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Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from na.ve libraries for specific targets. However, they often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, a process known as maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analysing the structure-activity relationship using X-ray crystallography, cryo-electron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein-nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with high binding affinity for the spike protein., These Cryo-EM based structural ensembles of spike-nanobody complexes generated by the EMMI method.