Hi Catherine,
As you said, not all spots were indexed. Maybe lowering your signal/noise could
make the trick. As Alejandro said, your structure has good stats for this given
resolution, but I understand this feeling inside us to model every single
density. Processing in P1 might help, although it might be painful to model all
the density you have. I would also model this "extra-density" with ala,
replacing them with the right amino acid whenever is possible. So, in case
there is proteolysis, you will know which part of the protein they are (if you
still have some crystals, you can collect them and run a SDS-PAGE. You will be
able to see if your sample is homogeneous and, if degraded, you can cut the gel
and send it to mass spec to find out where this degradation is occuring).
Best wishes
______________________________________________________
Rafael Marques da Silva
PhD Student – Structural Biology
University of Leicester
Mestre em Física Biomolecular
Universidade de São Paulo
Bacharel em Ciências Biológicas
Universidade Federal de São Carlos
phone: +44 07861 273773
"A sorte acompanha uma mente bem treinada"
________________________________________________
________________________________
De: CCP4 bulletin board <[email protected]> em nome de Catherine Back
<[email protected]>
Enviado: sexta-feira, 6 de fevereiro de 2026 14:12
Para: [email protected] <[email protected]>
Assunto: Re: [ccp4bb] Crystal pathology diagnosis please
Hi All,
Thank you so much to everyone for all the suggestions so far. In reply:
*
The density appears to form a contiguous gap in the lattice (see image
attached). The channel just looks too big to be real (in my opinion).
*
I didn't loop/freeze the crystals so I can't tell you if they were
soft/fragile, but no one mentioned that to me.
*
Apologies for not mentioning - the res of the initially solved structure = 2.3
Å. R factors for the almost complete structure were 0.22/0.27.
*
I tried forcing it to integrate in P1 in xia2/dials, MR solution was the same.
*
Xtriage (in Phenix - I'm happy to use either software packages!) said all was
fine, bar potentially some ice rings.
*
Zanuda agreed P212121.
*
Matthew's coefficient predicted 3 ish copies in the AU...but wasn't certain.
Though good point about the solvent content - the coiled coils do have low
solvent generally.
*
If you look carefully at the density the 'blue/green' density repeats every
width of the unit cell (34.8 A), so you cannot fit another molecule into the
density, because it is essentially only about a quarter of the actual protein,
just repeated.
*
I did try Modelcraft, which tried to model in bits into the density, but it was
all incorrect - I'm guessing because the density isn't for the whole molecule.
*
Similarly with MR - I tried to force Phaser to find more molecules, but to no
avail.
*
Contour level was probably around 1.
*
I have also looked at the indexing for each of the reprocessing outputs - and
as you say Graeme - not all the spots have been picked (see image).
*
Looking at the lattice viewer of the indexed spots there are a lot of white
crosses and two overlapping patterns? See image attached. Though as you say -
could be ice or another xtal?
[To confuse us even more we did collect data from another crystal (weirdly the
same condition) which had different packing - hexagonal, like the 'missing'
molecules in this one. The res and R factors were really bad though, so more
data was collected. There was no sign of this lattice formation in that data.]
So as you can see this is taking me to (and beyond) the edge of my
crystallography expertise! However, it's good to be challenged every now and
then, and my brain is certainly being stretched.
Cheers,
Catherine
Dr Catherine R. Back (she/her)
Senior Post-doctoral Research Associate
School of Biochemistry
University of Bristol
UK
[email protected]
________________________________
From: Graeme Winter <[email protected]>
Sent: 06 February 2026 13:27
To: Catherine Back <[email protected]>
Cc: [email protected] <[email protected]>
Subject: Re: [ccp4bb] Crystal pathology diagnosis please
In a case like this I would not trust the auto processing without question
(says the original author of said auto processing)
At the very least -
- check over the indexing results - has it caught all the spots or > 90% of them
- look at the indexed and unindexed spots in the reciprocal lattice viewer - if
the latter are unrelated to the former (e.g. secondary crystal, ice, …) then
it’s fine - but could be weak spots between strong ones indicating a larger
cell with near-symmetry
- look at the images with the integration results overlaid - are all the spots
measured? Check at various points in the data set
If all the spots are integrated, then xtriage etc. as others have suggested,
and taking your model and trying refinement in P1 (e.g. pull integrated data
down, scale in P1 or just use zanuda - also another’s suggestion) - also: check
that the symmetry determination (logs should be there) show the P2?2?2? option
as being just as good as the 3 x P2? options - if one P2 option is much better
could be that the true symmetry is different
Essentially looking over all the processing “decisions” with a critical eye
will help you a lot here, happy to help (off list) if you would like more
pointers
All the best Graeme
On Feb 6, 2026, at 5:48 AM, Catherine Back
<[email protected]> wrote:
Morning All,
My colleague and I are trying to solve the structure of a long, alpha helical
bundle protein. We thought it had solved well with 2 molecules in the AU (which
had decent stats), but upon a closer look there are very large 'solvent
channels' in the lattice, with green/blue density within. It looks like there
are protein molecules, positioned lengthways to the molecules in the AU, within
these channels which the autoprocessing (at Diamond Light Source) is not
picking up. See images attached.
I now have the image files and have been reprocessing them using xia2-DIALS in
ccp4i2.
I am unsure what kind of crystal pathology we're dealing with here. There is no
twinning warnings or anything else really that the processing has identified.
The space group is always identified as P212121, unit cell 34.8, 184.8, 202.3,
90.0, 90.0, 90.0.
I've tried quite a few different things to get the software to fit molecules
into the density, including manually extending the unit cell, but all the
outputs have been wrong or had really bad stats. I'm wondering if the indexing
is wrong, but I'm not sure how to sort this. There is clearly something going
on here, which is either really obvious (that I'm not identifying because I'm
an idiot) or it's something more complex.
Any ideas/help will be much appreciated.
Best wishes,
Catherine Back
Dr Catherine R. Back (she/her)
Senior Post-doctoral Research Associate
School of Biochemistry
University of Bristol
UK
[email protected]<mailto:[email protected]>
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