Dear all,
Apparently, a similar procedure was used in solving structure of trp RNA
regulator:
Nature. 1995 Apr 20;374(6524):693-700.
The structure of trp RNA-binding attenuation protein.
Antson AA, Otridge J, Brzozowski AM, Dodson EJ, Dodson GG, Wilson KS,
Smith TM,
Yang M, Kurecki T, Gollnick P.
Department of Chemistry, University of York, UK.
The extracted isomorphous and anomalous differences were used in a
"molecular replacement" procedure with Amore. The authors were intended to
publish a separate paper with the detailed description of the method, but
I could not find a reference. I'm currently trying to apply this method to
my data and would be interested if somebody can provide details of making
an "anomalous difference" dataset.
Possibly Eleanor Dodson can shed some light on the subject?
Best regards,
Konstantin Korotkov
Dr. K.Korotkov
Senior Fellow
Department of Biochemistry
University of Washington
Box 357742
Seattle, WA 98195-7742
Phone: (206)779-3749
E-mail: [EMAIL PROTECTED]
On Wed, 30 Aug 2006, Philippe DUMAS wrote:
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Bill
I already had reasoned exactly as you, but I never tried it: should be
possible to run molecular replacement only with anomalous data and only with
bromine positions from a reasonable model (because, as you said, one may
expect Aform helices). But PC refinement should probably be used. And I
guess, some luck is needed...
Apart that, don't forget bromine cleavage under X-rays ...
Ennifar et al. ActaCryst D58 (2002) 1262
Schiltz et al. ActaCryst D60 (2004) 1024
I suggest you to ask Clemens Vonrhein the new version of Sharp that is able
to handle varying bromine occupancies during data collection.
(as described in Schiltz et al)
Good luck
Philippe Dumas
IBMC-CNRS, UPR9002
15, rue René Descartes 67084 Strasbourg cedex
tel: +33 (0)3 88 41 70 02
[EMAIL PROTECTED]
-----Message d'origine-----
De : [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] la part de
William Scott
Envoyé : mercredi 30 aout 2006 20:16
A : [email protected]
Objet : [ccp4bb]: a MAD molecular replacement question
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Hi folks:
I have a question regarding a Br mad data set on RNA.
We have a crystal of an RNA in the lab in which the molecule has been
transcribed and incorporates 11 Br-U residues at known positions in
the sequence, and we have a MAD data set on this to about 3 A
resolution (as well as a native data set).
mmtbx.xtriage reports that we may have up to 3 molecules in the
asymmetric unit (of P21), meaning 11, 22 and 33 Br sites are possible:
----------------------------------------------------------------
| Copies | Solvent content | Matthews Coef. | P(solvent cont.) |
|--------|-----------------|----------------|------------------|
| 1 | 0.828 | 7.146 | 0.012 |
| 2 | 0.656 | 3.573 | 0.238 |
| 3 | 0.484 | 2.382 | 0.652 |
| 4 | 0.312 | 1.786 | 0.091 |
| 5 | 0.140 | 1.429 | 0.007 |
----------------------------------------------------------------
| Best guess : 3 copies in the asu |
----------------------------------------------------------------
I've been using ShelxD to find Br sites, and the maps, though quite
promising, still need significant improvement. I also haven't
convinced myself that there is a clear NCS 2-fold or 3-fold, and so
have been trying with 1,2, and 3 molecules.
Since there are regions of this molecule that (presumably) have
canonical A-form RNA helices, and these have Br-U in them at known
positions, I'd like to make use of the distance constraints these put
on the Br sites, as well as the Br absorption in the mad peak data.
Is it possible to do molecular replacement using the Br anomalous
signal and model helices to help pin down the locations of the Br atoms?
Thanks in advance.
Bill Scott
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