Traditional Biomolecular Structure Determination by NMR Spectroscopy Allows for Major Errors

One of the major goals of structural genomics projects is to determine the three-dimensional structure of representative members of as many different fold families as possible. Comparative modeling is expected to fill the remaining gaps by providing structural models of homologs of the experimentally determined proteins. However, for such an approach to be successful it is essential that the quality of the experimentally determined structures is adequate. In an attempt to build a homology model for the protein dynein light chain 2A (DLC2A) we found two potential templates, both experimentally determined nuclear magnetic resonance (NMR) structures originating from structural genomics efforts. Despite their high sequence identity (96%), the folds of the two structures are markedly different. This urged us to perform in-depth analyses of both structure ensembles and the deposited experimental data, the results of which clearly identify one of the two models as largely incorrect. Next, we analyzed the quality of a large set of recent NMR-derived structure ensembles originating from both structural genomics projects and individual structure determination groups. Unfortunately, a visual inspection of structures exhibiting lower quality scores than DLC2A reveals that the seriously flawed DLC2A structure is not an isolated incident. Overall, our results illustrate that the quality of NMR structures cannot be reliably evaluated using only traditional experimental input data and overall quality indicators as a reference and clearly demonstrate the urgent need for a tight integration of more sophisticated structure validation tools in NMR structure determination projects. In contrast to common methodologies where structures are typically evaluated as a whole, such tools should preferentially operate on a per-residue basis

Archiving and disseminating integrative structure models.

Limitations in the applicability, accuracy, and precision of individual structure characterization methods can sometimes be overcome via an integrative modeling approach that relies on information from all available sources, including all available experimental data and prior models. The open-source Integrative Modeling Platform (IMP) is one piece of software that implements all computational aspects of integrative modeling. To maximize the impact of integrative structures, the coordinates should be made publicly available, as is already the case for structures based on X-ray crystallography, NMR spectroscopy, and electron microscopy. Moreover, the associated experimental data and modeling protocols should also be archived, such that the original results can easily be reproduced. Finally, it is essential that the integrative structures are validated as part of their publication and deposition. A number of research groups have already developed software to implement integrative modeling and have generated a number of structures, prompting the formation of an Integrative/Hybrid Methods Task Force. Following the recommendations of this task force, the existing PDBx/mmCIF data representation used for atomic PDB structures has been extended to address the requirements for archiving integrative structural models. This IHM-dictionary adds a flexible model representation, including coarse graining, models in multiple states and/or related by time or other order, and multiple input experimental information sources. A prototype archiving system called PDB-Dev ( https://pdb-dev.wwpdb.org ) has also been created to archive integrative structural models, together with a Python library to facilitate handling of integrative models in PDBx/mmCIF format

Conceptual-level workflow modeling of scientific experiments using NMR as a case study

BACKGROUND: Scientific workflows improve the process of scientific experiments by making computations explicit, underscoring data flow, and emphasizing the participation of humans in the process when intuition and human reasoning are required. Workflows for experiments also highlight transitions among experimental phases, allowing intermediate results to be verified and supporting the proper handling of semantic mismatches and different file formats among the various tools used in the scientific process. Thus, scientific workflows are important for the modeling and subsequent capture of bioinformatics-related data. While much research has been conducted on the implementation of scientific workflows, the initial process of actually designing and generating the workflow at the conceptual level has received little consideration. RESULTS: We propose a structured process to capture scientific workflows at the conceptual level that allows workflows to be documented efficiently, results in concise models of the workflow and more-correct workflow implementations, and provides insight into the scientific process itself. The approach uses three modeling techniques to model the structural, data flow, and control flow aspects of the workflow. The domain of biomolecular structure determination using Nuclear Magnetic Resonance spectroscopy is used to demonstrate the process. Specifically, we show the application of the approach to capture the workflow for the process of conducting biomolecular analysis using Nuclear Magnetic Resonance (NMR) spectroscopy. CONCLUSION: Using the approach, we were able to accurately document, in a short amount of time, numerous steps in the process of conducting an experiment using NMR spectroscopy. The resulting models are correct and precise, as outside validation of the models identified only minor omissions in the models. In addition, the models provide an accurate visual description of the control flow for conducting biomolecular analysis using NMR spectroscopy experiment

