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Re: [ccp4bb]: Refmac vs. cns: nucleic acids?
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Hi everybody,
> >
> > I'd be interested in people's humble opinions on their experiences with
> > nucleic acids in particular. Many of the backbone torsion angles aren't
> > well defined in moderate-resolution maps (2.5ish), but they do matter.
> > Especially for non-canonical structures, I've wondered if we're really
> > dealing with them properly.
>
> Since nobody picked up on that yet, here I go ... i hope I will be
> humble enough [;-)]
>
> The word is that XPLOR/CNS is far better than REFMAC on refining nucleic
> acids. REFMAC (.. and PROLSQ back then) make the sugar backbone 'funny'
> according to some, the bases non-planar according to others.
>
> Partially true - I think.
Well, IMVHO (V stands for Very) "partially" is the good adverb here. It was
not the case for me: Refmac (at that time it was Refmac4/Protin) did a very
good job with my DNA structure (a Holliday junction including two GA
mismatches) For a summary, the crystals were quite anisotropic
(diamond-like plates diffracting poorly in the short side direction) and
some of the diffraction spots a little bit splitted, but the data was
reasonable, and we managed to solve the structure by MR and MAD. It was my
first structure (this is why my opinion must be considered VH), so I read
the (friendly?) manual of XPLOR, I asked around and started to refine the
whole thing. But, as you can imagine at least some parts of my structure,
those nucleotides laying in the crossing point, could not easily been
considered as "canonical". I was not able (obviously, not meaning it was
impossible) to completely abolish the torsion restrains on the sugars to
free the puckering (as Tasssos mention in his email) nor to find the right
weights for the so-called "b-restraints" on hydrogen bonding among the
complementary bases (when working at medium resolution, it was some 2.2A in
my case, and with not-so-good data you must use them, otherwise the
force-field tends to separate the bases)
So, I did a first attempt with SHELXL, I read the very friendly manual,
asked a lot of questions to Isabel Usón (thank you!) but I was in the
"twilight zone"... the maps looked somehow better, but my refinement
didn't progress. And then, I found refmac. At the first reading of the
manual, I did not understand hardly a word. But, as Kavafis poetically
mentioned, it was the rough long way which gave Ulysses' life a sense...
The manual did not gave any sense to my own's, but I was forced to learn a
lot more about what I was trying to do.
> For the backbone though there is a point. I vaguely recall the York
> refinement workshop at '97. After some smart participant (Miquel Ortiz
> ?) pointed it out, I recall Eleanor having a look at the all-famous
> PROTIN code around midnight and figuring that 'main chain' could only
> have one chiral center - which is fine with proteins but a bit of a
> disaster with nucleic acids if the ribose is considered the main chain !
> There was a single line fixed that I used ever since but I doubt if that
> ever made it in the wide public.
Yes, it was me (nice from you to remember, Tassos) Eleanor helped me a lot,
and I am in debt with her and with Garib, for their patience and their
suggestions. Following them, and adding some restraints to link, in protin,
one nucleotide to the next one (it was Phil Evans who provided me with,
then I changed them a little bit according to the "standard" DNA
parameters) the refinement with refmac4/protin was quite easy, and needed
not too hard manual rebuilding. BTW, as far as I remember, Eleanor's
protin fix was included in the next release of CCP4.
What at that time I found great in refmac/protin was it flexibility. I know
now that CNS/XPLOR can also be quite flexible, but as Tassos, I am a bit
biased toward refmac. I think it is useful to use as many as possible
approaches to solve a problem, that's why diversity is great. But maybe
this is not the "politically-correct" way in the "high-throughput" times...
Sorry for this long post. I hope not to have been too boring!
Cheers,
Miguel
--
Miguel Ortiz Lombardía | Tel: +33-1 45 68 86 08
Institut Pasteur | Fax: +33-1 45 68 86 04
25, rue du Dr. Roux | email: ortiz@pasteur.fr
75724 Paris Cedex 15 | http://www.pangea.org/ai-cat
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bonhomme. Mais les braves gens n'aiment pas que l'on suive une autre
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