SSEThread: Integrative threading of the DNA-PKcs sequence based on data from chemical cross-linking and hydrogen deuterium exchange

Abstract

X-ray crystallography and electron microscopy maps resolved to 3–8 angstroms are generally sufficient for tracing the path of the polypeptide chain in space, while often insufficient for unambiguously registering the sequence on the path (i.e., threading). Frequently, however, additional information is available from other biophysical experiments, physical principles, statistical analyses, and other prior models. Here, we formulate an integrative approach for sequence assignment to a partial backbone model as an optimization problem, which requires three main components: the representation of the system, the scoring function, and the optimization method. The method is implemented in the open source Integrative Modeling Platform (IMP) (https://integrativemodeling.org), allowing a number of different terms in the scoring function. We apply this method to localizing the sequence assignment within a 199-residue disordered region of three structured and sequence unassigned helices in the DNA-PKcs crystallographic structure, using chemical crosslinks, hydrogen deuterium exchange, and sequence connectivity. The resulting ensemble of threading models provides two major solutions, one of which suggests that the crucial ABCDE cluster of phosphorylation sites cannot undergo intra-molecular autophosphorylation without a conformational rearrangement. The ensemble of solutions embodies the most accurate and precise sequence threading given the available information.

From integrative structural biology to cell biology

“…A number of schemes for searching for good-scoring models are available, including local refinement methods and stochastic sampling methods in the continuous space of Cartesian coordinates as well as enumeration schemes in the discrete spaces of both Cartesian and internal coordinates. Finally, IMP implements protocols for analyzing and validating models, including methods for model clustering based on their structural similarity ( 4 , 5 , 9 , 17 , 19 , 29 , 33 , 34 , 36 , 45 , 66 , 68 , 86 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 131 , 132 , 132 , 133 , 134 , 135 ). IMP, Integrative Modeling Platform .…”

Comprehensive Structure and Functional Adaptations of the Yeast Nuclear Pore Complex

“…Integrative threading of Nup sequences into spoke orphan structure elements. A novel integrative threading approach (7,15,43,92,93) was applied to determine the protein, its copy identifier, and the sequence of orphan structure elements (SE) in the inner ring. All possible arrangements derived from the spoke C2-symmetry were considered, but only one resulted in a model that sufficiently satisfied the input data: this option splits the 30 orphan into two symmetric subsets of 15 orphan densities each.…”

“…The scoring function is a sum of terms, each one of which restrains some aspect of the model based on a subset of input information (e.g., stereochemistry, cross-links, sequences). We impose three scoring terms that restrain the four degrees of freedom (43) for each of the 30 SEs. First, the secondary structure restraint is computed by comparing the secondary structure assignment in the model with the secondary structure propensity predicted from the input sequence.…”

“…An accurate identification and topology map of these connectors is needed to understand how the OSD and the ISD are tied together. To solve this problem, we applied an integrative threading approach (43) to compute Nup identity, copy identifier, and sequence segments for each of the structure elements (SEs) that occupy orphan densities (Fig. S7, Methods, Table S2).…”

IMProv: A Resource for Cross-link-Driven Structure Modeling that Accommodates Protein Dynamics

“…As solvent accessibility is not the primary influencer of exchange rates, it must be used with caution in modeling protein interactions, as binding events can also induce distal changes in protein stability ( 43 , 44 ). However, it excels at detecting stability at high resolution and has been used to map and model secondary structure and protein folding ( 45 , 46 ). These capabilities stimulated us to evaluate its utility in determining the underlying structural stability of cross-linked sites.…”