Hi Daan, your project shouts "use random microseeding!!" You should make a seedstock by crushing those crystals in the solution that they grew in. Then you should add them to several random screens. The conventional volumes to use are 300 nL macromolecule, 200 nL screen, and 100 nL seedstock. If you're working by hand you can increase the volumes but keep the 3: 2: 1 ratio. You'll probably find several new crystallization conditions if you do that, and they're more likely to be in the metastable zone of the phase diagram, which will give you more control and increase the chance that they will diffract well. Bear in mind that you're doing an ADDITIVE experiment at the same time as a seeding experiment because roughly one-third of the precipitant in your drops comes from the seedstock - you are adding something to all your wells that has already crystallized your DNA.
I've pasted a couple of very good papers on this method below. Matthew, MPD isn't very volatile - its boiling point is 197°C. However, if you do have trouble with evaporation, Daan, you should move to a microbatch-under-oil setup. You put diluted MPD in the reservoir of a Vapor Batch plate, which will diffuse through the oil and equilibrate with the drop. Then you can harvest from microbatch without the MPD evaporating - the method works very well with really volatile alcohols such as isopropanol. Let me know if you'd like some sample Vapor Batch plates. Good luck with the project! Patrick _____________________ D'Arcy, Allan, Frederic Villard, and May Marsh. "An automated microseed matrix-screening method for protein crystallization." *Biological Crystallography* 63.4 (2007): 550-554. https://doi.org/10.1107/S0907444907007652 Obmolova, Galina, et al. "Protein crystallization with microseed matrix screening: application to human germline antibody Fabs." *Structural Biology and Crystallization Communications* 70.8 (2014): 1107-1115. https://doi.org/10.1107/S2053230X14012552 This is how we make the seedstock - there are some common mistakes, so we recommend this: https://www.douglas.co.uk/MMS_proc.htm Harvesting with volatile alcohols - presentation from Lesley Haire, who won our competition: https://www.douglas.co.uk/winner1.htm On Mon, Aug 11, 2025 at 9:01 PM Matthew Snee < [email protected]> wrote: > Hi > > if you can get the MPD concentration in the screen up a bit you should not > need additional Cryo, if you find that it causes too much precipitation, > you can try seeding into large drops with an asymmetric mixing ratio (I’ve > found this can be useful for getting larger crystals but you have to > carefully titrate the seed stock). > > Also, be aware that MDP is volatile, so the drop conditions will change > rapidly once the well is unsealed especially if you are trying to harvest > at warmer temperatures. > It’s possible that this was what destroyed the diffraction (or the PEG). > > Best wishes > > Matthew. > > > > > Sent from Outlook for iOS <https://aka.ms/o0ukef> > ------------------------------ > *From:* CCP4 bulletin board <[email protected]> on behalf of MEURS > Daan <[email protected]> > *Sent:* Monday, August 11, 2025 3:58:00 PM > *To:* [email protected] <[email protected]> > *Subject:* [ccp4bb] Pt-DNA dodecamer crystallization issues > > 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 > [rcsb.org] > <https://urldefense.com/v3/__https://www.rcsb.org/structure/3CO3__;!!PDiH4ENfjr2_Jw!GhtQuFyljouJz6zpLwcENCc3mPA6-rFM0ZKZu_m7bIJXozQDhLVxoUTaX3BierHMpwxp2KgyeJAICS17UFfNWv8w8LYeAzc0W95lZZidU6e3lA$>, > 1LU5 [rcsb.org] > <https://urldefense.com/v3/__https://www.rcsb.org/structure/1LU5__;!!PDiH4ENfjr2_Jw!GhtQuFyljouJz6zpLwcENCc3mPA6-rFM0ZKZu_m7bIJXozQDhLVxoUTaX3BierHMpwxp2KgyeJAICS17UFfNWv8w8LYeAzc0W95lZZjJnPIq2g$>, > and 1IHH [rcsb.org] > <https://urldefense.com/v3/__https://www.rcsb.org/structure/1IHH__;!!PDiH4ENfjr2_Jw!GhtQuFyljouJz6zpLwcENCc3mPA6-rFM0ZKZu_m7bIJXozQDhLVxoUTaX3BierHMpwxp2KgyeJAICS17UFfNWv8w8LYeAzc0W95lZZhfxing8w$>. > 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 > [jiscmail.ac.uk] > <https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1__;!!PDiH4ENfjr2_Jw!GhtQuFyljouJz6zpLwcENCc3mPA6-rFM0ZKZu_m7bIJXozQDhLVxoUTaX3BierHMpwxp2KgyeJAICS17UFfNWv8w8LYeAzc0W95lZZjIHMVcUQ$> > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > -- [email protected] Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Patrick Shaw Stewart, Peter Baldock, Stefan Kolek http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 ######################################################################## 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/
