Temperature-dependent macromolecular X-ray crystallography

Weik, Martin H.; Colletier, J.P.

doi:10.1107/s0907444910002702

Cited by 67 publications

(90 citation statements)

References 127 publications

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“…They may be particularly useful in studies of the protein glass transition, in mechanistic studies of protein function and in the developing field of kinetic crystallography (Bourgeois & Royant, 2005;Colletier et al, 2008;Weik & Colletier, 2010), in which temperature can be used as a parameter to control reaction rates within crystals.…”

Section: Resultsmentioning

confidence: 99%

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Glass transition in thaumatin crystals revealed through temperature-dependent radiation-sensitivity measurements

Warkentin

Thorne

2010

Acta Crystallogr D Biol Crystallogr

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The temperature-dependence of radiation damage to thaumatin crystals between T = 300 and 100 K is reported. The amount of damage for a given dose decreases sharply as the temperature decreases from 300 to 220 K and then decreases more gradually on further cooling below the protein-solvent glass transition. Two regimes of temperature-activated behavior were observed. At temperatures above $200 K the activation energy of 18.0 kJ mol À1 indicates that radiation damage is dominated by diffusive motions in the protein and solvent. At temperatures below $200 K the activation energy is only 1.00 kJ mol À1 , which is of the order of the thermal energy. Similar activation energies describe the temperaturedependence of radiation damage to a variety of solvent-free small-molecule organic crystals over the temperature range T = 300-80 K. It is suggested that radiation damage in this regime is vibrationally assisted and that the freezing-out of amino-acid scale vibrations contributes to the very weak temperature-dependence of radiation damage below $80 K. Analysis using the radiation-damage model of Blake and Phillips [Blake & Phillips (1962), Biological Effects of Ionizing Radiation at the Molecular Level, indicates that large-scale conformational and molecular motions are frozen out below T = 200 K but become increasingly prevalent and make an increasing contribution to damage at higher temperatures. Possible alternative mechanisms for radiation damage involving the formation of hydrogen-gas bubbles are discussed and discounted. These results have implications for mechanistic studies of proteins and for studies of the protein glass transition. They also suggest that data collection at T ' 220 K may provide a viable alternative for structure determination when cooling-induced disorder at T = 100 is excessive.

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Section: Resultsmentioning

confidence: 99%

“…As a result, radiation damage at temperatures spanning the full range T = 300-100 K remains a major issue in structural biology (Weik & Colletier, 2010). The mechanisms by which X-rays degrade diffraction quality throughout this temperature range remain poorly understood.…”

Section: Introductionmentioning

confidence: 99%

Glass transition in thaumatin crystals revealed through temperature-dependent radiation-sensitivity measurements

Warkentin

Thorne

2010

Acta Crystallogr D Biol Crystallogr

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“…In contrast to these small conformational perturbations, the dynamic characteristics of proteins in crystals are exquisitely sensitive to temperature (11,12). After accounting for static structural differences between molecules, model errors, and crystal lattice defects in myoglobin crystals, Frauenfelder and Petsko demonstrated widespread temperature-dependent decreases in the harmonic atomic vibrations measured by B-factors.…”

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confidence: 99%

“…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 increases lattice contacts and reduces protein volumes but causes only small changes in crystallographic models (10).In contrast to these small conformational perturbations, the dynamic characteristics of proteins in crystals are exquisitely sensitive to temperature (11,12). After accounting for static structural differences between molecules, model errors, and crystal lattice defects in myoglobin crystals, Frauenfelder and Petsko demonstrated widespread temperature-dependent decreases in the harmonic atomic vibrations measured by B-factors.…”

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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.

549

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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“…Typically, X-ray crystallographic data are measured from crystals maintained at 100 K in order to immobilize X-raygenerated free radicals and damaged protein and minimize radiation damage, leading an increase in crystal lifetime of up to two orders of magnitude relative to RT (Southworth-Davies et al, 2007). Between RT and 100 K, protein crystals undergo at least one and possibly several temperature-dependent transitions (Weik & Colletier, 2010;Lewandowski et al, 2015;Ringe & Petsko, 2003;Keedy et al, 2015). Notably, anharmonic macromolecular motions resume above the glass transition that occurs in the range 180-220 K, where solvent viscosity is greatly reduced.…”

Section: Introductionmentioning

confidence: 99%

Active-site protein dynamics and solvent accessibility in nativeAchromobacter cycloclastescopper nitrite reductase

Sen

Horrell

Kekilli

et al. 2017

Int Union Crystallogr J

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Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (Asp CAT and His CAT ) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the Asp CAT protonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site 'capping residue' (Ile CAT ), a determinant of ligand binding, are influenced both by temperature and by the protonation state of Asp CAT . A previously unobserved conformation of Ile CAT is seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.

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Temperature-dependent macromolecular X-ray crystallography

Cited by 67 publications

References 127 publications

Glass transition in thaumatin crystals revealed through temperature-dependent radiation-sensitivity measurements

Glass transition in thaumatin crystals revealed through temperature-dependent radiation-sensitivity measurements

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

Active-site protein dynamics and solvent accessibility in nativeAchromobacter cycloclastescopper nitrite reductase

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