Dear all,
Thank you for your response.
I think, Phoebe is right. I should add some more information, maybe it
will be useful:
- the molecule of interest is a single DNA strand 11mer with sequence
5'-GGTGGGGGTGG-3', what theoretically can formed G-tetraplex, but this
kind of structure can be formed by one, two, or four molecules,
- crystals was obtained after long time (almost three years, now I will
try to reduce this time) from conditions contained CaCl2, PEG400, with
addition of spermine. Crystals are long, thin needles. I collected two
useful datasets:
1) 200 x 0.5 deg, above 1.00A, processed in I222/I212121 with cell
dim. (20.64, 22.95, 43.36, 90, 90, 90)
2) 720 x 0.5 deg, 1.10A, but only first 640 img are useful, the rest
are blank.
I tried reprocess data to other proposed lower symmetry SG: C2 and P1,
but I observed that Rmeas goes strongly up, with cut-off of useful
resolution and completeness,
- there is no evidence of twinning, but tNCS is present. Output from
phenix.xtriage is below:
Frac. coord. : 0.000 0.500 0.000
Distance to origin : 11.475
Height relative to origin : 52.460 %
p_value(height) : 4.848e-05
- I tried shelxt on this I222 dataset - obtained SG is Ibca as output
- I back to images. I tried to index it again under iMOSFLM. Of course,
the most probably solutions are identical with those from XDS, but
predictions don't cover all spots on images. In addition I observed two
or three spots in the line on some images, with short distances between
them, so for me it can suggests much longer cell dimension in one
direction. Maybe there is an explanation. But when I tried add some
spots manually, usually iMOSFLM couldn't estimate cell parameters...
Best regards,
Rafal
---
|----------------------------------------------|
|Rafal Dolot, Ph.D. |
| |
|Polish Academy of Sciences |
|Centre of Molecular and Macromolecular Studies|
|Department of Bioorganic Chemistry |
|Macromolecular Crystallography Team |
|Sienkiewicza 112 |
|90-363 Lodz, Poland |
|Phone: +48(42)6803215 |
|Cell: +48 502897781 |
|----------------------------------------------|
W dniu 2018-04-21 23:02, Phoebe A. Rice napisał(a):
> Sadly Rafal is right the unit cell dimensions don't make sense -
> either the space group is wrong or the contents have been digested
> before crystallization.
> The size of the overall unit cell is ~21 x 23 x 43. Given a
> (DNA-centric but close enough) view that a duplex is ~25A wide and
> there are 3.4A per bp (and 43A/3.4 gives 12bp) , if this 11mer was a
> simple symmetric duplex, there could only be 2 chains, max, but I222
> has 8 asymmetric units.
>
> This handy tool gives the same answer in a more accurate manner:
> http://csb.wfu.edu/tools/vmcalc/vm.html
> It says that if the RNA is intact and the space group is P2, there is
> about 56% solvent. If the space group is really I222, the solvent
> content would be a rather fascinating negative 75%.
>
> -Phoebe
>
> On 4/20/18, 2:37 PM, "CCP4 bulletin board on behalf of Randy Read"
> <[log in to unmask] on behalf of [log in to unmask]> wrote:
>
> I think that starting with a direct methods program like shelxt
> would be fun. If that doesn’t work, it could be interesting to try to
> solve it by molecular replacement with fragments varying from a
> tetraplex, a base pair or even just single bases. (Assuming that
> Phoebe’s concern about twinning does not turn out to be correct…)
>
> Best wishes,
>
> Randy Read
>
> -----
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical Research Tel: +44 1223 336500
> Wellcome Trust/MRC Building Fax: +44 1223
> 336827
> Hills Road
> E-mail: [log in to unmask]
> Cambridge CB2 0XY, U.K.
> www-structmed.cimr.cam.ac.uk
>
> > On 20 Apr 2018, at 20:09, Tim Gruene <[log in to unmask]> wrote:
> >
> > Dear Rafal,
> >
> > shelxt does not require the space group, it only needs the Laue
> group. If it
> > finds a decent solution, it'll find also the space group for you.
> >
> > Best,
> > Tim
> >
> > On Friday, April 20, 2018 3:30:36 PM CEST Rafal Dolot wrote:
> >> Dear CCP4BB,
> >>
> >> I've recently collected data for 11mer build of DNA (9xG, 2xT).
> XDS, and
> >> DIALS gave me similar solution - SG: I2(1)2(1)2(1) or I222, with
> cell
> >> dimension 20.65, 22.96, 43.37, 90, 90, 90, what is too small for
> this
> >> size of the molecule. 11mer is rich in G, so we expect the
> G-tetraplex
> >> formation. Data were collected to almost 1 A, so it should be
> enough for
> >> trials with direct methods/ab initio solution. What I should do
> first to
> >> find correct SG and/or cell parameters?
> >>
> >> Best regards,
> >>
> >> Rafal
> >
> > --
> > --
> > Paul Scherrer Institut
> > Tim Gruene
> > - persoenlich -
> > OSUA/204
> > CH-5232 Villigen PSI
> > phone: +41 (0)56 310 5297
> >
> > GPG Key ID = A46BEE1A
