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The current standard method for amino acid signal identification in protein NMR spectra is sequential assignment using triple-resonance experiments. Good software and elaborate heuristics exist, but the process remains laboriously manual. Machine learning does help, but its training databases need millions of samples that cover all relevant physics and every kind of instrumental artifact. In this communication, we offer a solution to this problem. We propose polyadic decompositions to store millions of simulated three-dimensional NMR spectra, on-the-fly generation of artifacts during training, a probabilistic way to incorporate prior and posterior information, and integration with the industry standard CcpNmr software framework. The resulting neural nets take [1H,13C] slices of mixed pyruvate-labeled HNCA spectra (different CA signal shapes for different residue types) and return an amino acid probability table. In combination with primary sequence information, backbones of common proteins (GB1, MBP, and INMT) are rapidly assigned from just the HNCA spectrum.

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Biomolecular structure analysis from experimental NMR studies generally relies on restraints derived from a combination of experimental and knowledge-based data. A challenge for the structural biology community has been a lack of standards for representing these restraints, preventing the establishment of uniform methods of model-vs-data structure validation against restraints and limiting interoperability between restraint-based structure modeling programs. The NEF and NMR-STAR formats provide a standardized approach for representing commonly used NMR restraints. Using these restraint formats, a standardized validation system for assessing structural models of biopolymers against restraints has been developed and implemented in the wwPDB OneDep data deposition-validation-biocuration system. The resulting wwPDB restraint violation report provides a model vs. data assessment of biomolecule structures determined using distance and dihedral restraints, with extensions to other restraint types currently being implemented. These tools are useful for assessing NMR models, as well as for assessing biomolecular structure predictions based on distance restraints.

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Biomolecular structure analysis from experimental NMR studies generally relies on restraints derived from a combination of experimental and knowledge-based data. A challenge for the structural biology community has been a lack of standards for representing these restraints, preventing the establishment of uniform methods of model-vs-data structure validation against restraints and limiting interoperability between restraint-based structure modeling programs. The NMR exchange (NEF) and NMR-STAR formats provide a standardized approach for representing commonly used NMR restraints. Using these restraint formats, a standardized validation system for assessing structural models of biopolymers against restraints has been developed and implemented in the wwPDB OneDep data deposition-validation-biocuration system. The resulting wwPDB Restraint Violation Report provides a model vs. data assessment of biomolecule structures determined using distance and dihedral restraints, with extensions to other restraint types currently being implemented. These tools are useful for assessing NMR models, as well as for assessing biomolecular structure predictions based on distance restraints.

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TRPV6 is a Transient Receptor Potential Vanilloid (TRPV) cation channel with high selectivity for Ca2+ ions. First identified in 1999 in a search for the gene which mediates intestinal Ca2+ absorption, its far more extensive repertoire as a guardian of intracellular Ca2+ has since become apparent. Studies on TRPV6-deficient mice demonstrated additional important roles in placental Ca2+ transport, fetal bone development and male fertility. The first reports of inherited deficiency in newborn babies appeared in 2018, revealing its physiological importance in humans. There is currently strong evidence that TRPV6 also contributes to the pathogenesis of some common cancers. The recently reported association of TRPV6 deficiency with non-alcoholic chronic pancreatitis suggests a role in normal pancreatic function. Over time and with greater awareness of TRPV6, other disease-associations are likely to emerge. Powerful analytical tools have provided invaluable insights into the structure and operation of TRPV6. Its roles in Ca2+ signaling and carcinogenesis, and the use of channel inhibitors in cancer treatment are being intensively investigated. This review first briefly describes the biochemistry and physiology of the channel, and analytical methods used to investigate these. The focus subsequently shifts to the clinical disorders associated with abnormal expression and the underlying pathophysiology. The aims of this review are to increase awareness of this channel, and to draw together findings from a wide range of sources which may help to formulate new ideas for further studies.

