To evaluate the ability of AMOEBA to model typical intramolecular fields in CypA, we first calculated electric fields present in structural ensembles of the WT, cis-proline, CypA complex (PDB 1M9Y, prepared and simulated as per the Methods). For each simulation frame from the triplicate simulations performed with AMOEBA, the Cartesian x, y, and z components of environmental electric field were calculated at the C and N atoms of the substrate peptide bond. Each atom-centered field was projected along the vector perpendicular to the plane of the peptide proline ring, which reflects the magnitude of the field aligned with the carbonyl group of the peptide bond when rotated during the substrate isomerization. Projections were then linearly averaged to estimate the field magnitude at the peptide bond midpoint (Fig. ). After projection, intramolecular electric field magnitudes ranged from -40 to +40 MV cm -1 (Fig. ), even in these short simulations. However, the distributions overlapped substantially and the mean field strengths from each independent simulation spanned a much smaller range, from -6.9 to -16.8 MV cm -1 . distributions observed for the structures of the AMOEBA Run 1 ensemble (black, identical simulation to Run 1 in panel A), but with fields recalculated using either the Amber (orange), and Charmm (blue) force field. In both panels, ticks on the x-axis denote the mean of each distribution Part of the variation in field strength originates from the projection of the Cartesian field components along a vector perpendicular to the proline ring at each timestep. The direction of this vector is not identical for every MD snapshot, as torsional motions and out-of-plane bending subtly affect the conformation of the substrate proline ring. Thus, even if the CypA environment provided a constant electric field, a distribution of field strengths would be observed owing to the conformational fluctuation of the peptide. To interrogate the effects of substrate conformational fluctuation, for the first AMOEBA replicate we visualized the Cartesian components of the vector perpendicular to the proline ring, and compared them with the Cartesian components of the calculated electric fields at the substrate peptide N and C atoms (Movies S1 and S2). Even after structural superimposition of the frames, the electric field vectors were distributed across a wide range of orientations. However, the vectors perpendicular to the proline ring remained in a compact distribution of orientations, indicating that intramolecular fluctuations of the substrate proline were not the dominant source of the observed variance in electric field.