Hi Eleanor,
On Fri, Jul 27, 2007 at 12:56:07PM +0100, Eleanor Dodson wrote:
> Phasing tasks) , worrying a lot if they are not! then using the 2.3A
> data for phase extension from the 2.7A exptl phases. DM with averaging
> and phase extension will improve your map a lot, and maybe you can build
> the other 30%
> Eleanor
>
> PS - as a crystallographic educator I am a bit upset that this isnt the
> norm for experimental phasing - phase extension is brilliant!! and not
> using it is rather reminiscent of self flagelation..
It is the default/norm - at least for SHARP/autoSHARP (and most likely
for all other modern program suites). Ok, the NCS-averaging part
hasn't been automated (yet) completely, but the phase-extension is:
all reflections of all datasets will be used within our SOLOMON
density-modification approach.
It works really well for me - at least if there is meaningful
experimental phase information to a certain resolution. 2.7 to 2.3 is
easy ... the tricky ones are when you have poor 6A phases and some
really borderline 3.5A dataset ... which seems the norm for the kind
of datasets coming my way these days ... sigh.
Cheers
Clemens
>
> Anastassis Perrakis wrote:
> >Hi -
> >
> >Its not clear to me if the 2.3 A dataset is from a different crystal,
> >if it has the same space group as the 2.7 A dataset and if it is
> >isomorphous, and how the 'jump' from the 2.7 A dataset to the 2.3 A
> >dataset has been done. I can think of quite a few ways to do that
> >transition, and each way can carry its own problems.
> >
> >Maybe its worth clarifying these in more detail.
> >
> >I also have to say that (for once!) I dont agree with Kay. I have
> >tried in the past to refine against datasets processed with different
> >programs, and I know people that do that a lot; the differences are
> >minor at best (1-2 % Rfree, mostly differences are in the anomalous
> >signal quality). Thus I dont think its processing but a mistake in the
> >settings or somewhere else something that went clearly wrong
> >(different origin ?). Anyway, i am not sure about that either, since
> >Satinder says that the model does fit the density (which density
> >though ? Also not clear).
> >
> >BTW, assuming that the two datasets are from different non-isomorpous
> >crystals, thats what I would be tempted to try and do:
> >
> >1. Use 'phaser' to do a molecular replacement of the 70% complete
> >model to the 2.3 A dataset
> >2. Use 'cad' to combine the output mtz file of 'phaser' with phases
> >and weights, with you SAD phases and weights, plus Fobs of both datasets.
> >3. Use dmmulti to do 'multi-crystal averaging' using the molecular
> >replacement phases of the 2.3 A dataset and the 2.7 A SAD phases. Also
> >use the NCS. That must result to a good 2.3 A map.
> >4. Use ARP/wARP to autotrace the 2.3 A map with the 2.3 A fobs.
> >
> >I should also add that if I had a 2.3 A native and a 2.7 A SAD map in
> >NON-isomorphous crystals in the first place, I would use DM-multi
> >directly,
> >even without phases for the 2.3 A map. That has worked beautifully for
> >me in the past. Anyway - too late for that now since you have a model.
> >
> >Last but not least if the 2.3 A dataset and the 2.7 A dataset are
> >isomorphous, I would most definitely have tried phasing using both of them
> >Especially if one had eg Se and one did not (the best SAD experiment
> >will give you 6 to 7 e to use; Se-S has 18 e. why not use them if you
> >can ??
> >yes, SAD might sometimes be the best map as many people have shown
> >over the last few years,
> >but also sometimes the Se-derivative - Native has lots and lots of
> >info and using all info can give better maps when non-isomorphism is
> >not a problem.)
> >
> >May I also assume that when you are sure of the space group that means
> >you put it through x-triage and its not a disguised P21 twin,
> >as many P21212 cases I have seen.
> >
> >Tassos
> >
> >On Jul 27, 2007, at 2:19, Satinder K. Singh wrote:
> >
> >>Hello,
> >>
> >>I have a SAD data set to 2.9A and a native data set on the same
> >>protein to 2.3A. I have built 70% of the model (polyalanine) into the
> >>SAD experimental map that has been subjected to 2-fold NCS averaging
> >>and phase-extended to 2.3 A. From the maps, the model looks like it
> >>fits the density fine, but when I try to refine (either rigid-body or
> >>positional refinement in REFMAC or simulated annealing in CNS), I get
> >>an R-factor of ~60%, worse than random. Similarly, a SigmaA run
> >>("combine isomorphous phase with partial structure") on the model
> >>yields an R-factor of ~60%. However, when I run DM on the
> >>original phase-combined file (before NCS averaging and phase
> >>extension) in omit mode, I get an R-free of 34.6%, which suggests
> >>that the map files itself is okay.
> >>
> >>I also checked the space group of the processed data, and it is
> >>definitely P21212.
> >>
> >>Does anyone have any suggestions of what I may be doing wrong?
> >>
> >>Thanks in advance for your help.
> >>
> >>Kind regards,
> >>Satinder
> >>
> >
>
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