Dear Vito
AMPLE is another option to easily and automatically find conserved cores
among a set of distant homologues. You point it to a directory
containing your homologues and use the
-homologs True
flag. You can use the command line or CCP4i. It will then use GESAMT to
find the multiple structural superposition and then generate a series of
ensemble search models containing 100, 95, 90...% of the superimposable
set of residues shared among the structures. The processing uses
information about structural variance to trim progressively down to
smaller but more structurally homogeneous cores. Very often this further
processing, more dramatic than would be attempted manually, is necessary
for success.
If you only have a single homologous structure we have had some success
using it as the basis to generate ensembles using distance geometry
(CONCOORD) and treating the result in the same way as a set of ab initio
models.
These approaches work best where resolution allows for automated Shelxe
main chain tracing and phase modification since the resulting statistics
very clearly indicate success or failure.
These methods will be submitted for publication in Study Weekend papers.
In the meantime you can hear some explanation here
https://www.youtube.com/watch?v=3B1-Qr00zXk&t=2s
from about 19:37
There's also a multiple homolog tutorial here
https://amplemr.wordpress.com/programs/ample/ample-tutorials/ample-tutorial-3/
Do feel free to contact us if you need a hand trying this approach.
Best wishes
Daniel Rigden
On 22/06/17 16:37, Randy Read wrote:
Yes, I agree that MR is worth a shot, though depending on the
resolution your life may be vastly easier with experimental phases!
We've had several cases where the following kind of procedure works:
find related structures with a very sensitive homology search (we like
HHPRED, though other options probably work), use the corresponding
sequence alignment to prune the models (with sculptor or chainsaw or
other tools) and make a trimmed ensemble with ensembler. The last
step can be very important, in trimming off any bits of the collection
of models that do not constitute a conserved core. Then provide the
ensemble to Phaser as a molecular replacement model. The trimmed
ensemble is usually the best model, in our experience, but one of the
individual models may be better in some cases, so that's also worth
testing.
Also, the MR-Rosetta pipeline has had a substantial number of
successes when the highest sequence identity was in the range of 15-25%.
Best wishes,
Randy Read
On 21 Jun 2017, at 17:08, Mark J van Raaij <mjvanra...@cnb.csic.es
<mailto:mjvanra...@cnb.csic.es>> wrote:
If your data is good enough, your SeMets alone might well be enough.
Soaking native crystals in Hg compounds may also work, avoiding SeMet
altogether. We have had a lot of success with methylmercury chloride
binding to free Cys. You may have to experiment with different
soaking times and protocols.
Finally, don't give up on MR too early, what matters is the
structural similarity, not directly the sequence identity. We've had
success once with 19% identity.
Native protein may be much easier to produce than the SeMet and
SeMet/SeCys versions (and may differ a lot between proteins).
Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
<http://wwwuser.cnb.csic.es/%7Emjvanraaij>
On 21 Jun 2017, at 17:46, Vito Calderone <calder...@cerm.unifi.it
<mailto:calder...@cerm.unifi.it>> wrote:
I am working on a protein having 360 residues. In its sequence there
are 3
Met and 5 free Cys.
I will need MAD to solve the structure since based on the sequence the
closest homologue has 20% identity匢 suppose MR would be very
unlikely to
work卻o I would like to express a selenium derivative to exploit MAD.
Looking in the literature 1 Se-Met every 120 residues seems not to
comply
the threshold to get a good anomalous signal. For this reason I
would like
to exploit both Met and Cys so I would have 8 seleniums per 360
residues.
Could somenone suggest a reference to a protocol to express the double
mutant protein in NON auxotrophic strains of E. coli which you have
experienced working efficiently?
Thanks
------
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: rj...@cam.ac.uk <mailto:rj...@cam.ac.uk>
Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
<http://www-structmed.cimr.cam.ac.uk>
--
Dr Daniel John Rigden Tel:(+44) 151 795 4467
Institute of Integrative Biology FAX:(+44) 151 795 4406
Room 101, Biosciences Building
University of Liverpool http://pcwww.liverpool.ac.uk/~drigden/
Crown St.,
Liverpool L69 7ZB, U.K.
