Dear Joseph, I fully agree with Daniel Himmel's answer but you might be able to "index" your reflections and get a approximate cell parameters. I did that in the past with crystals that diffracted very poorly up to 15 angst. I used HKL200 at that time, cheated with the distance (HKL2000 won't accept indexing with too low resolution spots) and selected individual spots manually.It was enough for making Matthews analysis for each putative space group.The complex formation between the protein and the RNA was confirmed by a stoechiometric amount of each macromolecule resulting from OD spectra deconvolution taken on dissolved crystals, using a spectrum for the protein alone and a spectrum for the RNA alone as references. Best regards, Philippe Philippe BENAS, Ph.D.
Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS Faculté de Pharmacie, Université Paris Descartes Case 48 Av, de l'Observatoire F-75270 PARIS cedex 06 +33.1.5373.1599 E-mails: philippe.be...@parisdescartes.fr, philippe_be...@yahoo.fr URLs: http://lcrbw.pharmacie.univ-paris5.fr/ , http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18 De : Joseph Ho <sbddintai...@gmail.com> À : CCP4BB@JISCMAIL.AC.UK Envoyé le : Lundi 12 mars 2018 12h54 Objet : [ccp4bb] Strange Diffraction pattern! Protein/DNA complex or DNA alone crystal? Dear all: I would like to seek your wisdom on our latest diffraction pattern. We have been working on protein/DNA complex. The protein and DNA have similar MW. By binding assay, we know the minimal length of DNA. (The Kd is 0.1-1 microMolar and we can see the complex formation in size exclusion chromatography up to 200mM NaCl but also some unbound form) After trying different length of DNA, we recently obtained many crystal hits (the percipient is either PEG400 or MPD). The final ratio (prior to protein crystallization) between protein and DNA is 1:1.6 considering some loss of protein during concentration. The crystal is birefringent. Since high conc. of PEG400 (MPD), the crystals were directly frozen in liquid N2. However, crystals only diffract to 8-10 angstrom (anisotropic) and also weird striking line are present (please see attachment). Do you think if it is DNA alone crystal or protein/DNA complex crystal? How should I improve the diffraction quality? PS. We have done some tests. For example, set up the same conditions with DNA alone. I also tried to dissolve crystals in Bradford assay solution and I believe I saw some blueish color. But none of these tests are conclusive. Thanks for your suggestion. Joseph
