Hi, I agree with Rafael, if you’ve grown the plate crystals with MPD, then you should be trying to use an increased amount of MPD as cryoprotectant for those crystals.
I also agree that perhaps the easiest crystals to handle will be the ones grown in PEG 4,000, and you can increase your chances of getting good crystals by seeding. It sounds as though there is no precipitation involved in this condition, and in fact the publication for PDB 3CO3 indicates that they were eventually able to reach 2.17 Å at a later date. However, your cryoprotectant for the crystals grown in PEG 4,000 are not similar to the conditions reported in 3CO3. They report: Crystallization condition: 120 mM Mg(OAc)2, 50 mM sodium cacodylate pH 6.5, 1 mM spermine, 28% (w/v) PEG 4,000. Cryoprotectant condition: 120 mM Mg(OAc)2, 50 mM sodium cacodylate pH 6.5, 1 mM spermine, 30% (w/v) PEG 4,000, 15% (v/v) glycerol. You report: Crystallization condition: 120 mM Mg(OAc)2, 50 mM sodium cacodylate pH ?, 2-12 mM spermine, 15-30% (w/v) PEG 4,000. Cryoprotectant condition: 70% well solution + 30% of 50%(w/v) PEG 4,000 which works out to Cryoprotectant condition: 84 mM Mg(OAc)2, 35 mM sodium cacodylate pH ?, 1.4 - 8.4 mM spermine, 25.5 - 36% PEG 4,000 What you have done with this cryoprotectant formulation is you have REDUCED the salt concentrations while very drastically increased the PEG 4,000 concentration. Many protein crystal lattices would not easily withstand these drastic changes. You’ll note that they do not reduce the salt or buffer concentrations, and only increase the PEG 4,000 concentration by 2%. In addition, by not using glycerol, I would bet that you see ice formation. Try something more along the lines of the published conditions for 3CO3. Diana ************************************************** Diana R. Tomchick Professor Departments of Biophysics and Biochemistry UT Southwestern Medical Center 5323 Harry Hines Blvd. Rm. ND10.214A Dallas, TX 75390-8816 [email protected] (214) 645-6383 (phone) (214) 645-6353 (fax) On Aug 11, 2025, at 9:58 AM, MEURS Daan <[email protected]> wrote: External Mail This email originated from outside of UT Southwestern. Please be cautious. <https://us-phishalarm-ewt.proofpoint.com/EWT/v1/MznTZTSvDXGV0Co!WpxBdbYeK1t9xpzuTNJS71H351rJ74JE68A1dSTSQShDJKMEffWZqB1GmujRaFmhYo2ytOb7TC-GZ3Ahon62zxsRowjKBlJrETqrt3INUIYTb24s0suY9RXJJooutta8sdnyho8fSOzXJeKRa-7PkyY$> Report Suspicious Dear members of the CCP4 BB, I am writing you for help with obtaining diffracting crystals of a dsDNA molecule, complexed to a cytotoxic phenyl alkyl Pt(II) complex. We would like to solve a very similar type of structure as PDB codes 3CO3<https://urldefense.com/v3/__https://www.rcsb.org/structure/3CO3__;!!MznTZTSvDXGV0Co!GikcCUwjpjNE4TaIUgjqNIbqceSeyP6I82-C5rairaVjPKNrBnfUECb2aj5GIfiUOxW7YacU7JcwRUPyMfrLUCp3eWPILJdwWBLKBedfWzBtidY$>, 1LU5<https://urldefense.com/v3/__https://www.rcsb.org/structure/1LU5__;!!MznTZTSvDXGV0Co!GikcCUwjpjNE4TaIUgjqNIbqceSeyP6I82-C5rairaVjPKNrBnfUECb2aj5GIfiUOxW7YacU7JcwRUPyMfrLUCp3eWPILJdwWBLKBedfXTJxCxs$>, and 1IHH<https://urldefense.com/v3/__https://www.rcsb.org/structure/1IHH__;!!MznTZTSvDXGV0Co!GikcCUwjpjNE4TaIUgjqNIbqceSeyP6I82-C5rairaVjPKNrBnfUECb2aj5GIfiUOxW7YacU7JcwRUPyMfrLUCp3eWPILJdwWBLKBedfjF_EXxU$>. We use the exact same DNA sequence (CCTCTGGTCTCC - GGAGACCAGAGG), only our Pt(II) complex is different. Our method is based on these literature sources, and we obtained crystals. However, they are not diffracting. In short, I react pure aquated Pt(II) complex with the single strand containing the central GG. I purify this strand by IPRP-HPLC (Ion Pairing Reversed Phase). I lyophilize the product, redissolve in annealing buffer (100 mM HEPES pH 7, 500 mM LiCl, and 100 mM MgCl2 in fresh mQ) and add the complementary strand in excess. I heat to 95 °C and cool down slowly using a thermocycler. Next, I purify again by IPRP-HPLC, lyo, redissolve in fresh mQ (DNase free), and quantify by nanodrop. In our hanging drop crystallization screen at 4 °C, based on the conditions of the PDB codes above (120 mM Mg(OAc)2, 50 mM NaCaC, 2-12 mM Spermine, 15-30% PEG 4000 OR Drop: 20 mM NaCaco 6 mM Spermine, 2.5-5 % MPD, 10 mM BaCl2, 2.5 % EtOAc equilibrated against well: 25% MPD, 5% EtOAc), we obtain needle crystals with an orange shade or colorless plate crystals. The conditions with MPD yielded the most crystals. Fishing proved to be difficult. The crystals were fragile and surrounded by debris and precipitation, especially for the plates. The crystals were transferred to a cryoprotectant solution containing 70% solution of the well and 30% of 50% PEG 4000. However, we noticed a lot of (external) ice at the synchrotron. Characterizing the crystals by X-rays from a synchrotron (13 keV, T = 8%) shows no diffraction in any of the crystals. Increasing the transmission to T = 50% gave us an artifact: WAXS of the nylon loop. Performing XRF and an energy scan were positive for the presence of Pt (L3). We feel like we obtained crystals of the Pt-DNA complex (Pt positive XRF and Energy Scan), but the molecules are not ordered well, so we don't see diffraction? Do you happen to know any practical tricks/considerations, or do you see where there is room for improvement in our methods? Some other conditions we can try that worked well for you? We are also wondering if we could use the dsDNA without the Pt complex to optimize the crystallization parameters. Could the crystallization conditions of pure DNA sequence be comparable with the Pt-DNA? Is it really a process of trial and error, or could we take some concrete steps with these observations? I am new to macromolecular crystallography, and I appreciate all the help I can get. I thank you in advance for your responses. Please feel free to send me a personal email, and I will be happy to compile the answers in a future overview post. Daan Meurs PhD Student - FNRS | Télévie Université Libre de Bruxelles (ULB), Belgium ________________________________ 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__;!!MznTZTSvDXGV0Co!GikcCUwjpjNE4TaIUgjqNIbqceSeyP6I82-C5rairaVjPKNrBnfUECb2aj5GIfiUOxW7YacU7JcwRUPyMfrLUCp3eWPILJdwWBLKBedfWwinpEQ$> <img-188bb70e-b628-427b-b1d3-336fd3264f38> ________________________________ UT Southwestern Medical Center The future of medicine, today. ######################################################################## 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/
