[sharp-discuss] Re: Heavy Atom Negative Density

[Paul Hubbard]

Hello,

Thanks for the response. I guess it's pretty obvious now you've said it -
however, I have a few questions, and excuses!

1) The Os and Hg derivative data have really weak anomalous signals (hence no use
of anomalous data), so why would not including the Fe positions in these datasets
have such a big impact?

2) I don't understand what you mean by residual maps not using phase. I thought
residual maps were:

|Fph - Fp|exp[i.phi(best)]

Thanks for the help. Hope this works!

Paul Hubbard

Clemens Vonrhein wrote:

> Hi Paul,
>
> > Attached is a copy of my input file. It's setup just like the example - and
> > I use the in-house data set as the reference.
>
> Aha!!! Now things are clearer: you actually have a MAD + native + two
> derivatives. Your setup looks like this
>
>   C-1        protein + Fe-S cluster(s)
>     X-1        ? crystal
>       W-1        in-house data
>         B-1
>       W-2        wavelength 1
>         B-1
>       W-3        wavelength 2
>         B-1
>       W-4        wavelength 3
>         B-1
>   C-2        protein + Hg
>     X-1
>       W-1
>         B-1
>   C-3        protein + Os
>     X-1
>       W-1
>         B-1
>
> which is ok if
>
>   1. you measured your in-house data on the same crystal as your MAD
>      data
>
>   2. your 2nd and 3rd compound do not have Fe-S clusters
>
> (one note: you want to take the best of your MAD wavelengths as a
> reference wavelength to reduce corrleated non-isomorphism).
>
> However, if
>
>   1. all your crystals contain the Fe-S clusters
>
>   2. the in-house was collected on a different crystal
>
> the way to set it up is the following:
>
>   C-1        protein + Fe-S cluster(s)
>     X-1        MAD crystal
>       W-1        wavelength 1
>         B-1
>       W-2        wavelength 2
>         B-1
>       W-3        wavelength 3
>         B-1
>     X-2        in-house crystal
>       W-1
>         B-1
>   C-2        protein + Fe-S cluster(s) + Hg
>     X-1
>       W-1
>         B-1
>   C-3        protein + Fe-S cluster(s) + Os
>     X-1
>       W-1
>         B-1
>
> At least that is what I suspect your chemical composition in the
> various compounds are. The important bit is, that you have to declare
> the Fe atoms in ALL compounds: although they are common to all
> compounds, you have to declare them for the MAD - and therefore for
> all others too.
>
> In the two derivatives you then have to also declare the Os/Hg sites.
>
> > No - I'll give it a try. However, why then do I get nice positive density in
> > the residual map when I use just one Iron atom? Wouldn't these be negative
> > too?
>
> Residual maps don't use phases - they'll look identical for both hands
> (well apart from inverted coordinates of course). you also don't need
> to re-refine things in the inverted hand - just phase calculation will
> do.
>
> > The residual map from the 4Fe phased data set still has alot of strong
> > positive density round the 4Fe-4S cluster . I assumed this was from the 4
> > sulfurs and noise.
>
> If nothing is AT the sites (positive or negative) then your f'/f''
> seem to be ok. Beware when fixing the HAT_OCC if your data is not on
> roughly absolute scale and/or your f' are wrong. And 3A is definitely
> not going to be on absolute scale ...
>
> Unless these features are still > 6 sigma I would ignore them -
> otherwise you might want to put S atoms in - or model things with
> anisotropic B or ...
>
> > I used the EXAFS values for inflection and peak - and table values for
> > in-house and remote. They were all kept fixed.
>
> If you fix HAT_OCC you might want to refine f' values. But I rather
> suggest fixing the f'/f'' values (if your EXAFS is good) and refine
> HAT_OCC - even if you 'know' they should be 1.0 (assuming your data is
> on absolute scale - which it never really is).
>
> Some additional remakrs to your SIN file:
>
> 1. NANO_CLOC is HUGE for your in-house data and VERY large for your
>    MAD data: your residual maps are probably rather messy - or you've
>    missed several minor sites? Also, use one of the MAD wavelengths as
>    reference (see above).
>
> 2. how do you know that your Os and Hg derivatives have HAT_OCC of
>    1.0? I would always refine occupancies of soaks.
>
> 3. no anomalous data for the Os and Hg?
>
> I would actually first stick with the 3 wavelength MAD, try to get the
> residual maps clean etc. Then add another CRYSTAL (the in-house data)
> below the exisiting one. A last resort would be to add the Hg/Os
> compounds. But maybe/probably the MAD and/or MAD+native will be
> enough?
>
> Hope that helps
>
> Cheers
>
> Clemens
>
> --
>
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> * Clemens Vonrhein, Ph.D.          vonrhein@GlobalPhasing.com
> *
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--
Paul Hubbard
Dept. of Biochemistry
Medical College of Wisconsin
Phone: 414-456 4305
Fax: 414-456 6510
URL: iris9.biochem.mcw.edu