Structural Biology by NMR: Structure, Dynamics, and Interactions

The function of bio-macromolecules is determined by both their 3D structure and conformational dynamics. These molecules are inherently flexible systems displaying a broad range of dynamics on time-scales from picoseconds to seconds. Nuclear Magnetic Resonance (NMR) spectroscopy has emerged as the method of choice for studying both protein structure and dynamics in solution. Typically, NMR experiments are sensitive both to structural features and to dynamics, and hence the measured data contain information on both. Despite major progress in both experimental approaches and computational methods, obtaining a consistent view of structure and dynamics from experimental NMR data remains a challenge. Molecular dynamics simulations have emerged as an indispensable tool in the analysis of NMR data

Automatic \u3csup\u3e13\u3c/sup\u3eC Chemical Shift Reference Correction of Protein NMR Spectral Data Using Data Mining and Bayesian Statistical Modeling

Nuclear magnetic resonance (NMR) is a highly versatile analytical technique for studying molecular configuration, conformation, and dynamics, especially of biomacromolecules such as proteins. However, due to the intrinsic properties of NMR experiments, results from the NMR instruments require a refencing step before the down-the-line analysis. Poor chemical shift referencing, especially for 13C in protein Nuclear Magnetic Resonance (NMR) experiments, fundamentally limits and even prevents effective study of biomacromolecules via NMR. There is no available method that can rereference carbon chemical shifts from protein NMR without secondary experimental information such as structure or resonance assignment. To solve this problem, we constructed a Bayesian probabilistic framework that circumvents the limitations of previous reference correction methods that required protein resonance assignment and/or three-dimensional protein structure. Our algorithm named Bayesian Model Optimized Reference Correction (BaMORC) can detect and correct 13C chemical shift referencing errors before the protein resonance assignment step of analysis and without a three-dimensional structure. By combining the BaMORC methodology with a new intra-peaklist grouping algorithm, we created a combined method called Unassigned BaMORC that utilizes only unassigned experimental peak lists and the amino acid sequence. Unassigned BaMORC kept all experimental three-dimensional HN(CO)CACB-type peak lists tested within ± 0.4 ppm of the correct 13C reference value. On a much larger unassigned chemical shift test set, the base method kept 13C chemical shift referencing errors to within ± 0.45 ppm at a 90% confidence interval. With chemical shift assignments, Assigned BaMORC can detect and correct 13C chemical shift referencing errors to within ± 0.22 at a 90% confidence interval. Therefore, Unassigned BaMORC can correct 13C chemical shift referencing errors when it will have the most impact, right before protein resonance assignment and other downstream analyses are started. After assignment, chemical shift reference correction can be further refined with Assigned BaMORC. To further support a broader usage of these new methods, we also created a software package with web-based interface for the NMR community. This software will allow non-NMR experts to detect and correct 13C referencing errors at critical early data analysis steps, lowering the bar of NMR expertise required for effective protein NMR analysis

The eNMR platform for structural biology

The e-NMR project is a European cooperation initiative that aims at providing the bio-NMR user community with a software platform integrating and streamlining the computational approaches necessary for the analysis of bio-NMR data. The e-NMR platform is based on a Grid computational infrastructure. A main focus of the current implementation of the e-NMR platform is on streamlining structure determination protocols. Indeed, to facilitate the use of NMR spectroscopy in the life sciences, the eNMR consortium has set out to provide protocolized services through easy-to-use web interfaces, while still retaining sufficient flexibility to handle specific requests by expert users. Various programs relevant for structural biology applications are already available through the e-NMR portal, including HADDOCK, XPLOR-NIH, CYANA and csRosetta. The implementation of these services, and in particular the distribution of calculations to the GRID infrastructure, has required the development of specific tools. However, the GRID infrastructure is maintained completely transparent to the users. With more than 150 registered users, eNMR is currently the second largest European Virtual Organization in the life sciences

Novel weak alignment techniques for nuclear magnetic resonance spectroscopy and applications to biomolecular structure determination

