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

Area of Science:

• Structural Biology
• Biophysics
• Computational Biology

Background:

• X-ray crystallography typically uses cryogenic temperatures (100 K) for data collection.
• Cryocooling is assumed to minimally perturb protein structures, preserving functional interpretations.

Purpose of the Study:

• To investigate the potential bias introduced by cryocooling in protein crystal structures.
• To analyze the impact of cryocooling on protein conformational ensembles and functional motions.

Main Methods:

• Analysis of X-ray data from 30 diverse proteins.
• Application of novel computational tools for electron-density sampling and model refinement.
• Molecular packing analysis to assess crystal lattice interactions.

Main Results:

• Cryocooling was found to bias structural ensembles in over 35% of side chains.
• Essential packing defects required for functional motions were eliminated by cryocooling.
• Room-temperature data for H-Ras revealed an allosteric network not visible at cryogenic temperatures.

Conclusions:

• Cryocooling introduces significant bias, favoring smaller, overpacked, and less dynamic protein models.
• Monitoring room-temperature conformational ensembles via X-ray crystallography is vital for understanding protein dynamics.
• This approach can uncover motions critical for catalysis, ligand binding, and allosteric regulation.