I think the reference of T=8% refers to the strength of the incident X-ray beam, i.e. only 8% of the full-strength beam hit the sample. Increasing the T to 50% for most modern synchrotron beam lines is something that one often does just as a “last gasp” effort to see if there is any diffraction at all.
XRF and an energy scan will be positive for Pt at the L3 edge if there is sufficient Pt compound just in the surrounding buffer/cryoprotectant. Unless you back soaked your sample into a cryoprotectant that had no Pt compound, you wouldn’t know if the compound was bound to the DNA or whether it was just in the beam because some of it was still in the cryoprotectant (or in the solvent channels of the crystal). 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 13, 2025, at 4:58 AM, John R Helliwell <[email protected]> wrote: Unknown Sender You have not previously corresponded with this sender; be cautious of requests. <https://us-phishalarm-ewt.proofpoint.com/EWT/v1/MznTZTSvDXGV0Co!VXxD-bYca_TzZ7EA41PcLf-FlMJqIt8i5DB36cakwQCPsN25kdEVRFpVAuOwzcGSuVlTUcQaWx4FdoEG6q2XBzs-7xh4jhKQhmsphQHpvp2YOQ$> Report Suspicious Dear Daan You have had very pertinent suggestions already. Mine is more of a query:- In your details you mention a value of “T=8% which you increased to T=50%”. If I understand you correctly then your two crystals absorbed 92% and 50% respectively. Both values would be very high levels of absorption of X-rays by your crystal samples. What thickness were those two crystals? Or were you using a beam line absorber, of different attenuation factors, to reduce the incident Xray flux? Best wishes, John Emeritus Professor John R Helliwell DSc On 11 Aug 2025, at 16:08, MEURS Daan <[email protected]<mailto:[email protected]>> wrote: Dear members of the CCP4 BB, I am writing you for help with obtaining diffracting crystals of a dsDNA molecule, complexed to a cytotoxic phenyl alkyl Pt(II) complex. We would like to solve a very similar type of structure as PDB codes 3CO3<https://urldefense.com/v3/__https://www.rcsb.org/structure/3CO3__;!!MznTZTSvDXGV0Co!EWBsX86kaAhLypyZGOzxb_rsvrHg8Y5z8M0cYJdNFreBVHWoPB0NdlQTAdpFEWkpPA2INrLkIjX5LmU_vr14CvqRIk4swhDeew$>, 1LU5<https://urldefense.com/v3/__https://www.rcsb.org/structure/1LU5__;!!MznTZTSvDXGV0Co!EWBsX86kaAhLypyZGOzxb_rsvrHg8Y5z8M0cYJdNFreBVHWoPB0NdlQTAdpFEWkpPA2INrLkIjX5LmU_vr14CvqRIk5LJblzzA$>, and 1IHH<https://urldefense.com/v3/__https://www.rcsb.org/structure/1IHH__;!!MznTZTSvDXGV0Co!EWBsX86kaAhLypyZGOzxb_rsvrHg8Y5z8M0cYJdNFreBVHWoPB0NdlQTAdpFEWkpPA2INrLkIjX5LmU_vr14CvqRIk7zu5b-XQ$>. 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!EWBsX86kaAhLypyZGOzxb_rsvrHg8Y5z8M0cYJdNFreBVHWoPB0NdlQTAdpFEWkpPA2INrLkIjX5LmU_vr14CvqRIk6JAeS0CQ$> <img-188bb70e-b628-427b-b1d3-336fd3264f38> ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1<https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1__;!!MznTZTSvDXGV0Co!EWBsX86kaAhLypyZGOzxb_rsvrHg8Y5z8M0cYJdNFreBVHWoPB0NdlQTAdpFEWkpPA2INrLkIjX5LmU_vr14CvqRIk6JAeS0CQ$> ________________________________ 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/
