Hi,
In the past I had two cases where seemingly unsuccessful MR became
successful simply by collecting the missing cusp, which is due to exist
in your P1 case if you collected your data by rotation of the crystal
around a single orientation.
However, I don't know if modern MR programs use techniques that overcome
that problem.
Carlos
G. Sridhar Prasad wrote:
It will be useful if you share the unit cell dimensions, may be it
belongs to a higher symmetry, given the low resolution, you may have
missed it out.
Sridhar
From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of
Eugene Valkov
Sent: Monday, January 20, 2014 6:37 AM
To: [email protected]
Subject: Re: [ccp4bb] Phasing with Many Monomers/AU
What is the sequence identity of your best search model? Finding that
many copies in P1 with 3A data is a challenge but certainly not
impossible if there is a reasonably close (>20-25% identity) search
model available. I would suggest spending some time on preparing a
very good search model with tools like Sculptor as well as manually
trimming loops and using ensembles of conserved folds from several
homologous structures.
It is also worth remembering that there are a number of different
programs available for molecular replacement and it is worth investing
some time in learning how to use Molrep and Amore as well as Phaser as
they all have different strengths and weaknesses.
Is your data otherwise devoid of any other problems like
pseudo-translational symmetry? These can be readily identified with
tools like phenix.xtriage. PST can complicate matters quite
considerably in molecular replacement.
SAD phases, even if obtained at low-resolution, can still be very
useful if combined with molecular replacement, so it is well worth
pursuing all lines of attack simultaneously.
Hope this helps.
Eugene
On 19 January 2014 19:30, Chris Fage <[email protected]
<mailto:[email protected]>> wrote:
Thank you all for your responses. I already have a few ideas about
how to approach the problem.
One of my concerns with so monomers per asymmetric unit at lower
resolution was the failure of MR software. Neither PHENIX nor
Phaser MR have made progress. I am fairly new to anomalous
methods, having solved only two structures by SeMet-based SAD.
I've certainly picked up on a number of tricks from the recent
messages on heavy atoms, but I thought my case might be a little
unusual. I am confident the space group is P1, as it was the only
viable option when I indexed four clean albeit low-res datasets.
The monomers are ~38 kDa, and the crystals diffracted to 3.4-3.0
at a synchrotron.
The conditions for both native and SeMet crystals are:
8-12% PEG 2000 MME, 0.2 M ammonium sulfate, 0.1 M sodium acetate
pH 5.5.
Macromolecular seeding of native crystals into SeMet drops yields
the needle-like crystals.
Any further input is greatly appreciated!
Regards,
Chris
On Sat, Jan 18, 2014 at 11:14 AM, Chris Fage <[email protected]
<mailto:[email protected]>> wrote:
Hello Everyone,
I am currently trying to phase a structure with an asymmetric
unit predicted to contain 20-24 monomers (space group P1). The
native crystals, while beautiful in appearance (see attached),
only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived
crystals grow with poor morphology (small needles). Also,
based a fluorescence scan, I know that mercury does not bind
appreciably. Other than screening for a new space group, what
options might I have for phasing this many monomers at lower
resolution? Is there any real chance of solving the structure
in this space group?
Thank you in advance for any suggestions!
Regards,
Chris
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MRC Laboratory of Molecular Biology
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