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Over the last century, the definitions of pharmaceutical drug and drug discovery have changed considerably. Evolving from an almost exclusively serendipitous approach, drug discovery nowadays involves several distinct, yet sometimes interconnected stages aimed at obtaining molecules able to interact with a defined biomolecular target, and triggering a suitable biological response. At each of the stages, a wide range of techniques are typically employed to obtain the results required to move the project into the next stage. High Throughput Screening (HTS) and Fragment Based Drug Design (FBDD) are the two main approaches used to identify drug-like candidates in the early stages of drug discovery. Nuclear Magnetic Resonance (NMR) spectroscopy has many applications in FBDD and is used extensively in industry as well as in academia. In this manuscript, we discuss the paths of both successful and unsuccessful molecules where NMR had a crucial part in their development. We specifically focus on the techniques used and describe strengths and weaknesses of each stage by examining several case studies. More precisely, we examine the development history from the primary screening to the final lead optimisation of AZD3839 interacting with BACE-1, ABT-199 interacting with BCL2/XL and S64315 interacting with MCL-1. Based on these studies, we derive observations and conclusions regarding the FBDD process by NMR and discuss its potential improvements.

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Fragment-based drug discovery or FBDD is one of the main methods used by industry and academia for identifying drug-like candidates in early stages of drug discovery. NMR has a significant impact at any stage of the drug discovery process, from primary identification of small molecules to the elucidation of binding modes for guiding optimisations. The essence of NMR as an analytical tool, however, requires the processing and analysis of relatively large amounts of single data items, e.g. spectra, which can be daunting when managed manually. One bottleneck in FBDD by NMR is a lack of adequate and well-integrated resources for NMR data analysis that are freely available to the community. Thus, scientists typically resort to manually inspecting large datasets and relying predominantly on subjective interpretations. In this manuscript, we present CcpNmr AnalysisScreen, a software package that provides computational tools for automated analysis of FBDD data by NMR. We outline how the quality of collected spectra can be evaluated quickly, and how robust workflows can be optimised for reliable and rapid hit identification. With an intuitive graphical user interface and powerful algorithms, AnalysisScreen enables easy analysis of the large datasets needed in the early process of drug discovery by NMR.

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Structures of biomolecular systems are increasingly computed by integrative modeling. In this approach, a structural model is constructed by combining information from multiple sources, including varied experimental methods and prior models. In 2019, a Workshop was held as a Biophysical Society Satellite Meeting to assess progress and discuss further requirements for archiving integrative structures. The primary goal of the Workshop was to build consensus for addressing the challenges involved in creating common data standards, building methods for federated data exchange, and developing mechanisms for validating integrative structures. The summary of the Workshop and the recommendations that emerged are presented here.

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NMR is one of the major techniques for investigating the structure, dynamics and interactions between biomolecules. However, non-experts often experience NMR experimentation and data analysis as intimidating. We discuss a simple yet powerful NMR technique, the so-called chemical shift perturbation (CSP) analysis, as a tool to elucidate macromolecular interactions in small- and medium-sized complexes, including protein-protein, protein-drug, and protein-DNA/RNA interactions. We discuss current software packages for NMR data analysis and present a new interactive graphical tool implemented in CcpNmr AnalysisAssign version-3, which can drastically reduce the time required for the CSP analysis. Lastly, we illustrate the usefulness of a protein three-dimensional structure for interpretation of the CSP data.

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The Sin3A complex acts as a transcriptional hub, integrating the function of diverse transcription factors with histone modifying enzymes, notably, histone deacetylases (HDAC) 1 and 2. The Sin3A protein sits at the centre of the complex, mediating multiple simultaneous protein-protein interactions via its four paired-amphipathic helix (PAH) domains (PAH1-4). The PAH domains contain a conserved four helical bundle, generating a hydrophobic cleft into which the single-helix of a Sin3-interaction domain (SID) is able to insert and bind with high affinity. Although they share a similar mode of interaction, the SIDs of different repressor proteins bind to only one of four potential PAH domains, due to the specific combination of hydrophobic residues at the interface. Here we report the identification of a highly conserved SID in the 5-methylcytosine dioxygenase, Tet1 (Tet1-SID), which interacts directly with the PAH1 domain of Sin3A. Using a combination of NMR spectroscopy and homology modelling we present a model of the PAH1/Tet1-SID complex, which binds in a Type-II orientation similar to Sap25. Mutagenesis of key residues show that the 11-amino acid Tet1-SID is necessary and sufficient for the interaction with Sin3A and is absolutely required for Tet1 to repress transcription in cells.

