Using the NetMHCpan predictor to cluster SLA-1*0401 and representative molecules of 12 human HLA supertypes according to predicted peptide-binding specificities, the SLA-1*0401 specificity closely resembled that of HLA-A*01:01 (IEDB, http://www.immuneepitope.org/MHCalleleId/142, accessed March 9th 2011). This result was also obvious from an inspection of the PSCPL analysis of the SLA-1*0401. The PSCPL analysis of the P9 specificity of the SLA-1*0401 and the two chimeric molecules suggested that the P9 specificity primarily was determined by the α 1 domain. This contention was further strengthened by a NetMHCpan-driven in silico analysis of the residues delineating the F pocket, which interacts with P9. This suggests that NetMHCpan can be used to design and interpret detailed experiments addressing the structurefunction relationship of peptide-MHC-I interaction. In the case of SLA-1*0401, NetMHCpan suggests that Y74, G77, and S97 play a prominent role in defining the P9 F pocket. Whereas the NetMHCpan readily captured the P9 anchor residue of SLA-1*0401, it did not capture the P3 anchor (at least not in the 2.4 version). We surmise that this shortcoming is due to insufficient examples of the use of P3 anchors within the currently available peptide-MHC-I binding data. Inspecting the pseudo-sequence of SLA-1*0401 and HLA-A*01:01 vs. HLA-A*11:01 suggests that the presence of an arginine in position 156 might Fig. 8 Comparison of specific in silico mutations of the SLA-1*0401 molecule and comparison with the two HLA molecules: HLA-A*11:01 and HLA-A*01:01. The distance between any two MHC molecules and the consensus tree is calculated as described in "Materials and methods". All branch points in the tree have bootstrap values of 100%. The SLA-1*0401 mutations are indicated as Y74D, G77D, and S97I, where the letter before the position number indicates the SLA-1*0401 single letter residue and the letter after indicates the HLA-A*11:01 residue. YGS-DDI is the corresponding triple substitution. Sequence logos are calculated and visualized as described in Fig. 7 . The axis of the LOGOs indicates in all case positions one through nine of the motif, and the y-axis the information content (see Materials and methods) explain the preference for negatively charged amino acid residues in P3. Future NetMHCpan-guided experiments could pointedly address this question, and the resulting data could complement existing data and be used to update and improve the NetMHCpan predictor.