Amatoxins are strong inhibitors of RNA polymerase II, and cause cell death. Because of their cytotoxicity they are candidates for anti-cancer drugs, and understanding their structure-activity relationship is crucial. Amatoxins have a rigid bicyclic scaffold which consists of a cyclic octapeptide bridged by cysteine and tryptophan side chain forming a tryptathionine bridge. Here we show the influence of the N-methylation on the amatoxin scaffold by studying Gly5Sar-amanullin with MD simulations and NMR experiments. Since we have shown recently that the amatoxin scaffold allows for two isomeric forms (ansamers), we studied both isomers of Gly5Sar-amanullin. We found that both isomers of Gly5Sar-amanullin form two long-living conformations which is unusual for amatoxins, and that they are differently affected by the N-methylation. The natural Gly5Sar-amanullin forfeits the hydrogen bonds to Gly5 due to the N-methylation, which is expected from existing crystal structures for alpha-amanitin. Our results however indicate that this does not cause more flexibility due to a shift in the hydrogen bond pattern. In the unnatural isomer, we observe an interesting cis-trans-isomerisation of the backbone angles in Trp4 and Gly7, which is enabled by the N-methylation. We expect that our perspective on the effect of N-methylation in amatoxins could be a starting point for further SAR-studies which are urgently needed for the design of better anti-cancer agents.