Hi Kevin,

A pragmatic way to solve the problem is to index your data in P1 if you have 
the unmerged data, solve the structure, then revert to the correct space group 
for refinement. Yes you will have to find 8 copies of your dimer instead of 
two, but that is not such a big problem, I imagine. You may need to do rigid 
body refinement too, to be on the safe side, although Phaser does that. I would 
say your residuals are reasonable for the resolution you quote, but lower 
numbers would be preferable. The poor density for the second copy would be 
perhaps due to the approximate location. But if you persist with refinement, 
you might improve on the situation. Also, the packing pattern might show that 
the second copy has fewer lattice contacts, causing this 'disorder', something 
you cannot avoid.

The Patterson peak at 0.5, 0, 0.034 means the second copy is shifted by 2.5A 
from the ideal position along Z (.034 x 147.2= ~5A, which is the separation in 
Patterson space, but is halved for real space). From the other Patterson peak, 
the centre of gravity of the first solution would be at 0.25, 0 or 0.5, 0.065 
(fractional coordinates), and the second would be at 0.75, 0.5 or 0.0, 0.60. 
You might see weak fringes in the diffraction pattern if you view it in 
VIEWHKL, with the l axis in the display plane. The 5A modulation means the weak 
fringes in the even layers appear around l=30 or 60 (you can't see the 90th 
order as it is beyond the resolution limit), and in the odd layers they will be 
at ~15 and ~45 (order 75 might be on the edge of the pattern). Also, the N(z) 
plot in TRUNCATE should show a distinct shift of the observed curves to the 
left of the theoretical curves. I had a similar situation way back when, 3DZW, 
which I never got to publication despite the coordinates release, but the 
diagnosis followed the above.

Good luck.

Dr Pierre Rizkallah, Senior Lecturer Structural Biology
Institute of Infection & Immunology, Wales Heart Research Institute,
School of Medicine, Heath Campus, Cardiff, CF14 4XN
email: rizkall...@cardiff.ac.uk
From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Dr. Kevin M Jude
Sent: Monday, November 13, 2023 10:23 PM
Subject: [ccp4bb] How to describe/correct modulated diffraction?

External email to Cardiff University - Take care when replying/opening 
attachments or links.
Nid ebost mewnol o Brifysgol Caerdydd yw hwn - Cymerwch ofal wrth ateb/agor 
atodiadau neu ddolenni.

tl;dr: My data has native Patterson peaks but no tNCS; I can solve the 
structure and refine to reasonable R/Rfree, but much of the map is poor quality.

More details:
I have crystallized a heterodimer of Z domains (small three-helix bundles, like 
pdb id 8DA3) in a few different crystal forms that all diffracted in the 2-2.5 
Å resolution range, though in different space groups and different asymmetric 
unit volumes. I'll focus on the most successful crystal, space group C2221 a = 
39.9 b = 74.8 c = 147.2 Å. All of these data sets have native Patterson peaks 
(for the crystal in question, there are peaks at [0.5, 0, 0.034] and [0.127, 0, 
0.131] that are both 23% of the origin height) but tNCS search mode in phaser 

Turning off tNCS and searching separately for chain A and chain B of the 
heterodimer, I am able to find two heterodimers - that are not related by 
simple translation. TFZ for the first heterodimer is 14.6, which decreases to 
13.1 after finding the second heterodimer, though LLG increases. Electron 
density for the first heterodimer (AB) is very good, with easily identifiable 
side chains. Density for the second heterodimer (CD) is pretty dodgy; though 
some side chains are identifiable, chain D has tube-like helices. Using NCS 
restraints, I'm able to refine to Rfree/Rwork 0.27/0.24, but can only place 3 
water molecules (with 240 protein residues in the ASU).

All of this suggests to me that there must be some kind of modulation of my 
reflection intensities, though nothing is apparent to me in studying raw or 
pseudoprecession images. If possible I'd like to figure out how to correct it, 
but barring that I would be happy to describe it and possibly relate it to my 
structure. I'm at a loss for the next step, though, and would appreciate any 
advice from the community on how to approach this.

Best wishes to all,
Kevin M. Jude, PhD
Structural Biology Research Specialist, Garcia Lab
Howard Hughes Medical Institute
Stanford University School of Medicine
Beckman B177, 279 Campus Drive, Stanford CA 94305


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