Automated electron‐density sampling reveals widespread conformational polymorphism in proteins

Lang, P. Therese; Ng, Ho Leung; Fraser, James S.; Corn, Jacob E.; Echols, Nathaniel; Sales, Mark; Holton, James M.; Alber, Tom

doi:10.1002/pro.423

Cited by 163 publications

(217 citation statements)

References 40 publications

(42 reference statements)

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“…The more realistic description of the atomic thermal motion means, however, that the ADPs will not be able to compensate so well for unmodelled disorder and other problems. There is good evidence (MacArthur & Thornton, 1999;Lang et al, 2010) that protein structures possess many more alternative lowoccupancy side-chain conformations than are usually modelled. The extra (unrestrained) isotropic displacement parameters in the widely used TLS models for ADP refinement of proteins are, in contrast to the rigid-bond restraints, well able to compensate for the resulting unequal occupancies of the atoms involved.…”

Section: Discussionmentioning

confidence: 99%

Enhanced rigid-bond restraints

Thorn

Dittrich

Sheldrick

2012

Acta Crystallogr A Found Crystallogr

193

158

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The rigid‐bond model [Hirshfeld (1976). Acta Cryst. A32, 239–244] states that the mean‐square displacements of two atoms are equal in the direction of the bond joining them. This criterion is widely used for verification (as intended by Hirshfeld) and also as a restraint in structure refinement as suggested by Rollett [Crystallographic Computing (1970), edited by F. R. Ahmed et al., pp. 167–181. Copenhagen: Munksgaard]. By reformulating this condition, so that the relative motion of the two atoms is required to be perpendicular to the bond, the number of restraints that can be applied per anisotropic atom is increased from about one to about three. Application of this condition to 1,3‐distances in addition to the 1,2‐distances means that on average just over six restraints can be applied to the six anisotropic displacement parameters of each atom. This concept is tested against very high resolution data of a small peptide and employed as a restraint for protein refinement at more modest resolution (e.g. 1.7 Å).

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Section: Discussionmentioning

confidence: 99%

Enhanced rigid-bond restraints

Thorn

Dittrich

Sheldrick

2012

Acta Crystallogr A Found Crystallogr

193

158

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“…Much of this work has focused on incorporating dynamical information during NMR structure determination (5,6) or the extraction of multiple conformers from X-ray diffraction data (7,8). While these techniques are powerful, they share difficulties in data collection, the unified treatment of heterogeneous experimental data, and data sparseness relative to the number of degrees of freedom.…”

Section: Introductionmentioning

confidence: 99%

Bayesian Energy Landscape Tilting: Towards Concordant Models of Molecular Ensembles

Beauchamp¹,

Pande²,

Das³

2014

Preprint

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Predicting biological structure has remained challenging for systems such as disordered proteins that take on myriad conformations. Hybrid simulation/experiment strategies have been undermined by difficulties in evaluating errors from computational model inaccuracies and data uncertainties. Building on recent proposals from maximum entropy theory and nonequilibrium thermodynamics, we address these issues through a Bayesian Energy Landscape Tilting (BELT) scheme for computing Bayesian "hyperensembles" over conformational ensembles. BELT uses Markov chain Monte Carlo to directly sample maximum-entropy conformational ensembles consistent with a set of input experimental observables. To test this framework, we apply BELT to model trialanine, starting from disagreeing simulations with the force fields ff96, ff99, ff99sbnmr-ildn, CHARMM27, and OPLS-AA. BELT incorporation of limited chemical shift and 3 J measurements gives convergent values of the peptide's α, β, and P P II conformational populations in all cases. As a test of predictive power, all five BELT hyperensembles recover set-aside measurements not used in the fitting and report accurate errors, even when starting from highly inaccurate simulations. BELT's principled framework thus enables practical predictions for complex biomolecular systems from discordant simulations and sparse data.

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“…To access the conformational ensembles, we computationally sampled the electron-density maps using the programs qFit, to build and refine multiconformer models (17), and Ringer, to define unmodeled alternative side-chain conformations (18). These methods sample values of electron density that are usually ignored during model building to discover alternative conformations and distinguish these substates from harmonic motions of the average structure.…”

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.

Self Cite

554

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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Automated electron‐density sampling reveals widespread conformational polymorphism in proteins

Cited by 163 publications

References 40 publications

Enhanced rigid-bond restraints

Enhanced rigid-bond restraints

Bayesian Energy Landscape Tilting: Towards Concordant Models of Molecular Ensembles

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

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