Abstract
Ice formation within protein crystals is a major obstacle to the cryocrystallographic study of protein structure, and has limited studies of how the structural ensemble of a protein evolves with temperature in the biophysically interesting range from ∼260 K to the protein-solvent glass transition near 200 K. Using protein crystals with solvent cavities as large as ∼70 Å, time-resolved X-ray diffraction was used to study the response of protein and internal solvent during rapid cooling. Solvent nanoconfinement suppresses freezing temperatures and ice-nucleation rates so that ice-free, low-mosaicity diffraction data can be reliably collected down to 200 K without the use of cryoprotectants. Hexagonal ice (Ih) forms in external solvent, but internal crystal solvent forms stacking-disordered ice (Isd) with a near-random stacking of cubic and hexagonal planes. Analysis of powder diffraction from internal ice and single-crystal diffraction from the host protein structure shows that the maximum crystallizable solvent fraction decreases with decreasing crystal solvent-cavity size, and that an ∼6 Å thick layer of solvent adjacent to the protein surface cannot crystallize. These results establish protein crystals as excellent model systems for the study of nanoconfined solvent. By combining fast cooling, intense X-ray beams and fast X-ray detectors, complete structural data sets for high-value targets, including membrane proteins and large complexes, may be collected at ∼220-240 K that have much lower mosaicities and comparable B factors, and that may allow more confident identification of ligand binding than in current cryocrystallographic practice.
Highlights
IntroductionIce formation and its prevention are key issues in many areas of bioscience and biotechnology, including the cold-hardiness of microorganisms, animals and agriculturally relevant plants; the cryopreservation of cells, tissues and organs; the cold storage of proteins and biologics; and biomolecular and cellular structure determination using electrons and X-rays
Ice formation and its prevention are key issues in many areas of bioscience and biotechnology, including the cold-hardiness of microorganisms, animals and agriculturally relevant plants; the cryopreservation of cells, tissues and organs; the cold storage of proteins and biologics; and biomolecular and cellular structure determination using electrons and X-rays.X-ray crystallography is our primary tool for probing biomolecular structure
We show that protein crystals enable new quantitative approaches to probing the effects of nanoconfinement on ice formation
Summary
Ice formation and its prevention are key issues in many areas of bioscience and biotechnology, including the cold-hardiness of microorganisms, animals and agriculturally relevant plants; the cryopreservation of cells, tissues and organs; the cold storage of proteins and biologics; and biomolecular and cellular structure determination using electrons and X-rays. X-ray crystallography is our primary tool for probing biomolecular structure. Once synchrotron X-ray sources became widely available in the 1990s, data collection shifted to the near-exclusive use of cryogenically cooled crystals. Cryocooling to $100 K reduces the rate at which diffraction properties degrade with X-ray dose by a factor of $50, increasing the amount of data that can be collected per crystal, and reduces thermal motions, often increasing the resolution (Rupp, 2009; Pflugrath, 2015). Crystallography beamlines can collect diffraction data sets sufficient for protein structure determination in less than one second
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