Modeling discrete heterogeneity in X-ray diffraction data by fitting multi-conformers

Bedem, Henry van den; Dhanik, Ankur; Latombe, Jean‐Claude; Deacon, Ashley M.

doi:10.1107/s0907444909030613

Cited by 118 publications

(116 citation statements)

References 39 publications

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“…To reduce biases introduced by different refinement programs and model builders, we rebuilt all 60 models using qFit (17), which automates the discovery and building of alternative polypeptide conformations, allowing for coupled side-chain-backbone flexibility. The multiconformer qFit models were further refined with phenix.refine (19) to ensure that all models experienced similar restraints on geometry, B-factors and solvent, improving R free values by an average of 1.0% relative to the deposited structures.…”

Section: Resultsmentioning

confidence: 99%

“…When no R free set was deposited or could be extracted, we chose a test set using the standard parameters in phenix.refine (19). Structures were rebuilt using qFit (17) and then refined for five additional cycles in phenix.refine, with occupancy refinement and automated solvent picking. For all models, we included riding protein hydrogen atoms in refinement.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

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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.

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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: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

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.

Self Cite

562

454

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“…21,22 A computational method that explores large numbers of side-chain conformations to more fully explain electron density has also been described. 23 These methods, however, are subject to model bias and local errors in individual models. 24 In addition, despite the addition of numerous atoms to the structural model, these methods generally afford only limited improvements in the R cryst and R free values.…”

Section: Introductionmentioning

confidence: 99%

Automated electron‐density sampling reveals widespread conformational polymorphism in proteins

et al. 2010

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Although proteins populate large structural ensembles, X-ray diffraction data are traditionally interpreted using a single model. To search for evidence of alternate conformers, we developed a program, Ringer, which systematically samples electron density around the dihedral angles of protein side chains. In a diverse set of 402 structures, Ringer identified weak, nonrandom electron-density features that suggest of the presence of hidden, lowly populated conformations for >18% of uniquely modeled residues. Although these peaks occur at electron-density levels traditionally regarded as noise, statistically significant (P < 10 25 ) enrichment of peaks at successive rotameric v angles validates the assignment of these features as unmodeled conformations. Weak electron density corresponding to alternate rotamers also was detected in an accurate electron density map free of model bias. Ringer analysis of the high-resolution structures of free and peptide-bound calmodulin identified shifts in ensembles and connected the alternate conformations to ligand recognition. These results show that the signal in high-resolution electron density maps extends below the traditional 1 r cutoff, and crystalline proteins are more polymorphic than current crystallographic models. Ringer provides an objective, systematic method to identify previously undiscovered alternate conformations that can mediate protein folding and function.

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“…Of the 14 residues, 8 show at least two alternate conformations in the apo SpA C crystal structure and have dynamics in χ 1 or χ 2 by NMR. Two of these, L17 and I31, are found at the F c interface and lose their conformational heterogeneity in the crystal structure of the complex, as judged by the lack of alternate side-chain conformations detected by qFit (24) and by their low ρ ratios (0.24 and 0.13, respectively). Unfortunately, the 4 remaining interfacial residues were not among the 14 side chains whose dynamics were observable in our NMR experiments, so the loss in heterogeneity observed in the crystal structures for these residues has not been confirmed by NMR.…”

Section: Residues At the Helix 1/2 Interface Undergo Concerted Conformentioning

confidence: 99%

Suppression of conformational heterogeneity at a protein–protein interface

Deis

Wang

et al. 2015

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

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Staphylococcal protein A (SpA) is an important virulence factor from Staphylococcus aureus responsible for the bacterium’s evasion of the host immune system. SpA includes five small three-helix–bundle domains that can each bind with high affinity to many host proteins such as antibodies. The interaction between a SpA domain and the Fc fragment of IgG was partially elucidated previously in the crystal structure 1FC2. Although informative, the previous structure was not properly folded and left many substantial questions unanswered, such as a detailed description of the tertiary structure of SpA domains in complex with Fc and the structural changes that take place upon binding. Here we report the 2.3-Å structure of a fully folded SpA domain in complex with Fc. Our structure indicates that there are extensive structural rearrangements necessary for binding Fc, including a general reduction in SpA conformational heterogeneity, freezing out of polyrotameric interfacial residues, and displacement of a SpA side chain by an Fc side chain in a molecular-recognition pocket. Such a loss of conformational heterogeneity upon formation of the protein–protein interface may occur when SpA binds its multiple binding partners. Suppression of conformational heterogeneity may be an important structural paradigm in functionally plastic proteins.

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Modeling discrete heterogeneity in X-ray diffraction data by fitting multi-conformers

Cited by 118 publications

References 39 publications

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

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

Automated electron‐density sampling reveals widespread conformational polymorphism in proteins

Suppression of conformational heterogeneity at a protein–protein interface

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