Paramagnetic metal ions generate pseudocontact shifts (PCSs) in nuclear magnetic resonance spectra that are manifested as easily measurable changes in chemical shifts. Metals can be incorporated into ...proteins through metal binding tags, and PCS data constitute powerful long-range restraints on the positions of nuclear spins relative to the coordinate system of the magnetic susceptibility anisotropy tensor (Δχ-tensor) of the metal ion. We show that three-dimensional structures of proteins can reliably be determined using PCS data from a single metal binding site combined with backbone chemical shifts. The program PCS-ROSETTA automatically determines the Δχ-tensor and metal position from the PCS data during the structure calculations, without any prior knowledge of the protein structure. The program can determine structures accurately for proteins of up to 150 residues, offering a powerful new approach to protein structure determination that relies exclusively on readily measurable backbone chemical shifts and easily discriminates between correctly and incorrectly folded conformations.
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► PCS-ROSETTA offers a powerful new approach to protein structure determination. ► Three-dimensional structures of proteins up to 150 residues can reliably be determined using backbone PCS data only. ► PCS-ROSETTA automatically determines protein structure, the Δχ-tensor and metal position from the PCS data.
Pseudocontact shift (PCS) effects induced by a paramagnetic lanthanide bound to a protein have become increasingly popular in NMR spectroscopy as they yield a complementary set of orientational and ...long-range structural restraints. PCS are a manifestation of the χ-tensor anisotropy, the Δχ-tensor, which in turn can be determined from the PCS. Once the Δχ-tensor has been determined, PCS become powerful long-range restraints for the study of protein structure and protein-ligand complexes. Here we present the newly developed package Numbat (New User-friendly Method Built for Automatic Δχ-Tensor determination). With a Graphical User Interface (GUI) that allows a high degree of interactivity, Numbat is specifically designed for the computation of the complete set of Δχ-tensor parameters (including shape, location and orientation with respect to the protein) from a set of experimentally measured PCS and the protein structure coordinates. Use of the program for Linux and Windows operating systems is illustrated by building a model of the complex between the E. coli DNA polymerase III subunits ε186 and θ using PCS.
A new lanthanide tag was designed for site-specific labeling of proteins with paramagnetic lanthanide ions. The tag, 4-mercaptomethyl-dipicolinic acid, binds lanthanide ions with nanomolar affinity, ...is readily attached to proteins via a disulfide bond, and avoids the problems of diastereomer formation associated with most of the conventional lanthanide tags. The high lanthanide affinity of the tag opens the possibility to measure residual dipolar couplings in a single sample containing a mixture of paramagnetic and diamagnetic lanthanides. Using the DNA-binding domain of the E. coli arginine repressor as an example, it is demonstrated that the tag allows immobilization of the lanthanide ion in close proximity of the protein by additional coordination of the lanthanide by a carboxyl group of the protein. The close proximity of the lanthanide ion promotes accurate determinations of magnetic susceptibility anisotropy tensors. In addition, the small size of the tag makes it highly suitable for studies of intermolecular interactions.
In order to enhance the structure determination process of macromolecular assemblies by NMR, we have implemented long-range pseudocontact shift (PCS) restraints into the data-driven protein docking ...package HADDOCK. We demonstrate the efficiency of the method on a synthetic, yet realistic case based on the lanthanide-labeled N-terminal
ε
domain of the
E. coli
DNA polymerase III (
ε
186
)
in complex with the HOT domain. Docking from the bound form of the two partners is swiftly executed (interface RMSDs < 1 Å) even with addition of very large amount of noise, while the conformational changes of the free form still present some challenges (interface RMSDs in a 3.1–3.9 Å range for the ten lowest energy complexes). Finally, using exclusively PCS as experimental information, we determine the structure of
ε
186 in complex with the HOT-homologue
θ
subunit of the
E. coli
DNA polymerase III.
WeNMR: Structural Biology on the Grid Wassenaar, Tsjerk A.; van Dijk, Marc; Loureiro-Ferreira, Nuno ...
Journal of grid computing,
12/2012, Letnik:
10, Številka:
4
Journal Article
Odprti dostop
The WeNMR (
http://www.wenmr.eu
) project is a European Union funded international effort to streamline and automate analysis of Nuclear Magnetic Resonance (NMR) and Small Angle X-Ray scattering ...(SAXS) imaging data for atomic and near-atomic resolution molecular structures. Conventional calculation of structure requires the use of various software packages, considerable user expertise and ample computational resources. To facilitate the use of NMR spectroscopy and SAXS in life sciences the WeNMR consortium has established standard computational workflows and services through easy-to-use web interfaces, while still retaining sufficient flexibility to handle more specific requests. Thus far, a number of programs often used in structural biology have been made available through application portals. The implementation of these services, in particular the distribution of calculations to a Grid computing infrastructure, involves a novel mechanism for submission and handling of jobs that is independent of the type of job being run. With over 450 registered users (September 2012), WeNMR is currently the largest Virtual Organization (VO) in life sciences. With its large and worldwide user community, WeNMR has become the first Virtual Research Community officially recognized by the European Grid Infrastructure (EGI).
Pseudocontact shifts (PCSs) induced by a site-specifically bound paramagnetic lanthanide ion are shown to provide fast access to sequence-specific resonance assignments of methyl groups in proteins ...of known three-dimensional structure. Stereospecific assignments of Val and Leu methyls are obtained as well as resonance assignments of all other methyls, including Met εCH3 groups. No prior assignments of the diamagnetic protein are required nor are experiments that transfer magnetization between the methyl groups and the protein backbone. Methyl C z -exchange experiments were designed to provide convenient access to PCS measurements in situations where a paramagnetic lanthanide is in exchange with a diamagnetic lanthanide. In the absence of exchange, simultaneous 13C-HSQC assignments and PCS measurements are delivered by the newly developed program Possum. The approaches are demonstrated with the complex between the N-terminal domain of the subunit ε and the subunit θ of the Escherichia coli DNA polymerase III.
Anisotropic magnetic susceptibility tensors chi of paramagnetic metal ions are manifested in pseudocontact shifts, residual dipolar couplings, and other paramagnetic observables that present valuable ...long-range information for structure determinations of protein-ligand complexes. A program was developed for automatic determination of the chi-tensor anisotropy parameters and amide resonance assignments in proteins labeled with paramagnetic metal ions. The program requires knowledge of the three-dimensional structure of the protein, the backbone resonance assignments of the diamagnetic protein, and a pair of 2D 15N-HSQC or 3D HNCO spectra recorded with and without paramagnetic metal ion. It allows the determination of reliable chi-tensor anisotropy parameters from 2D spectra of uniformly 15N-labeled proteins of fairly high molecular weight. Examples are shown for the 185-residue N-terminal domain of the subunit epsilon from E. coli DNA polymerase III in complex with the subunit theta and La3+ in its diamagnetic and Dy3+, Tb3+, and Er3+ in its paramagnetic form.
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