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yes, at lowish resolution considering potential H-bonds is often the only way to orient the His, but also Asn and Gln side-chains correctly. Molprobity (http://molprobity.biochem.duke.edu/) and other programs also check this for you, suggesting side-chain "flips" where necessary or advised. And Coot has a special "side-chain 180º flip" function" for you to flip them easily. At higher resolution you can look at the refined B-factors, if the more electron-rich atoms (N in His, O in Asn/Gln) have a much higher B than the less electron-rich ones (C in His, N in Asn/Gln), this can also mean the side-chain needs flipping. At even higher solution you may see difference density in wrongly oriented His/Asn/Gln side-chains. Mark J van Raaij Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 20 May 2015, at 14:10, Smith Liu wrote: > Dear All, > > Suppose the protein crystal resolution is about 2-3A, then in the map it should be rather difficult to distinguish the C and N in the sidechain of HIS. In this way we may regard the sidechain of HIS is flippable. But suppose in one flipped conformation of the HIS, the free N in the sidechain of HIS can form H-bond with the neighbour H of the OH of the Thr, in the other flipped conformation of the HIS, the free N in the sidechain of HIS cannot form H-bond with the neighbour H of the OH of the Thr (caused by distance issue). > > Suppose whether the H-bond forms between the free N in the sidechain of HIS and the neighbour H of the OH of the Thr was very important biologically, in this situation how can we distinguish whether the H-bond forms? > > Smith > >