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://www.rcsb.org/structure/3CO3>, 1LU5<https://www.rcsb.org/structure/1LU5>, and 1IHH<https://www.rcsb.org/structure/1IHH>. 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 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/
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