Hi Careina, I have had similar issues in the past. The crystals of my complex (only protein, but it formed a hexamer and had a very mobile domain which was impairing diffraction) were also big and round in the beginning and had either no or very low diffraction. And I also got many different crystal shapes, though most did not diffract at all. In the end, I made a few changes which I think overall contributed to improving diffraction:
* Go back to the protein purification. I added an ion exchange column (Resource Q). This made the crystals go from round to hexagonal, although I could still see multiple overlaying lattices. The last step should be the size exclusion and be as conservative as possible by collecting only 'the peak of the peak'. The way to thaw your protein may also have an effect. Some proteins prefer a quick thaw, while others prefer to be left on ice until thawed. * Vary crystallization pH or exchange the buffer of your protein to a different pH prior to crystallization. * Try controlled dehydration to see if you can 'nudge' the crystal packing into higher order. * In the end, the crystals that provided the structure for me were not the initial large hexagons, but smaller needles that grew in a condition similar to yours. * You may already have tried it, but it may be worth testing additives. Best of luck to you! Sara ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of careinaedgo...@yahoo.com <000002531c126adf-dmarc-requ...@jiscmail.ac.uk> Sent: 24 November 2023 08:59 To: CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] What could these crystals be? CAUTION: This email originated from outside of the ICR. Do not click links or open attachments unless you recognize the sender's email address and know the content is safe. I wanted to thank everyone for their suggestions and ideas regarding the eyeball shaped crystals that I got at the beginning of the month. I can confirm that these crystals do indeed contain protein and DNA which is good news. I have tried a number of different buffers and salts since then. I have also tried seeding but nothing I have tried has changed the morphology of the crystals. They remain thin, flat and eyeball/pumpkin seed shaped. It is not difficult to make the crystals, they form quite easily under quite a few conditions. The difficulty is in the diffraction. By changing the buffer conditions we can now see some very weak diffraction (previously there was no diffraction at all). Are there any suggestions as to how to improve diffraction of these crystals? I did try different cryoprotectants, parabar, glycerol, PEG but no difference. I think perhaps the problem is heterogeneity considering my sample contains both protein and DNA. Any suggestions or thoughts are welcome. Thank you Careina On Wednesday, November 8, 2023 at 06:18:46 PM GMT+2, careinaedgo...@yahoo.com <000002531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote: The reservior solution is 0.2 M NaCl2, 0.1 M HEPES pH 7.5, and 25% 3350 PEG Protein buffer is 300 mM NaCl, 20 mM HEPES pH 7.5, 3mM TCEP The drop contains 1.5ul protein-DNA complex and 1.5 ul reservior solution On Wednesday, November 8, 2023 at 05:12:25 PM GMT+2, stephen.c...@rc-harwell.ac.uk <stephen.c...@rc-harwell.ac.uk> wrote: Hi Careina, Without knowing what's in your protein buffer or crystallisation condition it's hard to comment. Best wishes, Steve Dr Stephen Carr Research Complex at Harwell (RCaH) Rutherford Appleton Laboratory Harwell Oxford Didcot Oxon OX11 0FA United Kingdom Email stephen.c...@rc-harwell.ac.uk tel 01235 567717 ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of careinaedgo...@yahoo.com <000002531c126adf-dmarc-requ...@jiscmail.ac.uk> Sent: 08 November 2023 15:00 To: ccp4bb <ccp4bb@jiscmail.ac.uk> Subject: [ccp4bb] What could these crystals be? Hi all We have been trying with no success to crystalize a protein. Recently we got these strange shape "crystals". They are hard and flat but they do not diffract at all. Any ideas as to what could cause this? 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