The interactions of the 50S subunit of bacterial ribosome with antibiotic sparsomycin (SPS) and five analogs (AN) are investigated through the calculation of the standard (absolute) binding free energy and the characterization of conformational dynamics. The standard binding free energies of the complexes are computed using free energy perturbation molecular dynamics (FEP/MD) simulations with explicit solvent. Restraining potentials are applied and then released during the simulation to efficiently sample the changes in translational, orientational, and conformational freedom of the ligand and receptor upon binding. The biasing effects of the restraining potentials are rigorously removed. The loss of conformational freedom of the ligand upon binding is determined by introducing a potential of mean force (PMF) as a function of the root-mean-square deviation (rmsd) of the ligand relative to its conformation in the bound state. To reduce the size of the simulated system, the binding pocket of the ribosome is simulated in the framework of the generalized solvent boundary potential (GSBP). The number of solvent molecules in the buried binding site is treated via grand canonical Monte Carlo (GCMC) during the FEP/MD simulations. The correlation coefficient between the calculated and measured binding free energies is 0.96, and the experimentally observed ranking order for the binding affinities of the six ligands is reproduced. However, while the calculated affinities of the strong binders agree well with the experimental values, those for the weak binders are underestimated.