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Re: [ccp4bb] bigger size - > better diffraction? |
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CCP4bb navigationCCP4bb <-- 2007 <-- April 2007 <-- 04 April 2007Subject: Re: bigger size - > better diffraction? From: Leonard Thomas thomasle {- at -} ITS {- dot -} CALTECH {- dot -} EDU Date: 2007-04-04 with cryo is to take a shot at room temp. and see how your crystal diffracts in general. It is true it may be a cryo problem, but if the non cryo protected crystals do not diffract then why would one expect the cryo protected one to. As for controlling crystal growth. I would second what Shane wrote and try seeding. Also trying the usually additives and varying protein concentration/precipitant concentration should help also so. Len On Apr 4, 2007, at 8:56 AM, Shane Atwell wrote: > Streak seeding all your trays should give you a better handle on > nucleation. You might be too high in protein or precipitant w/o the > seeding, hence the showering and rare nice crystals. > > Varying cryos, or cryo concentrations, or how the cryo is added can > help > a lot. Try 5 or 6 different cryos at 3 concentrations each. When you > have the best nailed down then try sequential transfers of the > crystals > from low to target concentrations (e.g. into 5% for a couple minutes, > then 10, 20, 25%). Also, you can try growing the crystals w/ a bit > (5%) > or the final conc or cryo already present. Should help in getting them > habituated to the cryo. > > Shane Atwell > >> -----Original Message----- >> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On >> Behalf Of Jenny >> Sent: Wednesday, April 04, 2007 5:50 AM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: [ccp4bb] bigger size - > better diffraction? >> >> Hi, All, >> >> I got a crystal that diffracts at 3.3A in house.The crystal >> size is about 0.2mm* 0.1mm * 0.2mm. At first I thought the >> size is fine,but it turns out the smaller ones diffract >> worse.I guess the reason is that >> the cell unit is really big (126.292 126.292 134.904 p4212, >> pretty big for a 10kD protein, isn't it?) >> >> So looks like I need to grow bigger crystals in order to get >> better diffractions.The problems is ,every time when I set up >> trays, the growing conditions is not exactly the same, so I >> have to set up a whole tray or maybe even 2 trays , then 2 or >> 3 conditions will jump out with good crystals ( 2 or 3 >> nucleation site ) and some of the others will show lots lots >> of small crystals.I used NaCl as the salt, in a 4*6 tray, the >> [NaCl] is going from 2.0,2.05,2.1,2.15,....something like >> that and 0.05M does make big difference.I used Urea as the >> additive in this case ( 25 m ~ 100 mM) and tried >> 2+2,3+1,3+2, 3+1 ( 3 uL protein and 1 uL buffer ) is better >> than the other two cases.Right now it's growing in room temp >> in about a week.And crystals that not fresh got some bubbles >> around the edge and didn't diffract well. >> >> Does anyone have any suggestions that what I could do to >> improve the diffraction? >> >> Thanks a lot. >> >> Jenny >> CCP4bb navigationCCP4bb <-- 2007 <-- April 2007 <-- 04 April 2007 |
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