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:[EMAIL PROTECTED] 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
