Biomolecular cryocrystallography: Structural changes during flash-cooling

Halle, Bertil

doi:10.1073/pnas.0308315101

Cited by 189 publications

(151 citation statements)

References 55 publications

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“…Our results support the theoretical prediction that crystal cooling is too slow to trap the equilibrium side-chain distributions at both solvent-exposed and buried positions of proteins (15). Consistent with Halle's dynamic quenching theory (15), cryocrystallography causes anisotropic and idiosyncratic effects on the contraction of each protein and the quality of local packing (Fig. 3).…”

Section: Discussionsupporting

confidence: 89%

“…A theoretical analysis by Halle suggests that the crystal cryocooling process, which typically takes up to a second, is too slow to trap the room-temperature equilibrium distribution of protein and solvent configurations (15). According to Halle's dynamic quenching theory, changes in the energy landscape upon cooling can cause specific changes in the relative populations of alternative conformations that are not apparent from B-factor analysis or the standard refinement of unique models.…”

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confidence: 99%

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

Fraser

Bedem

Samelson

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

538

454

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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.protein conformational dynamics | energy landscape | Ringer | qFit M acromolecular X-ray crystallographic diffraction experiments provide powerful insights into the relationship between structure and biological function. Although macromolecules populate vast ensembles of alternative conformational substates (1), crystallographic models depicting the major average conformation have provided foundational ideas about the mechanisms of biochemical reactions. By slowing radiation damage to the sample, crystal cooling has catalyzed a revolution in structural biology, enabling structure determinations from tiny crystals using bright synchrotron X-ray sources (2-4). It is estimated that more than 95% of the >65;000 crystal structures deposited in the Protein Data Bank (PDB) are based on cryogenic data (5).Crystal cooling is generally thought to introduce little bias in the functional interpretation of structural results. Some investigators have suggested that the standard practice of plunging crystals into liquid nitrogen and collecting X-ray diffraction data at 100 K traps a representative set of conformations populated at room temperature (6, 7). In contrast, early structural comparisons by Petsko, Frauenfelder, and colleagues indicated that cooling myoglobin crystals causes a small reduction in the protein volume due to anisotropic displacements of atomic positions and subtle changes of contacts between α-helices (8). A landmark study of crystalline ribonuclease A (RNaseA) by Tilton, Petsko, and coworkers revealed diverse temperature-dependent changes in the average structure, ranging from shrinkage at low temperatures to increased loop disorder at 47°C (9). A recent analysis comparing 15 room-temperature and cryogenic crystal structures documented that cryocooling generally increas...

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Section: Discussionsupporting

confidence: 89%

mentioning

confidence: 99%

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

Fraser

Bedem

Samelson

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

538

454

View full text Add to dashboard Cite

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“…The state interconversion kinetics are modelled with an activation enthalpy of 40 kJ mol Ϫ1 , the diffusion coefficient of water, and a state lifetime of 10 ns at 293 K. The equilibrium is quenched after 47 ms when the crystal temperature is 202 K, corresponding to 95% population in the low-temperature state (compared with 15% at 293 K). The time-dependent temperature is averaged over the middle one-third of the crystal volume (Halle 2004). 200 K below the physiological temperature range (Garman 2003). Cryocrystallography evolved primarily as a means to combat radiation damage to crystals from intense synchrotron X-ray beams, based on the idea that radiation-induced free radicals cannot damage the biomolecule once they are trapped in the vitrified bulk solvent within the crystal (Garman & Schneider 1997).…”

Section: Structure Of Protein Hydrationmentioning

confidence: 99%

“…There is thus ample time for thermal averaging during the cooling process. The structural changes expected during flash cooling of a protein crystal have recently been calculated for a temperature-dependent two-state equilibrium (Halle 2004). This analysis indicates that many degrees of freedom are quenched at temperatures near 200 K, where local conformational and association equilibria may be strongly shifted towards lowenthalpy states (see figure 1).…”

