On the complementarity of X-ray and NMR data.

X-ray crystallography and NMR contain complementary information for the structural characterization of biological macromolecules. X-ray diffraction is primarily sensitive to the overall shape of the molecule, whereas NMR is mostly sensitive to the atomic detail. Their combination can therefore provide a stronger justification for the resulting structure. For their combination we have recently proposed REFMAC-NMR, which relies on primary data from both techniques for joint refinement. This possibility raises the compelling question of how far the complementarity can be extended. In this paper, we describe an integrative approach to the refinement with NMR data of four X-ray structures of hen-egg-white lysozyme, solved at atomic resolution in four different crystal forms, and we demonstrate that the outcome critically depends on the crystal form itself, reflecting the sensitivity of NMR to fine details.

Assessing Structural Preferences of Unstructured Protein Regions by NMR.

Biomacromolecules, such as proteins, often exhibit significant motions intimately associated with their function. Intrinsically disordered proteins and proteins with intrinsically disordered regions, although extremely important for a plethora of cellular functions, are difficult to structurally characterize at the atomic level because the experimental parameters report on ensemble and time averages. Here, we demonstrate for the C-terminal domain of the human immunodeficiency virus type 1 capsid protein that NMR and, in particular, residual dipolar couplings (RDCs) measured for the folded portion of the protein can inform on the structural preferences of the unstructured portion using RDC-prediction tools and the maximum occurrence approach.

Correction to: Joint X-ray/NMR structure refinement of multidomain/multisubunit systems.

The article "Joint X-ray/NMR structure refinement of multidomain/multisubunit systems" written by "Azzurra Carlon, Enrico Ravera, Giacomo Parigi, Garib N. Murshudov and Claudio Luchinat" was originally published electronically on the publisher's internet portal (currently SpringerLink) on 11 October 2018 without open access.

Joint X-ray/NMR structure refinement of multidomain/multisubunit systems.

Data integration in structural biology has become a paradigm for the characterization of biomolecular systems, and it is now accepted that combining different techniques can fill the gaps in each other's blind spots. In this frame, one of the combinations, which we have implemented in REFMAC-NMR, is residual dipolar couplings from NMR together with experimental data from X-ray diffraction. The first are exquisitely sensitive to the local details but does not give any information about overall shape, whereas the latter encodes more the information about the overall shape but at the same time tends to miss the local details even at the highest resolutions. Once crystals are obtained, it is often rather easy to obtain a complete X-ray dataset, however it is time-consuming to obtain an exhaustive NMR dataset. Here, we discuss the effect of including a-priori knowledge on the properties of the system to reduce the number of experimental data needed to obtain a more complete picture. We thus introduce a set of new features of REFMAC-NMR that allow for improved handling of RDC data for multidomain proteins and multisubunit biomolecular complexes, and encompasses the use of pseudo-contact shifts as an additional source of NMR-based information. The new feature may either help in improving the refinement, or assist in spotting differences between the crystal and the solution data. We show three different examples where NMR and X-ray data can be reconciled to a unique structural model without invoking mobility.

Characterization of PEGylated Asparaginase: New Opportunities from NMR Analysis of Large PEGylated Therapeutics.

Resonance assignment and structural characterization of pharmacologically relevant proteins promise to improve understanding and safety of these proteins by rational design. However, the PEG coating that is used to evade the immune system also causes these molecules to "evade" the standard structural biology methodologies. We here demonstrate that it is possible to obtain the resonance assignment and a reliable structural model of large PEGylated proteins through an integrated approach encompassing NMR and X-ray crystallography.

Overview of refinement procedures within REFMAC5: utilizing data from different sources.

Refinement is a process that involves bringing into agreement the structural model, available prior knowledge and experimental data. To achieve this, the refinement procedure optimizes a posterior conditional probability distribution of model parameters, including atomic coordinates, atomic displacement parameters (B factors), scale factors, parameters of the solvent model and twin fractions in the case of twinned crystals, given observed data such as observed amplitudes or intensities of structure factors. A library of chemical restraints is typically used to ensure consistency between the model and the prior knowledge of stereochemistry. If the observation-to-parameter ratio is small, for example when diffraction data only extend to low resolution, the Bayesian framework implemented in REFMAC5 uses external restraints to inject additional information extracted from structures of homologous proteins, prior knowledge about secondary-structure formation and even data obtained using different experimental methods, for example NMR. The refinement procedure also generates the `best' weighted electron-density maps, which are useful for further model (re)building. Here, the refinement of macromolecular structures using REFMAC5 and related tools distributed as part of the CCP4 suite is discussed.

Paramagnetic NMR as a new tool in structural biology.

NMR (nuclear magnetic resonance) investigation through the exploitation of paramagnetic effects is passing from an approach limited to few specialists in the field to a generally applicable method that must be considered, especially for the characterization of systems hardly affordable with other techniques. This is mostly due to the fact that paramagnetic data are long range in nature, thus providing information for the structural and dynamic characterization of complex biomolecular architectures in their native environment. On the other hand, this information usually needs to be complemented by data from other sources. Integration of paramagnetic NMR with other techniques, and the development of protocols for a joint analysis of all available data, is fundamental for achieving a comprehensive characterization of complex biological systems. We describe here a few examples of the new possibilities offered by paramagnetic data used in integrated structural approaches.

A rule-based model of insulin signalling pathway.

