NMR-Based Configurational Assignments of Natural Products: Gibbs Sampling and Bayesian Inference Using Floating Chirality Distance Geometry Calculations

Floating chirality restrained distance geometry (fc-rDG) calculations are used to directly evolve structures from NMR data such as NOE-derived intramolecular distances or anisotropic residual dipolar couplings (RDCs). In contrast to evaluating pre-calculated structures against NMR restraints, multiple configurations (diastereomers) and conformations are generated automatically within the experimental limits. In this report, we show that the “unphysical” rDG pseudo energies defined from NMR violations bear statistical significance, which allows assigning probabilities to configurational assignments made that are fully compatible with the method of Bayesian inference. These “diastereomeric differentiabilities” then even become almost independent of the actual values of the force constants used to model the restraints originating from NOE or RDC data.

NMR refinement and peptide folding using the GROMACS software

Nuclear magnetic resonance spectroscopy is used routinely for studying the three-dimensional structures and dynamics of proteins and nucleic acids. Structure determination is usually done by adding restraints based upon NMR data to a classical energy function and performing restrained molecular simulations. Here we report on the implementation of a script to extract NMR restraints from a NMR-STAR file and export it to the GROMACS software. With this package it is possible to model distance restraints, dihedral restraints and orientation restraints. The output from the script is validated by performing simulations with and without restraints, including the ab initio refinement of one peptide.

NMR Refinement and Peptide Folding Using the GROMACS Software

<div><div><div><p>Nuclear magnetic resonance spectroscopy is used routinely for studying the three-dimensional structures and dynamics of proteins. Structure determination is usually done by adding restraints based upon NMR data to a classical energy function and performing restrained molecular simulations. Here we report on the implementation of a script to extract NMR restraints from a NMR-STAR file and export it to the GROMACS software. With this package it is possible to model distance restraints, dihedral restraints and orientation restraints. The output from the script is validated by performing simulations with and without restraints, including the ab initio refinement of one peptide.</p></div></div></div>

NMR Refinement and Peptide Folding Using the GROMACS Software

<div><div><div><p>Nuclear magnetic resonance spectroscopy is used routinely for studying the three-dimensional structures and dynamics of proteins. Structure determination is usually done by adding restraints based upon NMR data to a classical energy function and performing restrained molecular simulations. Here we report on the implementation of a script to extract NMR restraints from a NMR-STAR file and export it to the GROMACS software. With this package it is possible to model distance restraints, dihedral restraints and orientation restraints. The output from the script is validated by performing simulations with and without restraints, including the ab initio refinement of one peptide.</p></div></div></div>

The MPS1 kinase NTE region has helical propensity and preferred conformations towards the TPR domain

The mitotic spindle assembly checkpoint (SAC) ensures accurate segregation of chromosomes by preventing onset of anaphase until all chromosomes are properly attached to spindle microtubules. The Monopolar spindle 1 (MPS1) kinase is one of the SAC components, localizing at unattached kinetochores by an N-terminal localization module. This module comprises a flexible NTE module and the TPR domain, which we previously characterized for their contribution to kinetochore binding. Here we discuss the conformations of the highly flexible NTE with respect to the TPR domain, using paramagnetic NMR. The distance restraints derived from paramagnetic relaxation enhancements (PREs) show that the mobile NTE can be found in proximity of a large but specific part of the surface area of the TPR domain. To sample the conformational space of the NTE in the context of the NTE-TPR module, we used the ab initio Rosetta approach supplemented by paramagnetic NMR restraints. We find that many NTE residues have a propensity to form helical structures and that the module localizes at the convex surface of the TPR domain. This work demonstrates the highly dynamic nature of the interactions between the NTE and TPR domains and it shows that the convex rather than the canonical concave TPR surface mediates interactions, leading to the auto-inhibition that the TPR exerts upon the NTE region in the context of SAC signaling.

Determining structures of biomacromolecular complexes from ambiguous NMR restraints and cryoEM data

Integrated structural biology aims at combining different techniques to tackle challenging systems. Where individual techniques are not delivering structures of suitable quality, harnessing the strengths of various methods can often overcome this problem. X-ray crystallography and NMR have been the two most widely applied structural biology disciplines. In recent years cryoelectron microscopy (cryoEM) has become ever more powerful and is now capable of providing structures at resolutions comparable to those common in X-ray crystallography. Unfortunately, both NMR and cryoEM have inherent limitations on the system under study. However, the two techniques can be considered somewhat complementary as NMR has an upper and cryoEM a lower molecular weight (MW) limit. Here, we present a joint NMR and cryoEM methodology for the determination of biomacromolecular structures at the boundary region between the MW limits of the two techniques. The method relies on measuring chemical shift perturbations, which is the most readily accessible NMR parameter for characterizing the interaction of biomacromolecular complexes. Low-resolution cryoEM information yields global information on the shape of the complex and is used for complementing the local NMR data. We have successfully applied this method to the model system histidine-containing phosphoprotein (HPr) in complex with the glucose-specific acceptor protein IIAGlc from Escherichia coli.

Integrative Approaches in Structural Biology: A More Complete Picture from the Combination of Individual Techniques

With the recent technological and computational advancements, structural biology has begun to tackle more and more difficult questions, including complex biochemical pathways and transient interactions among macromolecules. This has demonstrated that, to approach the complexity of biology, one single technique is largely insufficient and unable to yield thorough answers, whereas integrated approaches have been more and more adopted with successful results. Traditional structural techniques (X-ray crystallography and Nuclear Magnetic Resonance (NMR)) and the emerging ones (cryo-electron microscopy (cryo-EM), Small Angle X-ray Scattering (SAXS)), together with molecular modeling, have pros and cons which very nicely complement one another. In this review, three examples of synergistic approaches chosen from our previous research will be revisited. The first shows how the joint use of both solution and solid-state NMR (SSNMR), X-ray crystallography, and cryo-EM is crucial to elucidate the structure of polyethylene glycol (PEG)ylated asparaginase, which would not be obtainable through any of the techniques taken alone. The second deals with the integrated use of NMR, X-ray crystallography, and SAXS in order to elucidate the catalytic mechanism of an enzyme that is based on the flexibility of the enzyme itself. The third one shows how it is possible to put together experimental data from X-ray crystallography and NMR restraints in order to refine a protein model in order to obtain a structure which simultaneously satisfies both experimental datasets and is therefore closer to the ‘real structure’.