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Dear Wei, to get more information on this putative “butterfly-like” ligand, you can perform non-denaturing and denaturing mass spectrometry. This should give you the molecular weight of this compound, which most probably comes from E. coli and co-purifies with your protein of interest. In parallel, you could imagine to purify this compound by HPLC purification. Based on the shape and the details on the density, it might be rich in aromatic rings and then reversed-phase column might be useful. If you end up with enough material, you could try NMR to get info on this ligand. Before performing such experiments, you should definitely check whether this ligand binds to a conserved region of your protein such as a putative active site. If this molecule binds to a region formed by poorly conserved residues, you have to consider whether this is important or not to know precisely the identity of this compound. I mean biologically important. Ideally, this would of course be important to model the correct ligand in the electron density but I am sure it will not be the first structure deposited at the PDB with unattributed density or an unknown ligand. HTH, Best wishes, Marc > Le 29 nov. 2016 à 09:26, Wei Liu <[log in to unmask]> a écrit : > > Hi all, > > Thanks for all answers and suggestions to my question regarding the density for an unknown ligand. Many people raised a possibility that the density corresponding molecule might locate at a two-fold symmetry axis, but I am quite sure to rule out this possibility. I tried to placed a molecule of biphenyl sulfide, which can roughly fit into one half of the density in question, and performed one round of refinement. As shown in the attached picture, strong residual density still exist in the rest half, where no symmetry atoms can be seen. So apparently a real molecule rather than an artifact lied in this structure, but I am still wondering from where such a molecule comes and strongly binds to our protein, E. coli or impurities in the crystallization conditions, as Prem suggested, and if there is any experimental methods to identify this compound. > > Looking forward to more suggestions. > > Best > Wei > > <Snapshot20161128.png> Marc GRAILLE, PhD Directeur de recherche CNRS Laboratoire de Biochimie ECOLE POLYTECHNIQUE - UMR7654 CNRS 91128 PALAISEAU CEDEX Phone : +33 (0)1 69 33 48 90 – Fax : +33 (0)1 69 33 49 09 Email: [log in to unmask] Team: Translation and degradation of eukaryotic mRNAs https://portail.polytechnique.edu/bioc/recherche/couplage-entre-la-traduction-et-la-degradation-des-arnm-chez-les-eucaryotes Team supported by the ATIP-Avenir CNRS program