Addition of Missing Loops and Domains to Protein Models by X-Ray Solution Scattering

Abstract

Inherent flexibility and conformational heterogeneity in proteins can often result in the absence of loops and even entire domains in structures determined by x-ray crystallographic or NMR methods. X-ray solution scattering offers the possibility of obtaining complementary information regarding the structures of these disordered protein regions. Methods are presented for adding missing loops or domains by fixing a known structure and building the unknown regions to fit the experimental scattering data obtained from the entire particle. Simulated annealing was used to minimize a scoring function containing the discrepancy between the experimental and calculated patterns and the relevant penalty terms. In low-resolution models where interface location between known and unknown parts is not available, a gas of dummy residues represents the missing domain. In high-resolution models where the interface is known, loops or domains are represented as interconnected chains (or ensembles of residues with spring forces between the C(alpha) atoms), attached to known position(s) in the available structure. Native-like folds of missing fragments can be obtained by imposing residue-specific constraints. After validation in simulated examples, the methods have been applied to add missing loops or domains to several proteins where partial structures were available.

Structural analysis of flexible proteins in solution by small angle X-ray scattering combined with crystallography

“…SAXS can provide an effective complementary technique that can build low resolution models for these regions onto the known structure. Svergun and his coworkers have developed a suite of programs, including CREDO, based on their ab initio programs that can build dummy atoms or Cα chains onto the crystal structure to show the relative location of these "missing" regions (Petoukhov et al, 2002). The known structure is fixed and then dummy residues are placed randomly within the input Dmax region.…”

The impact of protein characterization in structural proteomics

“…(iii) If high-resolution methods yield an incomplete structure of the macromolecule, overall configurations of missing (flexible) loops and domains in solution can be reconstructed using the SAXS data (Petoukhov et al, 2002;Marquez et al, 2003).…”

“…These results indicate that, in contrast to Src and Abl, Btk might not require an assembled conformation for the regulation of its activity. The rigid-body model was constructed by a multi-step procedure involving interactive modelling with the program MASSHA (Konarev et al, 2001) with missing linkers added by the program CREDO (Petoukhov et al, 2002). The recently developed global modelling programs SASREF and BUNCH (Petoukhov & Svergun, 2005) allow the construction of models automatically (also by fitting simultaneously multiple scattering curves) and gave results similar to those in Fig.…”

Structure Analysis of Biological Macromolecules by Small-Angle X-ray Scattering

“…because of fl exibility). The missing portions are represented as chains of the so-called dummy residues [ 34 ] , and the known domains/ subunits can be translated and rotated as rigid bodies while simultaneously changing the local conformation of the chains representing the unknown fragments [ 23 ] . Numerous applications of rigid body modelling are reported to build structural models of complicated objects in solution (see e.g.…”