Dear Daan,
You can try to optimize your crystals by multiple rounds of seeding. Also,
what's the cryoprotectant you use? I hope it is not PEG 4000. But in short, it
might be that your crystal condition does not diffract. I would screen your
sample with different crystallization kits before going to hanging drop.
Best wishes
______________________________________________________
Rafael Marques da Silva
PhD Student – Structural Biology
University of Leicester
Mestre em Física Biomolecular
Universidade de São Paulo
Bacharel em Ciências Biológicas
Universidade Federal de São Carlos
phone: +44 07861 273773
"A sorte acompanha uma mente bem treinada"
________________________________________________
________________________________
De: CCP4 bulletin board <[email protected]> em nome de MEURS Daan
<[email protected]>
Enviado: segunda-feira, 11 de agosto de 2025 15:58
Para: [email protected] <[email protected]>
Assunto: [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<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
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