Dear Andy,
for the I222 crystal, you mention a large difference in diffracting
resolution, but staraniso doesn't help. Maybe you simply don't have
anisotropy, but rather lack of completeness in high resolution shells, a
different problem. How much is the anisotropy value given by Phaser?
I got confused by your description of what you have and don't have.
Maybe the simplest would be to build as good as you can in your P3212,
and use this as a MR search for the I222 crystal. Phaser will take care
of things for you.
Your anisotropy is strong but others have managed with these values so
keep faith, you can still get something out of it. Start building in
Calpha, and iterative cycles of manual building and refinement will get
you to a good place. If you have alpha helices, it is much easier than
beta sheets, so focus on those. I wouldn't give up on a 3A dataset, even
with large difference in diffracting resolutions.
Good luck.
Vincent
On 10/01/2019 12:05, Andrew Lovering wrote:
Ok – small oversight – I can see now that phenix has the atoms
placed/saved during cutout stage – so how best to use PHASER output to
shift this file?
Andy
*From:*Andrew Lovering (School of Biosciences)
*Sent:* 10 January 2019 10:48
*To:* 'ccp4bb@jiscmail.ac.uk'
*Subject:* RE: complex multi-crystal averaging
Thanks everyone for the pointers. I should clarify – my issue here is
the multiple stage “disconnect” between PDB in xtal1 -> cutout density
(into a “interim cell” just for this stage) -> phaser MR with density
search model into xtal2
Hence, do I just simply pop the model from (1) into the “interim cell”
(display cutout density, move molecule here in coot, bit of manual
shifting / rigid body, save interim PDB) then use PDBSET on saved PDB
with angles and shifts from phaser output?
To use clear language, “what’s the easiest way to get PDB xtal1 to
match placed density of MR in xtal2” J
Andy
*From:*Andrew Lovering (School of Biosciences)
*Sent:* 09 January 2019 16:08
*To:* 'ccp4bb@jiscmail.ac.uk'
*Subject:* complex multi-crystal averaging
Dear All,
I suspect the way out of this is a new crystal (!) but interested to
hear any advice.
I have two crystal forms of a 500aa protein, vaguely tube-shaped
1=P3121, diffracts to 4.1 ang, 3 copies in asu, 86% solvent; map
indicates it is a relative of other folds (but those are not so close
that they’d be a good guide to sequence register). I have selenomet
SAD phases which helps identify Met positions. The 3-fold and high
solvent give a great map but you wouldn’t want to build it and
buccanner and phenix think similar
2=I222, 2 copies per asu, 66% solvent. This has a cell that gives
wildly anisotropic diffraction – ~3, 3.5, 4.3 down different axes. Not
really rectified by staraniso. No phases
So I can cut the density out of form 1 map (using secondary structure
elements of a rough PDB as a mask), and use phaser with this density
as search model to find the two copies with a TFZ of about 10. The
phaser map shows a bit of detail and the solution has placed the
protomers such that they agree with a self-rotation function. Phenix
find_ncs on phaser map similarly agrees (as I would expect).
Now...I have an eye on multi-crystal averaging between the two forms.
BUT the phaser map isn’t good enough to manually place the PDB used in
cut-out density, and I can’t see a straightforward way of using the
angle info in phaser sol to perform a co-ordinate transformation (but
I think ccp4bb users will put me right on this – I’d imagine placing
the PDB into the cutout density mtz then using PDBset?). I tried
converting the phaser mtz to a map using FFT then using phased TF in
Molrep to place the PDB but this didn’t work, complaining about the
grid used.
One last caveat – I have multiple sets for the I222 that intriguingly
differ by 12 angstrom down a 240 ang axis: doing multi-crystal
averaging between these two forms achieves little when I would expect
otherwise (all the NCS correlations are good, high initial agreement)
Like I said...fingers crossed for a new crystal form!
Andy
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Vincent Chaptal, PhD
MMSB -UMR5086
Drug Resistance and Membrane Proteins Laboratory
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