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 <[email protected]> To: [email protected] 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 <[email protected]> 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 <[email protected]> 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 >> >> >
