (6-18 hours), which can be enough to get a useful RT dataset
on the rotating anode.
If you like, the crystal can also be recovered after RT diffraction
checking for addition of cryosolution and freezing.
Mark
Quoting "Clayton, Gina Martyn" <[log in to unmask]>:
>
> MiTeGen have instructions on their website for using their system. In
> practice I usually put a little silicon grease at the base of the
> capillary as sometimes the capillaries fall off plus you need reasonable
> steady hand eye coordination to put the capillary over the loop...I have
> found MiTeGen RT system very useful.
>
> Good luck!
>
>
>
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
> Ezra Peisach
> Sent: Wednesday, January 27, 2010 7:41 AM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] Crystal rescue
>
> MiTeGen (www.mitegen.com) sells a RT device - plastic capillary to put
> over loop... I do not know how well it would work for a long data
> collection - but people here have used it to evaluate their crystals....
>
> Ezra
>
>
> On 1/27/2010 3:58 AM, Flip Hoedemaeker wrote:
>> Zhiyi,
>>
>> You can use a thin cap over your cryo loop, just put a drop of mother
>> liquor in the top, place over the loop and make it airtight at the
>> base. Not sure who sells these things though, I guess you can make it
>
>> from a capillary too. Then remove the cryo stream or put it at a temp
>> above freezing, say 253K.
>>
>> Flip
>>
>> Zhiyi Wei wrote:
>>> Thanks for so many quick responses!
>>>
>>> Actually, I have test several different cryo-protectants, including
>>> glycerol, EG, and PEG400. I did not see much differences between
> these
>>> cryo conditions. So, I choose glycerol.
>>>
>>> I would like to test my crystals in RT. But I don't know how to do
>>> this. Just mount crystal to the X-ray machine without cryo stream? Or
>>> I should use capillaries?
>>>
>>> Zhiyi
>>>
>>> On Wed, Jan 27, 2010 at 5:45 AM, Kelly Daughtry <[log in to unmask]>
> wrote:
>>>> Tascimate can be used as the cryo as well. I have had experience
> with
>>>> crystals in similar condition and moved the crystals to a 20%
>>>> increased Tascimate solution and they froze well.
>>>>
>>>> I agree with Ezra, room temperature mount your crystal before
>>>> freezing. It is the only way to know the true problem.
>>>>
>>>>
>>>> Kelly
>>>> *******************************************************
>>>> Kelly Daughtry
>>>> PhD Candidate
>>>> Department of Physiology and Biophysics
>>>> Boston University School of Medicine
>>>> 590 Commonwealth Ave
>>>> R 390
>>>> Boston MA, 02215
>>>> (P) 617-358-5548
>>>> *******************************************************
>>>>
>>>>
>>>>
>>>> On Tue, Jan 26, 2010 at 1:25 PM, Marcus Winter
>>>> <[log in to unmask]> wrote:
>>>>> Dear Zhiyi,
>>>>>
>>>>>
>>>>> Ezra is exactly right, of course. The Oxford Diffraction PX
> Scanner
>>>>> system can assess the diffraction qualities of (putative) protein
>>>>> crystals in situ - in the crystallisation plate. So, directly, you
>>>>> would discover if your 'big and beautiful' crystals actually
> diffract
>>>>> well... in their mother liquor under ambient conditions and before
> the
>>>>> addition of any cryo-protect. Do you have a friend or neighbour
> with
>>>>> a PX Scanner ? If not, please feel most welcome to contact
>>>>> Oxford Diffraction: we would be pleased to assist if at all
> possible.
>>>>>
>>>>>
>>>>> Good Luck and Best Wishes,
>>>>>
>>>>> Marcus Winter.
>>>>>
>>>>> www.oxford-diffraction.com
>>>>>
>>>>>
>>>>>
>>>>>
>>>>> -----Original Message-----
>>>>> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf
> Of
>>>>> Ezra Peisach
>>>>> Sent: 26 January 2010 16:01
>>>>> To: [log in to unmask]
>>>>> Subject: Re: [ccp4bb] Crystal rescue
>>>>>
>>>>> First you need to establish if it is your cryo conditions or the
>>>>> crystals. Depending where you are - they might have the equipment
>>>>> to do
>>>>>
>>>>> a wet mount - without freezing. Yes the crystal will not last -
> but
>>>>> then you know if the problem is in the
>>>>> crystal. If it is - you need better crystals. If it is the cryo -
>
>>>>> you
>>>>> need to work on that. Tacsimate is mixture of alot of different
>>>>> compounds - but the smears are too close together to be a small
> salt
>>>>> crystal on top...
>>>>>
>>>>> Good luck,
>>>>>
>>>>> Ezra
>>>>>
>>>>> On 1/26/2010 10:42 AM, Zhiyi Wei wrote:
>>>>>> Dear all,
>>>>>>
>>>>>> I got a problem with my crystals. I have two total different
> proteins
>>>>>> that both can be crystallized in the condition with PEG3350 and
>>>>> Tacsimate
>>>>>> (although the concentrations are different) with different shapes.
>
>>>>>> The
>>>>>> crystals look big and beautiful. However, when I test them in
>>>>> synchrotron,
>>>>>> both of these two types of crystals showed poor diffractions. As
>>>>> showed in
>>>>>> the attached diffraction image, the diffraction is up to ~4 A but
>>>>> smear in
>>>>>> one direction while<8 A in the other direction. The interesting
> thing
>>>>> is
>>>>>> that the diffraction pattern is similar for all crystals (from two
>>>>> different
>>>>>> proteins) that I tested without exception although they belong to
>>>>> different
>>>>>> space groups. So, I wonder whether these kind of pattern is caused
> by
>>>>>> Tacsimate (I don't know what it is) and how to rescue these
> crystals.
>>>>> Any
>>>>>> suggestions or comments?
>>>>>>
>>>>>> Thanks a lot!
>>>>>>
>>>>>> Best,
>>>>>> Zhiyi
>>>>>>
>>>
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