Can there be some modifications in the inner space - like hydroxyprolines? Hydroxyls might also have a shifted pKa in a specific milieu and perhaps coordinate some metals... Or can there be some chemical cross-linking? The inner space of these bundles is generally "used by Nature" to stabilize the bundles... --- Michal Navrátil, PhD
ne 8. 2. 2026 v 3:31 odesílatel Santelli, Eugenio < [email protected]> napsal: > It does indeed look like you have extra helices sitting along the x > direction, especially in the corners of the channels, each spanning > multiple unit cells, and you see an average density over two directions and > all possible translations. Since each one of them only sees molecules in > the main lattice (except maybe within each individual channel), lowering > the symmetry or processing with a multiple of the unit cell may not help. > > If it really bothers you, you could try to soak the crystals in cryo for a > long time and hope they diffuse out without destroying the crystal. > > On Feb 6, 2026, at 6:12 AM, Catherine Back < > [email protected]> wrote: > > 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 > 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] > > > ------------------------------ > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1__;!!KOmnBZxC8_2BBQ!00ki0e9esRe5ordkP-uVU1groe7WEghUn2SwAkWgLwHjmaSfHOFQloFAVvQ7SXLuLrnDA7mg9y_gyx8BXcvQ04Gv-FJO_HPm2Kxkiw$> > <Screenshot 2026-01-22 at 10.39.07.png><Screenshot 2026-01-27 at 15.10.55 > (2).png><Screenshot 2026-01-27 at 15.12.12 (2).png><Screenshot 2026-01-27 > at 15.13.57 (2).png> > > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1__;!!KOmnBZxC8_2BBQ!00ki0e9esRe5ordkP-uVU1groe7WEghUn2SwAkWgLwHjmaSfHOFQloFAVvQ7SXLuLrnDA7mg9y_gyx8BXcvQ04Gv-FJO_HPm2Kxkiw$> > <Screenshot 2026-02-05 at 12.26.27 (2).png><Screenshot 2026-02-06 at > 12.51.50 (2).png><Screenshot 2026-02-06 at 13.00.02 (2).png><Screenshot > 2026-02-06 at 13.51.40.png> > > > > > IMPORTANT WARNING: This message is intended for the use of the person or > entity to which it is addressed and may contain information that is > privileged and confidential, the disclosure of which is governed by > applicable law. If the reader of this message is not the intended > recipient, or the employee or agent responsible for delivering it to the > intended recipient, you are hereby notified that any dissemination, > distribution or copying of this information is strictly prohibited. Thank > you for your cooperation. > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <https://www.avast.com/sig-email?utm_medium=email&utm_source=link&utm_campaign=sig-email&utm_content=webmail> Virenfrei.www.avast.com <https://www.avast.com/sig-email?utm_medium=email&utm_source=link&utm_campaign=sig-email&utm_content=webmail> <#DAB4FAD8-2DD7-40BB-A1B8-4E2AA1F9FDF2> ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
