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]<mailto:[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]<mailto:[email protected]> ________________________________ From: Graeme Winter <[email protected]<mailto:[email protected]>> Sent: 06 February 2026 13:27 To: Catherine Back <[email protected]<mailto:[email protected]>> Cc: [email protected]<mailto:[email protected]> <[email protected]<mailto:[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]<mailto:[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]> ________________________________ 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 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/
