We had a P1 case with 8 molecules/asu and the crystal diffracted to 2A
resolution. Initial indexing showed that the data could be indexed in P212121
space group but the data could not be merged in this space group (Rsym 60%). P1
was the only space group that we could merge the data (Rsym 10%).
After solving the structure, the major differences we saw in each monomer are
at the N and C termini. They adopt different conformations that prohibit the
molecules to follow the symmetry that you would otherwise have in P212121. The
reference is given below.
Structures of Mycobacterium tuberculosis DosR and DosR-DNA complex involved in
gene activation during adaptation to hypoxic latency.
Wisedchaisri G, Wu M, Rice AE, Roberts DM, Sherman DR, Hol WG. J Mol Biol. 2005 Dec 2;354(3):630-41.
PDB 1ZLJ
George Wisedchaisri
On Thu, 30 Sep 2010, Mark J van Raaij wrote:
we had a P1 case with 12 mols/au, about 30% sequence id, 2.4A resolution,
which was solved after a lot of trials with phaser. Other problems of these
crystals were that they took months to grow and invariable presented multiple
lattices (we did not try microbeam).
See:
Crystallization of the avian reovirus double-stranded RNA-binding and core
protein sigmaA.
Hermo-Parrado XL, Guardado-Calvo P, Llamas-Saiz AL, Fox GC, Vazquez-Iglesias
L, Martínez-Costas J, Benavente J, van Raaij MJ.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 May 1;63(Pt 5):426-9.
Epub 2007 Apr 20. PMID: 17565188
Crystal structure of the avian reovirus inner capsid protein sigmaA.
Guardado-Calvo P, Vazquez-Iglesias L, Martinez-Costas J, Llamas-Saiz AL,
Schoehn G, Fox GC, Hermo-Parrado XL, Benavente J, van Raaij MJ.
J Virol. 2008 Nov;82(22):11208-16. Epub 2008 Sep 17. PMID: 18799570
Quoting Mario Milani <[email protected]>:
Thank you for the suggestions. The data resolution is 1.9 so i can try
different heavy atoms techniques ... anyway i am really puzzled by the
peculiar assembly in the crystal and on the possible causes... does anyone
know about similar cases?
mario
Mario,
beside what you were mentioning, I would definitely try a quick soak
(10-30 seconds) of the crystals in cryo conditions supplemented with
halides such as NaBr, or NaI, at pretty high concentrations (say 0.5 M),
then directly freezing without backsoak.
If the crystals survive the treatment, with that amount of mols per unit
cells and surely some good NCS, you should be able to phase pretty easily.
Well, of course SeMet would be the other option...
ciao,
s
On Sep 30, 2010, at 1:54 PM, Mario Milani wrote:
Dear all,
i have a 30 kDa protein that crystallize so far in three different
conditions but with the same space group. It initially looks like
tetragonal (I4, a=141, b=141, c=208) and then results triclinic (P1,
a=141, b=141 c=144, alpha=119, beta=119, gamma=90), hosting about 24
mol. in the unit cell. Other data: self rotation shows the presence of 4
peaks with chi=180; molecular replacement shows the presence of a
pseudo-translation peak; DLS made at protein concentration close to
crystal growth conditions shows a Rh compatible with something like a
tetramer with low polydispersity (about 15%). Do you have any experience
with similar asymmetric associations? Do you have any suggestions,
beside the addition of ligands to the crystal growth conditions, in
order to get a simpler crystallographic assembly? I have some models
(with sequence identity less than 25%) in order to try MR but all trials
so far did not solve the structure (using balbes, molrep, phaser and
epmr). Any suggestion is welcome.
Thank you,
Mario Milani
--
Sebastiano Pasqualato, PhD
IFOM-IEO Campus
Dipartimento di Oncologia Sperimentale
Istituto Europeo di Oncologia
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Italy
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