Research Article: Identifying dynamic, partially occupied residues using anomalous scattering

Date Published: December 01, 2019

Publisher: International Union of Crystallography

Author(s): Serena Rocchio, Ramona Duman, Kamel El Omari, Vitaliy Mykhaylyk, Christian Orr, Zhen Yan, Loïc Salmon, Armin Wagner, James C. A. Bardwell, Scott Horowitz.


Structural studies of partially occupied, heterogeneous protein systems using crystallography are difficult. Here, methods are presented for detecting these states in crystals.

Partial Text

In crystallography, anomalous scattering is commonly used to help solve the phase problem (Hendrickson, 2014 ▸). A second, less well utilized aspect of anomalous scattering is its ability to selectively label and identify residues of interest. Crystallo­graphers can use anomalous maps to pinpoint metal ions (Handing et al., 2018 ▸) or to aid in model building or electron-density interpretation (Pflug et al., 2012 ▸; Wang et al., 2012 ▸). We recently introduced a method called residual electron and anomalous density (READ) that uses anomalous maps to allow the reconstruction of highly heterogeneous conformational ensembles in regions of the protein that are not sufficiently well ordered for traditional model building (Salmon et al., 2018 ▸; Horowitz et al., 2016 ▸). This method uses selective anomalous labeling with iodophenylalanine (pI-Phe) to generate multiple partially occupied iodine anomalous signals in the crystal corresponding to different protein conformations. Ensemble-selection techniques (Venditti et al., 2016 ▸; Salmon et al., 2018 ▸; Horowitz et al., 2016 ▸) are then used to create ensembles that are consistent with both the anomalous data and weak electron-density data for the disordered segment(s) of the crystal. The Im7 ensembles generated by READ were highly consistent with orthogonal experimental approaches that were also used to characterize Im7 binding and folding with Spy, including NMR spectroscopy and thermodynamic and kinetic analyses (Salmon et al., 2018 ▸; Horowitz et al., 2016 ▸; Koldewey et al., 2016 ▸; Stull et al., 2016 ▸; He & Hiller, 2018 ▸; He et al., 2016 ▸).

Our new, optimized data-collection strategy provides an improved method to obtain high-quality anomalous signals with less noise contamination; it also verifies the presence of several of the iodine anomalous signals identified in our previous data sets. Combined, the experiments demonstrate that the Im7 peptide binds to Spy in multiple different binding poses, which are detectable using anomalous scattering. Refining the occupancies of the iodines suggests that we are able to detect iodine anomalous signals at as low as ∼12% occupancy, even with a high temperature factor of 80 Å2. These low-occupancy states were confirmed via reproducibility through independent data collections at multiple angles and using separate crystals. The iodine positions are plausible, and on average are located around 3.25 Å from the closest atom (Supplementary Table S2). READ selections demonstrated that the new L19pI-Phe data produced a consistent ensemble with previous efforts, while the consistency of the L18pI-Phe and K20pI-Phe data could not be assessed properly with our initial sampling.




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