[ccp4bb]: SeMet

["David E. Timm" <dtimm@iupui.edu>]

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Hello,  Here is the list of responses to my question about SeMet crystal
stability:

We are looking for suggestions to try improving the stability of SeMet
substituted crystals.  The native crystals are stable to
handling,cryocooling, etc.  However, the SeMet substituted crystals are very
fragile and frequently crack when the coverslip is removed from the tray,
when the crystal is placed on a loop or when placed in cryo protectant
solution.  Out of about 50 crystals we have mananged to collect a 2.0 ang
data set in house using one of the SeMet substituted crystals, which shows
no significant cell or space group changes compared to the native crystals.
While we have kept this crystal in cryogenic storage, it would be nice to
have some backups. Any suggestions?

Cheers,

Dave Timm

Hi Dave,

A possible suggestion might be the method of Fernandez et al that was
recently published published J. Appl. Cryst. 33 (2000) 168-171. They had
similar problems but were using volatile organics as the precipitant.  Hope
this helps - I'm also trying to find a similar solution for my SeMet
crystals.

I have now settled in here in Missouri and am off to the APS next week
collecting data.

Regards

Chris
**********************************************
Chris F. Snook, Ph.D.


Try adding some glutaraldehyde or sim. crosslinking agents
(volatile ones!!) to the well.  It's been described by various authors
(Czworkowski, Wang Morre Steitz EMBO J 1994). However, changes in unit cell
dimensions often result from such experiments.

Can you grow SeMet protein xtals in sitting drops or in microbatch??

JJ

 Joe Jaeger, D.Phil.
 Lecturer & Research Fellow

We experienced similar disparity between the behavior of Native and Se-Met
crystals for a protein where most of the methionines are exposed to solvent.

i) The addition of 50mM mercaptoethylamine during purification and
crystallization helped minimize 'aggregation' problems, and helped stabilize
the crystals.

ii) The use of sitting drop, rather than hanging drop is perhaps a better
option for 'delicate' crystals. Why? The feeling that a drop on a 'flipped
over' coverslip is much more likely to evaporate faster - and hence cause
osmotic shock to the crystals - than a drop in a 'sitting drop' experiment
after the sealing tape has been cut open.

How about trying to grow the crystals in the presence of the
cryo-protectant

Chris Dealwis (Assistant Professor)

Hi Dave

I have one thought that may be helpful to you.  We've been
using 50% saturated sucrose as a cryoprotectant lately.  It
works for lots of crystals and seems to be very gentle.  If
tranferring to cryoprotectant is an issue, you might give it
a shot.

-Lesa

PS  just make a stock sat. sucrose solution with your well buffer, then
dilute it in half with well buffer again.  Can sometimes get away
with less than 50% depending on your well buffer composition.

**********************************************************************
* Lesa J. Beamer, PhD