One other thought following from Kelly's point about room temp check is that most likely these are thin plates or needles. And I think it was Frank VonD who pointed out that some of these thin crystals can be physically stressed by the shape of the frozen cryo formed in the cryoloop. So choose loops with care - maybe go with litholoops?
Martyn
Martyn Symmons
MRC-MBU
Cambridge
----- Original Message ----
From: Kelly Daughtry <[log in to unmask]>
To: [log in to unmask]
Sent: Thursday, 21 January, 2010 14:27:58
Subject: Re: [ccp4bb] smeared spot in diffraction
Fengxia,
To me the diffraction appears as if the crystals did not freeze well.
So the best option seems to be annealing (as already suggested) or to
try different cryo-protectants.
Did you perform a room-temperature mount? If so, were the spots nice
and round (suggesting the cryo is the cause of the smears)?
I would suggest growing your crystals in the presence of cryo as well.
Glycerol, PEG 400, all the usual suspects.
May I suggest 2-methyl-2,4-pentanediol (MPD). I have had success
adding to well (10 - 20 %) but not in crystal drop, then using 20% as
a cryo (MPD acting to slow diffusion to give better ordered crystals).
Kelly Daughtry
*******************************************************
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 Thu, Jan 21, 2010 at 9:07 AM, Brad Bennett <[log in to unmask]> wrote:
> Hi Fengxia-
> How about increasing the PEG% so you don't have to add as much "other"
> cryoprotectant?
>
> Also, have you tried annealing the crystals? I've had success with this when
> I've had samples diffract similarly. The simplest way is by blocking the
> cryostream for 2-3 seconds (repeat 1-2 times) and then shoot. More involved
> way is by actually dismounting your crystals back into cryo buffer for 20-30
> seconds, then remounting and shooting.
>
> HTH-
> Brad
>
> On Thu, Jan 21, 2010 at 1:54 AM, Fengxia Liu <[log in to unmask]> wrote:
>>
>> Dear all,
>>
>> I am trying to diffract one semet-protein, it gave me some clear spots and
>> some smeared spots in one diffration, you can find maps attached.
>> Mother liquor condition is 0.1MTris-HCL pH8.5+10% PEG4k + 0.2M Li2SO4, I
>> have tried mother liquor+25%glycerol, paratone, paratone+ mineral oil, but
>> they all gave me such pattern.
>> Does anyone know how to solve this? I appreciate any advice.
>>
>> Thank you in advance.
>>
>> Best wishes,
>> Fengxia
>>
>>
>
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