Accessing protein conformational ensembles using room-temperature X-ray crystallography

Abstract

Modern protein crystal structures are based nearly exclusively on X-ray data collected at cryogenic temperatures (generally 100 K). The cooling process is thought to introduce little bias in the functional interpretation of structural results, because cryogenic temperatures minimally perturb the overall protein backbone fold. In contrast, here we show that flash cooling biases previously hidden structural ensembles in protein crystals. By analyzing available data for 30 different proteins using new computational tools for electron-density sampling, model refinement, and molecular packing analysis, we found that crystal cryocooling remodels the conformational distributions of more than 35% of side chains and eliminates packing defects necessary for functional motions. In the signaling switch protein, H-Ras, an allosteric network consistent with fluctuations detected in solution by NMR was uncovered in the room-temperature, but not the cryogenic, electron-density maps. These results expose a bias in structural databases toward smaller, overpacked, and unrealistically unique models. Monitoring room-temperature conformational ensembles by X-ray crystallography can reveal motions crucial for catalysis, ligand binding, and allosteric regulation.

Fig. 1.

qFit and Ringer automate the discovery of alternative conformations. Automated qFit (top) and manual Ringer-guided (bottom) model building for CypA into room-temperature electron density reveals the temperature dependence of interconverting side-chain conformations linked to catalysis for CypA. Both procedures identify alternative conformations (colored in red, orange, and gray) for dynamic network residues using the room-temperature X-ray data (residues Leu98, Ser99, Phe113, Met61, Arg55). For some residues (for example Phe60), qFit builds alternative conformations to optimize geometry within a single rotameric substate.

Fig. 2.

Cryocooling changes vibrational amplitudes and conformational distributions. (A)Ringer plots and correlations of electron-density distributions reflect both vibrational and conformational changes induced by crystal cryocooling. Correlation coefficients of Ringer plots based on the room-temperature density (red) and the cryogenic density (blue) in cross-validated maximum-likelihood-weighted, 2mF_o-DF_c maps classify changes. Electron-density levels between 0.3–1σ (shaded green) are enriched in alternative conformations compared to lower electron-density levels (< 0.3σ, shaded gray). (i) Asn106 of asparaginase has a high (0.99) correlation coefficient with reduced thermal motion and no changes to alternative conformations. Intermediate correlations for (ii) Leu98 of CypA and (iii) Ser109 of superoxide reductase reflect differences in the relative populations of alternative conformations (arrows). (iv) The characteristic double peaks for Val53 Cγ1 and Cγ2 of trypsin in the complex with BPTI shift upon cryocooling. The negative correlation coefficient reflects the occupancy of a new rotamer. (B) A cumulative histogram of Ringer correlations for all residues (shaded gray area) and buried residues (black line) reveal that cryocooling alters the distribution of conformational substates. Of all residues, 18.9% have correlations coefficients less than 0.85 (red vertical line), indicating significant changes to χ1 electron-density distribution. (C) A cumulative histogram of the maximum absolute change in rotamer occupancy from qFit models for all residues (shaded gray area) and buried residues (black line) reveal that 37.7% of residues have a 20% or greater change in refined occupancy (red vertical line).

Fig. 3.

Crystal cooling contracts unit cells and proteins and increases packing quality. (A) The distribution of changes in unit-cell volumes and protein volumes reveals asymmetric alterations upon cryocooling. Protein volume decreases (cryogenic/room-temperature ratio < 1.0) are generally smaller than the changes in unit-cell volumes (gray shaded area). This suggests that unit-cell volume decreases (cryogenic/room-temperature ratio < 1.0) result from expulsion of solvent from channels in addition to protein compression. Expansions are created by cooling-induced crystal contacts or subdomain reorientations (Fig. S2). (B) Visualizing room-temperature (red) and cryogenic (blue) packing defects for CypA indicates common defects (indicated by letters) and defects unique to the room-temperature model (indicated by numbers). Defect A is present at both temperatures and is associated with a dynamic loop important for HIV-Capsid binding selectivity. Defect 1 is associated with an alterative conformation of Phe113. Defects 2 and 3 are associated with functional alternative conformations of Leu 98 and Ser99.

Fig. 4.

Cryocooling quenches an intrinsically flexible network in H-Ras. (A) Residues with detectable conformational exchange by NMR (26) (brown, orange, and red) mapped onto the structure of GMPPNP-bound H-Ras (PDB ID code 5P21). Residues colored red were fit to a collective exchange rate, and residues colored brown were too broadened to permit classification into a collective exchange process. Gln61 is thought to position a catalytic water molecule (H₂O^cat) during intrinsic hydrolysis of GTP. (B) Models built into room temperature (red and orange), but not the cryogenic (blue), electron-density maps reveal two conformations of Gln61. Gln61 fluctuates to a catalytically competent conformation resembling the GDP-AlF₃ transition state complex stabilized by GAP binding (magenta). Electron-density maps are shown at 1.0 (blue) and 0.3 (cyan) σ contours. The X-ray datasets are derived from a single study of H-Ras crystals grown in a single condition. (C) Ringer electron-density sampling reveals Gln61 and surrounding residues in the active site display conformational differences between the room-temperature structure (red) and cryogenic structure (blue).

Fig. 5.

Multiple conformations in the active site and allosteric cavity of H-Ras. (A) Room-temperature (red and orange), but not the cryogenic (blue), electron-density maps reveal two conformations of His94. Rotameric switching of His94 may influence the chemical shifts of surrounding residues, explaining how NMR relaxation dispersion signals across helix 3 can arise with minimal changes in backbone geometry. (B) An allosteric cavity (red spheres) at the C-terminal end of helix 3 is present only at room temperature. The cavity is transiently filled by alternative conformations (primary conformation in brown, alternative conformations in orange and gray) that become the major conformation and fill the cavity during crystal cryocooling. (C) Ringer electron-density sampling reveals temperature-dependent, side-chain conformational differences at the allosteric cavity, including alternative conformations of Arg97 and Lys101.