Section: Structure Of Protein Hydrationmentioning

confidence: 99%

Protein hydration dynamics in solution: a critical survey

Halle

2004

Phil. Trans. R. Soc. Lond. B

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492

576

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The properties of water in biological systems have been studied for well over a century by a wide range of physical techniques, but progress has been slow and erratic. Protein hydration-the perturbation of water structure and dynamics by the protein surface-has been a particularly rich source of controversy and confusion. Our aim here is to critically examine central concepts in the description of protein hydration, and to assess the experimental basis for the current view of protein hydration, with the focus on dynamic aspects. Recent oxygen-17 magnetic relaxation dispersion (MRD) experiments have shown that the vast majority of water molecules in the protein hydration layer suffer a mere twofold dynamic retardation compared with bulk water. The high mobility of hydration water ensures that all thermally activated processes at the protein-water interface, such as binding, recognition and catalysis, can proceed at high rates. The MRD-derived picture of a highly mobile hydration layer is consistent with recent molecular dynamics simulations, but is incompatible with results deduced from intermolecular nuclear Overhauser effect spectroscopy, dielectric relaxation and fluorescence spectroscopy. It is also inconsistent with the common view of hydration effects on protein hydrodynamics. Here, we show how these discrepancies can be resolved.

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“…(ii) At a typical resolution limit of Ϸ2 Å, a contaminating nonpolar ligand may be misinterpreted as a water cluster (9,10). (iii) For structures determined at cryogenic temperatures, the hydration status of the cavity may be altered by re-equilibration during the flash cooling process (11).…”

mentioning

confidence: 99%

A dry ligand-binding cavity in a solvated protein

Qvist

Davidovic

Hamelberg

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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129

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Ligands usually bind to proteins by displacing water from the binding site. The affinity and kinetics of binding therefore depend on the hydration characteristics of the site. Here, we show that the extreme case of a completely dehydrated free binding site is realized for the large nonpolar binding cavity in bovine ␤-lactoglobulin. Because spatially delocalized water molecules may escape detection by x-ray diffraction, we use water 17 O and 2 H magnetic relaxation dispersion (MRD), 13 C NMR spectroscopy, molecular dynamics simulations, and free energy calculations to establish the absence of water from the binding cavity. Whereas carbon nanotubes of the same diameter are filled by a hydrogenbonded water chain, the MRD data show that the binding pore in the apo protein is either empty or contains water molecules with subnanosecond residence times. However, the latter possibility is ruled out by the computed hydration free energies, so we conclude that the 315 Å 3 binding pore is completely empty. The apo protein is thus poised for efficient binding of fatty acids and other nonpolar ligands. The qualitatively different hydration of the ␤-lactoglobulin pore and carbon nanotubes is caused by subtle differences in water-wall interactions and water entropy.␤-lactoglobulin ͉ free energy simulation ͉ hydrophobic hydration ͉ magnetic relaxation dispersion G lobular proteins are stabilized by dense atomic packing and by water exclusion from the nonpolar core region. However, the packing density is not uniform and a typical protein contains approximately four cavities per 100 residues of sufficient size to accommodate at least one water molecule (1). Small nonpolar cavities are usually empty and can be regarded as packing defects, whereas small polar cavities tend to be occupied by structural water molecules that stabilize the folded protein by H-bonding to otherwise unsatisfied peptide partners (2). Large cavities are frequently linked to protein functions, such as ligand binding and transport, membrane translocation, and enzyme catalysis. Some of these large cavities are lined exclusively or predominantly by nonpolar sidechains. The question whether such large nonpolar cavities are empty or contain water molecules is of fundamental biological importance (3-8).The principal tool of structural biology, x-ray crystallography, cannot easily resolve this issue. There are three potential problems. (i) Because water molecules interact only weakly with the nonpolar cavity walls, they tend to be positionally disordered. As a result of such delocalization, the electron density of the water molecules may fall below the detection limit. (ii) At a typical resolution limit of Ϸ2 Å, a contaminating nonpolar ligand may be misinterpreted as a water cluster (9, 10). (iii) For structures determined at cryogenic temperatures, the hydration status of the cavity may be altered by re-equilibration during the flash cooling process (11).In favorable cases, water molecules in nonpolar protein cavities can be inferred from intermolecular nuclear O...

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Biomolecular cryocrystallography: Structural changes during flash-cooling

Cited by 189 publications

References 55 publications

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

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

Protein hydration dynamics in solution: a critical survey

A dry ligand-binding cavity in a solvated protein

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