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Re: [ccp4bb] bigger size - > better diffraction? |
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CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: bigger size - > better diffraction? From: Elspeth Garman elspeth {- at -} BIOP {- dot -} OX {- dot -} AC {- dot -} UK Date: 2007-04-04 For room temperature testing, it is much easuier to use a cryoloop enclosed in a capillary (with some mother liqour at the top) or the plastic tubes supplied by MiTeGen as per the method detailed in: Skrzypczak-Jankun, E., Bianchet, M. A., Amzel, L. M. and Funk Jr., M. O. (1996). /Acta Cryst./,/ /*D52*, 959-965. than to capillary mount. Also, if you do sequential transfer into cryo, try it `in situ' by pipetting on increasing concetrations and removal of liquid rather than moving the crystal between wells of higher concentration - mosaicity should be less. Good luck Elspeth Leonard Thomas wrote: > The first thing to try before going down the long road of fussing > 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 >>> > -- ------------------------------------------------- Dr. Elspeth F. Garman, Reader in Molecular Biophysics, Department of Biochemistry, Rex Richards Building, University of Oxford, Tel: (44)-1865-275398 South Parks Road, FAX: (44)-1865-275182 OXFORD, OX1 3QU, U.K. E-mail: elspeth@biop.ox.ac.uk ------------------------------------------------- CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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