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Automated electron-density sampling reveals widespread conformational polymorphism in proteins.

ABSTRACT: 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(-5)) enrichment of peaks at successive rotameric chi 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 sigma 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.

SUBMITTER: Lang PT

PROVIDER: S-EPMC2974833 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Automated electron-density sampling reveals widespread conformational polymorphism in proteins.

Lang P Therese PT Ng Ho-Leung HL Fraser James S JS Corn Jacob E JE Echols Nathaniel N Sales Mark M Holton James M JM Alber Tom T

Protein science : a publication of the Protein Society 20100701 7

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 modele ...[more]

PMID: 20499387

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Project description:Crystal and conformational polymorphisms play crucial roles in the physical and chemical properties of materials, impacting their stability, solubility, and bioavailability, which are essential for various applications in pharmaceuticals, materials science, and chemistry. Despite their significance, the structural analysis of these polymorphisms, particularly conformational polymorphisms, remains challenging due to the limited methodology that provides sufficient resolution for microcrystalline variants of polymorphs. Three-dimensional electron diffraction (3D ED) is an emerging technique with significant potential for elucidating the microcrystal structures of functional organic molecules, pharmaceuticals, and biomolecules. Despite this potential, there are limited instances of 3D ED structures for small molecules exhibiting the lowest crystallographic symmetry with a preferred orientation and possibly conformational variations of constituent molecules. A novel organic semiconductor, Ph-anti-benzothieno[5,6-b]benzothieno[3,2-b]thiophene-C10 (antiC10), is one of such examples. We successfully determined the 3D ED structure of this challenging molecule. The antiC10 crystal exhibited the lowest symmetry (space group P1), and the preferred orientations against the grid resulted in a missing cone. These challenges were surmounted by employing a sequential molecular replacement approach with an ab initio-generated search model. The resulting octameric antiC10 structure reveals a two-monolayer architecture and an antiparallel alkyl-interdigitated herringbone configuration in contrast to the all-parallel associations observed in its previously reported isomer. Concurrently, the alkyl chains are intricately interdigitated with each other and positioned between the adjacent π-core strata. Detailed analysis has elucidated the conformational polymorphism in herringbone packing between the two monolayers as well as in intramolecular conformations among monomers. The structure with conformational polymorphism is presumably in a metastable intermediate state, stabilized by twinning. These findings may provide critical insights into the crystallization mechanisms and rational design of organic semiconductors. This research demonstrates that advancements in 3D ED technology and sequential phasing methodologies have enabled the study of previously unreachable structures.

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Automated electron-density sampling reveals widespread conformational polymorphism in proteins.

Publications

Automated electron-density sampling reveals widespread conformational polymorphism in proteins.

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