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The paralogues TRPV5 and TRPV6 belong to the vanilloid subfamily of the transient receptor potential (TRP) superfamily of ion channels, and both play an important role in overall Ca2+ homeostasis. The functioning of the channels centers on a tightly controlled Ca2+-dependent feedback mechanism in which the direct binding of the universal Ca2+-binding protein calmodulin (CaM) to the channel's C-terminal tail is required for channel inactivation. We have investigated this interaction at the atomic level and propose that under basal cellular Ca2+ concentrations CaM is constitutively bound to the channel's C-tail via CaM C-lobe only contacts. When the cytosolic Ca2+ concentration increases charging the apo CaM N-lobe with Ca2+, the CaM:TRPV6 complex rearranges and the TRPV6 C-tail further engages the CaM N-lobe via a crucial interaction involving L707. In a cellular context, mutation of L707 significantly increased the rate of channel inactivation. Finally, we present a model for TRPV6 CaM-dependent inactivation, which involves a novel so-called "two-tail" mechanism whereby CaM bridges two TRPV6 monomers resulting in closure of the channel pore.

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The transient receptor potential vanilloid channel subfamily member 5 (TRPV5) is a highly selective calcium ion channel predominately expressed in the kidney epithelium that plays an essential role in calcium reabsorption from renal infiltrate. In order to maintain Ca2+ homeostasis, TRPV5 possesses a tightly regulated negative feedback mechanism, where the ubiquitous Ca2+ binding protein calmodulin (CaM) directly binds to the intracellular TRPV5 C-terminus, thus regulating TRPV5. Here we report on the characterization of the TRPV5 C-terminal CaM binding site and its interaction with CaM at an atomistic level. We have solved the de novo solution structure of the TRPV5 C-terminus in complex with a CaM mutant, creating conditions that mimic the cellular basal Ca2+ state. We demonstrate that under these conditions the TRPV5 C-terminus is exclusively bound to the CaM C-lobe only, while it confers conformational freedom to the CaM N-lobe. We also show that at elevated calcium levels, additional interactions between the TRPV5 C-terminus and CaM N-lobe occur, resulting in formation of a tight 1:1 complex, effectively making the N-lobe the calcium sensor. Together, these data are consistent with and support the novel model for Ca2+/CaM-dependent inactivation of TRPV channels as proposed by Bate and co-workers [ Bate , N. , et al. ( 2018 ) Biochemistry , ( 57), DOI: 10.1021/acs.biochem.7b01286 ].

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NMR spectroscopy is an indispensably powerful technique for the analysis of biomolecules under ambient conditions, both for structural- and functional studies. However, in practice the complexity of the technique has often frustrated its application by non-specialists. In this paper, we present CcpNmr version-3, the latest software release from the Collaborative Computational Project for NMR, for all aspects of NMR data analysis, including liquid- and solid-state NMR data. This software has been designed to be simple, functional and flexible, and aims to ensure that routine tasks can be performed in a straightforward manner. We have designed the software according to modern software engineering principles and leveraged the capabilities of modern graphics libraries to simplify a variety of data analysis tasks. We describe the process of backbone assignment as an example of the flexibility and simplicity of implementing workflows, as well as the toolkit used to create the necessary graphics for this workflow. The package can be downloaded from www.ccpn.ac.uk/v3-software/downloads and is freely available to all non-profit organisations.

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We performed a comprehensive structure validation of both automated and manually generated structures of the 10 targets of the CASD-NMR-2013 effort. We established that automated structure determination protocols are capable of reliably producing structures of comparable accuracy and quality to those generated by a skilled researcher, at least for small, single domain proteins such as the ten targets tested. The most robust results appear to be obtained when NOESY peak lists are used either as the primary input data or to augment chemical shift data without the need to manually filter such lists. A detailed analysis of the long-range NOE restraints generated by the different programs from the same data showed a surprisingly low degree of overlap. Additionally, we found that there was no significant correlation between the extent of the NOE restraint overlap and the accuracy of the structure. This result was surprising given the importance of NOE data in producing good quality structures. We suggest that this could be explained by the information redundancy present in NOEs between atoms contained within a fixed covalent network.