BACKGROUND: The insulin signalling pathway (ISP) is an important biochemical pathway, which regulates some fundamental biological functions such as glucose and lipid metabolism, protein synthesis, cell proliferation, cell differentiation and apoptosis. In the last years, different mathematical models based on ordinary differential equations have been proposed in the literature to describe specific features of the ISP, thus providing a description of the behaviour of the system and its emerging properties. However, protein-protein interactions potentially generate a multiplicity of distinct chemical species, an issue referred to as "combinatorial complexity", which results in defining a high number of state variables equal to the number of possible protein modifications. This often leads to complex, error prone and difficult to handle model definitions. RESULTS: In this work, we present a comprehensive model of the ISP, which integrates three models previously available in the literature by using the rule-based modelling (RBM) approach. RBM allows for a simple description of a number of signalling pathway characteristics, such as the phosphorylation of signalling proteins at multiple sites with different effects, the simultaneous interaction of many molecules of the signalling pathways with several binding partners, and the information about subcellular localization where reactions take place. Thanks to its modularity, it also allows an easy integration of different pathways. After RBM specification, we simulated the dynamic behaviour of the ISP model and validated it using experimental data. We the examined the predicted profiles of all the active species and clustered them in four clusters according to their dynamic behaviour. Finally, we used parametric sensitivity analysis to show the role of negative feedback loops in controlling the robustness of the system. CONCLUSIONS: The presented ISP model is a powerful tool for data simulation and can be used in combination with experimental approaches to guide the experimental design. The model is available at http://sysbiobig.dei.unipd.it/ was submitted to Biomodels Database ( https://www.ebi.ac.uk/biomodels-main/ # MODEL 1604100005).

How to tackle protein structural data from solution and solid state: An integrated approach.

Long-range NMR restraints, such as diamagnetic residual dipolar couplings and paramagnetic data, can be used to determine 3D structures of macromolecules. They are also used to monitor, and potentially to improve, the accuracy of a macromolecular structure in solution by validating or "correcting" a crystal model. Since crystal structures suffer from crystal packing forces they may not be accurate models for the macromolecular structures in solution. However, the presence of real differences should be tested for by simultaneous refinement of the structure using both crystal and solution NMR data. To achieve this, the program REFMAC5 from CCP4 was modified to allow the simultaneous use of X-ray crystallographic and paramagnetic NMR data and/or diamagnetic residual dipolar couplings. Inconsistencies between crystal structures and solution NMR data, if any, may be due either to structural rearrangements occurring on passing from the solution to solid state, or to a greater degree of conformational heterogeneity in solution with respect to the crystal. In the case of multidomain proteins, paramagnetic restraints can provide the correct mutual orientations and positions of domains in solution, as well as information on the conformational variability experienced by the macromolecule.

Improved Accuracy from Joint X-ray and NMR Refinement of a Protein-RNA Complex Structure.

Integrated experimental approaches play an increasingly important role in structural biology, taking advantage of the complementary information provided by different techniques. In particular, the combination of NMR data with X-ray diffraction patterns may provide accurate and precise information about local conformations not available from average-resolution X-ray structures alone. Here, we refined the structure of a ternary protein-protein-RNA complex comprising three domains, Sxl and Unr, bound to a single-stranded region derived in the msl2 mRNA. The joint X-ray and NMR refinement reveals that-despite the poor quality of the fit found for the original structural model-the NMR data can be largely accommodated within the uncertainty in the atom positioning (structural noise) from the primary X-ray data and that the overall domain arrangements and binding interfaces are preserved on passing from the crystalline state to the solution. The refinement highlights local conformational differences, which provide additional information on specific features of the structure. For example, conformational dynamics and heterogeneity observed at the interface between the CSD1 and the Sxl protein components in the ternary complex are revealed by the combination of NMR and crystallographic data. The joint refinement protocol offers unique opportunities to detect structural differences arising from various experimental conditions and reveals static or dynamic differences in the conformation of the biomolecule between the solution and the crystals.

FANTEN: a new web-based interface for the analysis of magnetic anisotropy-induced NMR data.

Pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) arising from the presence of paramagnetic metal ions in proteins as well as RDCs due to partial orientation induced by external orienting media are nowadays routinely measured as a part of the NMR characterization of biologically relevant systems. PCSs and RDCs are becoming more and more popular as restraints (1) to determine and/or refine protein structures in solution, (2) to monitor the extent of conformational heterogeneity in systems composed of rigid domains which can reorient with respect to one another, and (3) to obtain structural information in protein-protein complexes. The use of both PCSs and RDCs proceeds through the determination of the anisotropy tensors which are at the origin of these NMR observables. A new user-friendly web tool, called FANTEN (Finding ANisotropy TENsors), has been developed for the determination of the anisotropy tensors related to PCSs and RDCs and has been made freely available through the WeNMR ( http://fanten-enmr.cerm.unifi.it:8080 ) gateway. The program has many new features not available in other existing programs, among which the possibility of a joint analysis of several sets of PCS and RDC data and the possibility to perform rigid body minimizations.

Pairwise binding competition experiments for sorting hub-protein/effector interaction hierarchy and simultaneous equilibria.

NMR experiments on proteins in simultaneous equilibria with multiple binding partners can provide a tool to understand complex biological interaction networks. Competition among proteins for binding to signaling hubs is often at the basis of the information transmission across signaling networks in every organism. Changes in affinity towards one or more partners, as well as changes of the relative concentration of the competing partners, can determine pathways alterations that lead to pathological consequences. Overall, the knowledge of the interaction hierarchy of the multiple partners to a single signaling hub can lead to new therapeutic strategies. Smith and Ikura (Nat Chem Biol 10:223­230, 2014) have recently proposed pairwise competition NMR experiments to determine the binding hierarchy in network interactions. We have taken the moves from their approach to show how from pairwise competition NMR experiments the ratios between the equilibrium constants for multiple binding partners can be determined, and thus, given their concentration in solution, the concentrations of all the possible complexes can be obtained.