We study several free energy calculation methods in the dissociation process of lysozyme and its antibody. We introduce the multi-step targeted molecular dynamics (mTMD) method to determine the dissociation pathway. The dissociation free energy calculated along the mTMD dissociation pathway is significantly lower than that along the dissociation pathway determined by the steered molecular dynamics (SMD) method. This indicates that SMD leads to a meta-stable dissociation state. While the SMD restrains the distance between the two proteins, the mTMD restrains the internal structures additionally. We discuss the effect of fragility of the protein structures on the free energy calculations. (C) 2014 Elsevier B.V. All rights reserved.