Presently known flaviviruses belong to three major evolutionary branches: tick-borne viruses, mosquitoborne viruses and viruses with no known vector. Here we present the crystal structure of the Yokose virus methyltransferase at 1.7 angstrom resolution, the first structure of a methyltransferase of a Flavivirus with no known vector. Structural comparison of three methyltransferases representative of each of the Flavivirus branches shows that fold and structures are closely conserved, most differences being related to surface loops flexibility. Analysis of the conserved residues throughout all the sequenced flaviviral methyltransferases reveals that, besides the central cleft hosting the substrate and cofactor binding sites, a second, almost continuous, patch is conserved and points away from active site towards the back of the protein. The high level of structural conservation in this region could be functional for the methyl transferase/RNA interaction and stabilization of the ensuing complex. (C) 2009 Elsevier Inc. All rights reserved.