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The second round of the community-wide initiative Critical Assessment of automated Structure Determination of Proteins by NMR (CASD-NMR-2013) comprised ten blind target datasets, consisting of unprocessed spectral data, assigned chemical shift lists and unassigned NOESY peak and RDC lists, that were made available in both curated (i.e. manually refined) or un-curated (i.e. automatically generated) form. Ten structure calculation programs, using fully automated protocols only, generated a total of 164 three-dimensional structures (entries) for the ten targets, sometimes using both curated and un-curated lists to generate multiple entries for a single target. The accuracy of the entries could be established by comparing them to the corresponding manually solved structure of each target, which was not available at the time the data were provided. Across the entire data set, 71 % of all entries submitted achieved an accuracy relative to the reference NMR structure better than 1.5 Å. Methods based on NOESY peak lists achieved even better results with up to 100% of the entries within the 1.5 Å threshold for some programs. However, some methods did not converge for some targets using un-curated NOESY peak lists. Over 90% of the entries achieved an accuracy better than the more relaxed threshold of 2.5 Å that was used in the previous CASD-NMR-2010 round. Comparisons between entries generated with un-curated versus curated peaks show only marginal improvements for the latter in those cases where both calculations converged.

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CcpNmr Analysis provides a streamlined pipeline for both NMR chemical shift assignment and structure determination of biological macromolecules. In addition, it encompasses tools to analyse the many additional experiments that make NMR such a pivotal technique for research into complex biological questions. This report describes how CcpNmr Analysis can seamlessly link together all of the tasks in the NMR structure-determination process. It details each of the stages from generating NMR restraints [distance, dihedral, hydrogen bonds and residual dipolar couplings (RDCs)], exporting these to and subsequently re-importing them from structure-calculation software (such as the programs CYANA or ARIA) and analysing and validating the results obtained from the structure calculation to, ultimately, the streamlined deposition of the completed assignments and the refined ensemble of structures into the PDBe repository. Until recently, such solution-structure determination by NMR has been quite a laborious task, requiring multiple stages and programs. However, with the new enhancements to CcpNmr Analysis described here, this process is now much more intuitive and efficient and less error-prone.

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NMR is a well-established method to characterize the structure and dynamics of biomolecules in solution. High-quality structures can now be produced thanks to both experimental advances and computational developments that incorporate new NMR parameters and improved protocols and force fields in the structure calculation and refinement process. In this chapter, we give a short overview of the various types of NMR data that can provide structural information, and then focus on the structure calculation methodology itself. We discuss and illustrate with tutorial examples "classical" structure calculation, refinement, and structure validation approaches.

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ATP-sensitive potassium channels play key roles in many tissues by coupling metabolic status to membrane potential. In contrast with other potassium channels, the pore-forming Kir6 subunits must co-assemble in hetero-octameric complexes with ATP-binding cassette (ABC) family sulfonylurea receptor (SUR) subunits to facilitate cell surface expression. Binding of nucleotides and drugs to SUR regulates channel gating but how these responses are communicated within the complex has remained elusive to date. We have now identified an electrostatic interaction, forming part of a functional interface between the cytoplasmic nucleotide-binding domain-2 of SUR2 subunits and the distal C-terminus of Kir6 polypeptides that determines channel response to nucleotide, potassium channel opener and antagonist. Mutation of participating residues disrupted physical interaction and regulation of expressed channels, properties that were restored in paired charge-swap mutants. Equivalent interactions were identified in Kir6.1- and Kir6.2-containing channels suggesting a conserved mechanism of allosteric regulation.

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Biomolecular structures at atomic resolution present a valuable resource for the understanding of biology. NMR spectroscopy accounts for 11% of all structures in the PDB repository. In response to serious problems with the accuracy of some of the NMR-derived structures and in order to facilitate proper analysis of the experimental models, a number of program suites are available. We discuss nine of these tools in this review: PROCHECK-NMR, PSVS, GLM-RMSD, CING, Molprobity, Vivaldi, ResProx, NMR constraints analyzer and QMEAN. We evaluate these programs for their ability to assess the structural quality, restraints and their violations, chemical shifts, peaks and the handling of multi-model NMR ensembles. We document both the input required by the programs and output they generate. To discuss their relative merits we have applied the tools to two representative examples from the PDB: a small, globular monomeric protein (Staphylococcal nuclease from S. aureus, PDB entry 2kq3) and a small, symmetric homodimeric protein (a region of human myosin-X, PDB entry 2lw9).

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