Exposing hidden alternative backbone conformations 1 in X - ray crystallography using qFit 2 3

13 Proteins must move between different conformations of their native ensemble to perform their 14 functions. Crystal structures obtained from high-resolution X-ray diffraction data reflect this 15 heterogeneity as a spatial and temporal conformational average. Although movement between natively 16 populated alternative conformations can be critical for characterizing molecular mechanisms, it is 17 challenging to identify these conformations within electron density maps. Alternative side chain 18 conformations are generally well separated into distinct rotameric conformations, but alternative 19 backbone conformations can overlap at several atomic positions. Our model building program qFit uses 20 mixed integer quadratic programming (MIQP) to evaluate an extremely large number of combinations of 21 sidechain conformers and backbone fragments to locally explain the electron density. Here, we 22 describe two major modeling enhancements to qFit: peptide flips and alternative glycine conformations. 23 We find that peptide flips fall into four stereotypical clusters and are enriched in glycine residues at the 24 n+1 position. The potential for insights uncovered by new peptide flips and glycine conformations is 25 exemplified by HIV protease, where different inhibitors are associated with peptide flips in the " flap " 26 regions adjacent to the inhibitor binding site. Our results paint a picture of peptide flips as 27 conformational switches, often enabled by glycine flexibility, that result in dramatic local 28 rearrangements. Our results furthermore demonstrate the power of large-scale computational analysis 29 to provide new insights into conformational heterogeneity. Overall, improved modeling of backbone 30 heterogeneity with high-resolution X-ray data will connect dynamics to the structure-function 31 relationship and help drive new design strategies for inhibitors of biomedically important systems. 32 33 Author Summary 34 Describing the multiple conformations of proteins is important for understanding the relationship 35 between molecular flexibility and function. However, most methods for interpreting data from X-ray 36 crystallography focus on building a single structure of the protein, which limits the potential for biological 37 insights. Here we introduce an improved algorithm for using crystallographic data to model these 38 multiple conformations that addresses two previously overlooked types of protein backbone flexibility: 39 peptide flips and glycine movements. The method successfully models known examples of these types 40 of multiple conformations, and also identifies new cases that were previously unrecognized but are well 41 supported by the experimental data. For example, we discover glycine-driven peptide flips in the 42 inhibitor-gating " flaps " of the drug target …