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Cluster analysis of time-dependent crystallographic data: Direct identification of time-independent structural intermediates.

ABSTRACT: The initial output of a time-resolved macromolecular crystallography experiment is a time-dependent series of difference electron density maps that displays the time-dependent changes in underlying structure as a reaction progresses. The goal is to interpret such data in terms of a small number of crystallographically refinable, time-independent structures, each associated with a reaction intermediate; to establish the pathways and rate coefficients by which these intermediates interconvert; and thereby to elucidate a chemical kinetic mechanism. One strategy toward achieving this goal is to use cluster analysis, a statistical method that groups objects based on their similarity. If the difference electron density at a particular voxel in the time-dependent difference electron density (TDED) maps is sensitive to the presence of one and only one intermediate, then its temporal evolution will exactly parallel the concentration profile of that intermediate with time. The rationale is therefore to cluster voxels with respect to the shapes of their TDEDs, so that each group or cluster of voxels corresponds to one structural intermediate. Clusters of voxels whose TDEDs reflect the presence of two or more specific intermediates can also be identified. From such groupings one can then infer the number of intermediates, obtain their time-independent difference density characteristics, and refine the structure of each intermediate. We review the principles of cluster analysis and clustering algorithms in a crystallographic context, and describe the application of the method to simulated and experimental time-resolved crystallographic data for the photocycle of photoactive yellow protein.

SUBMITTER: Kostov KS

PROVIDER: S-EPMC3021666 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Cluster analysis of time-dependent crystallographic data: Direct identification of time-independent structural intermediates.

Kostov Konstantin S KS Moffat Keith K

Biophysical journal 20110101 2

The initial output of a time-resolved macromolecular crystallography experiment is a time-dependent series of difference electron density maps that displays the time-dependent changes in underlying structure as a reaction progresses. The goal is to interpret such data in terms of a small number of crystallographically refinable, time-independent structures, each associated with a reaction intermediate; to establish the pathways and rate coefficients by which these intermediates interconvert; and ...[more]

PMID: 21244840

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Project description:Accurate knowledge of retention time (RT) in liquid chromatography-based mass spectrometry data facilitates peptide identification, quantification, and multiplexing in targeted and discovery-based workflows. Retention time prediction is particularly important for peptide analysis in emerging data-independent acquisition (DIA) experiments such as SWATH-MS. The indexed RT approach, iRT, uses synthetic spiked-in peptide standards (SiRT) to set RT to a unit-less scale, allowing for normalization of peptide RT between different samples and chromatographic set-ups. The obligatory use of SiRTs can be costly and complicates comparisons and data integration if standards are not included in every sample. Reliance on SiRTs also prevents the inclusion of archived mass spectrometry data for generation of the peptide assay libraries central to targeted DIA-MS data analysis. We have identified a set of peptide sequences that are conserved across most eukaryotic species, termed Common internal Retention Time standards (CiRT). In a series of tests to support the appropriateness of the CiRT-based method, we show: (1) the CiRT peptides normalized RT in human, yeast, and mouse cell lysate derived peptide assay libraries and enabled merging of archived libraries for expanded DIA-MS quantitative applications; (2) CiRTs predicted RT in SWATH-MS data within a 2-min margin of error for the majority of peptides; and (3) normalization of RT using the CiRT peptides enabled the accurate SWATH-MS-based quantification of 340 synthetic isotopically labeled peptides that were spiked into either human or yeast cell lysate. To automate and facilitate the use of these CiRT peptide lists or other custom user-defined internal RT reference peptides in DIA workflows, an algorithm was designed to automatically select a high-quality subset of datapoints for robust linear alignment of RT for use. Implementations of this algorithm are available for the OpenSWATH and Skyline platforms. Thus, CiRT peptides can be used alone or as a complement to SiRTs for RT normalization across peptide spectral libraries and in quantitative DIA-MS studies.

Dataset Information

Cluster analysis of time-dependent crystallographic data: Direct identification of time-independent structural intermediates.

Publications

Cluster analysis of time-dependent crystallographic data: Direct identification of time-independent structural intermediates.

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