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Dear so-far-posters,
I do not know Tom Van den Bergh, nor do I know his background, nor the
history of the data, nor the reasons why he may have sent it to this
list (although I think he did it to ask for help), but I find these
answers irritatingly disrespectful and nasty.
No regards to the ones addressed,
Tim
On 03/27/2013 06:43 PM, Bosch, Juergen wrote:
> Sorry it has been accepted already, see attachment, it will soon be
> online see the date for that.
>
> Jürgen
>
> ...................... Jürgen Bosch Johns Hopkins University
> Bloomberg School of Public Health Department of Biochemistry &
> Molecular Biology Johns Hopkins Malaria Research Institute 615
> North Wolfe Street, W8708 Baltimore, MD 21205 Office:
> +1-410-614-4742 Lab: +1-410-614-4894 Fax:
> +1-410-955-2926 http://lupo.jhsph.edu
>
> [cid:C2A945C2-7702-4EAE-B6E5-2825754D728C@sph.ad.jhsph.edu] On Mar
> 27, 2013, at 1:30 PM, Steiner, Roberto wrote:
>
> we should all chip in a water molecule or two and the second author
> becomes "the CCP4 community"….
>
> On 27 Mar 2013, at 17:22, Antony Oliver
> <[log in to unmask]<mailto:[log in to unmask]>>
> wrote:
>
> At the risk of being somewhat cheeky - perhaps I could claim second
> author? I too have successfully solved the structure - and I
> totally concur with Phil. Placing a second molecule in the
> asymmetric unit, essentially resolves the perceived R-factor
> problem.
>
> A good thorough manual inspection and rebuilding is *ALWAYS* good
> practice for newcomers to the field.
>
> Tony.
>
>
> On 27 Mar 2013, at 17:14, Petr Leiman
> <[log in to unmask]<mailto:[log in to unmask]>> wrote:
>
> Since this is now public domain knowledge and if this gets ever
> published, Phil has my vote to be the first author!
>
> Petr
>
>
> On Mar 27, 2013, at 6:09 PM, Phil Jeffrey wrote:
>
> That's quite brave - shipping your entire structure to people that
> could be actual competitors. But it was fun to play at 1.4
> Angstrom over lunch.
>
> Practical points:
>
> * not everyone loves 12Mb of attachments in one email in their
> inbox, so if you do this again please put the files on a webserver
> and point us there
>
> Structural points:
>
> * the map looks pretty good, but I think the sequence is
> misassigned in some regions (e.g. A118-A122 etc). Automation is a
> good tool but a poor master, and extreme caution is required before
> taking the results too literally. Usually you'd expect a 1.4
> Angstrom to be easy to autobuild but I recently had a sequence
> misassignment at just that resolution. That map was trivial to
> interpret with the correct sequence however - one of the joys of
> working with Arp/wArp at 1.4 Angstrom.
>
> * the large number of positive difference density blobs and water
> molecules clustered in what otherwise would be the solvent void
> strongly suggest that there's a second molecule present.
>
>
> If I take redfluorescentprotein_refine_10.pdb (waters removed) and
> exptl_fobs_phases_freeR_flags.mtz and ask Phaser to look for two
> molecules, it finds them quite successfully. (for the record an
> LLG of 15111 using nominal sequence identity of 90%). I will send
> this to you off-list. Please note that Phaser is using a different
> origin for this molecular replacement solution so the coordinates
> and your previous map do not overlap.
>
> This rather nicely explains why your structure had an R-factor in
> the 40's despite being a half-way decent model. The new MR
> solution has an R-free in the 30's in the phenix.refine job I'm
> running right now.
>
>
> Going forward I suggest you utilize the Arp/wArp program to
> autobuild your structure for you, starting from the molecular
> replacement solution (or, perhaps with it stripped to ALA). While
> you could use Autobuild, this is the CCP4 list and so you should
> use CCP4 programs.
>
> Phil Jeffrey Princeton
>
>
> On 3/27/13 12:22 PM, Tom Van den Bergh wrote: Dear members of
> ccp4bb,
>
> I need some help with the refinement of my structure of a variant
> of mRFP (monomer red fluorescent protein, sequence in attachment).
> I have done molecular replacement with phaser with model 2VAD of
> protein database. Then i have done some model building
> phenix.autobuild. (2 pdb's (overall...), freeR flags and log file
> attached) When i refine with phenix.refine my structure i get a
> R-value of 0,42 which is still way too high. (redfluorescent
> protein.pdb, .mtz and logfile attached) When i look at the
> structure in coot i find many unmodelled blobs and many outliers in
> density analysis and rotamer analysis. The problem is that there
> are so many problems with my structure, that i dont know where to
> begin. Could you try some refinement for me, because this is first
> structure that i need to solve as a student and i dont have too
> many experience with it.
>
> Greetings,
>
> Tom
>
>
>
> Roberto A. Steiner Group Leader Randall Division of Cell and
> Molecular Biophysics King's College London
> [log in to unmask]<mailto:[log in to unmask]>
>
> Room 3.10A New Hunt's House Guy's Campus SE1 1UL London
>
> Phone 0044 20 78488216 Fax 0044 20 78486435
>
>
>
>
>
- --
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
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