Nuclear magnetic resonance spectroscopy has continuously been developing ever since its introduction as a structural method in bioscience. Recently established residual dipolar coupling techniques yield information on long-range order in weakly aligned samples as they define the orientation of vectors between nuclei in a common global reference frame. These data complement classical short-range information and have a unique potential especially for the characterization of non-globular states. This thesis describes the development of novel methods for the weak alignment of biomacromolecules in charged gels and for the measurement of long-range residual dipolar couplings in perdeuterated proteins. These weak alignment techniques and other nuclear magnetic resonance methods were applied to study the structure and folding of various proteins such as the fibritin folding nucleus, the minicollagen cysteine rich domain and human protein tyrosine phosphatase 1B

A Grid-enabled web portal for NMR structure refinement with AMBER

Abstract Motivation: The typical workflow for NMR structure determination involves collecting thousands of conformational restraints, calculating a bundle of 20–40 conformers in agreement with them and refining the energetics of these conformers. The structure calculation step employs simulated annealing based on molecular dynamics (MD) simulations with very simplified force fields. The value of refining the calculated conformers using restrained MD (rMD) simulations with state-of-art force fields is documented. This refinement however presents various subtleties, from the proper formatting of conformational restraints to the definition of suitable protocols. Results: We describe a web interface to set up and run calculations with the AMBER package, which we called AMPS-NMR (AMBER-based Portal Server for NMR structures). The interface allows the refinement of NMR structures through rMD. Some predefined protocols are provided for this purpose, which can be personalized; it is also possible to create an entirely new protocol. AMPS-NMR can handle various restraint types. Standard rMD refinement in explicit water of the structures of three different proteins are shown as examples. AMPS-NMR additionally includes a workspace for the user to store different calculations. As an ancillary service, a web interface to AnteChamber is available, enabling the calculation of force field parameters for organic molecules such as ligands in protein–ligand adducts. Availability and Implementation: AMPS-NMR is embedded within the NMR services of the WeNMR project and is available at http://py-enmr.cerm.unifi.it/access/index/amps-nmr; its use requires registration with a digital certificate. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online

Advances in Nuclear Magnetic Resonance for Drug Discovery

Background—Drug discovery is a complex and unpredictable endeavor with a high failure rate. Current trends in the pharmaceutical industry have exasperated these challenges and are contributing to the dramatic decline in productivity observed over the last decade. The industrialization of science by forcing the drug discovery process to adhere to assembly-line protocols is imposing unnecessary restrictions, such as short project time-lines. Recent advances in nuclear magnetic resonance are responding to these self-imposed limitations and are providing opportunities to increase the success rate of drug discovery. Objective/Method—A review of recent advancements in NMR technology that have the potential of significantly impacting and benefiting the drug discovery process will be presented. These include fast NMR data collection protocols and high-throughput protein structure determination, rapid protein-ligand co-structure determination, lead discovery using fragment-based NMR affinity screens, NMR metabolomics to monitor in vivo efficacy and toxicity for lead compounds, and the identification of new therapeutic targets through the functional annotation of proteins by FASTNMR. Conclusion—NMR is a critical component of the drug discovery process, where the versatility of the technique enables it to continually expand and evolve its role. NMR is expected to maintain this growth over the next decade with advancements in automation, speed of structure calculation, incell imaging techniques, and the expansion of NMR amenable targets

Structural investigation of the Bacillus subtilis morphogenic factor RodZ

A thesis to obtain a Master degree in Structural and Functional BiochemistryRodZ is a protein widely conserved in bacteria and a core component of the morphogenic apparatus of the cell. It is known to be required for assembly of the bacterial actin homologue, MreB, that controls cell wall synthesis and cell shape. The domain organization of RodZ consists of a well-conserved N-terminal (RodZn) with helix-turn-helix motif (HTH), a conserved transmembrane domain, and a conserved C-terminal domain (RodZc). RodZn, located in the cytoplasm, has been shown to interact with MreB actin-homologue by x-ray studies in T. maritima. However, the structure of RodZn from gram-positive B. subtilis showed low homology with the published one from gram-negative T. maritima. Here we present the solution structure of RodZn from B. subtilis determined for the first time, by NMR spectroscopy. Compared to previous structural data obtained from the crystallized RodZn from T. maritima and more recently from S. aureus, several differences could be observed, namely the length of the alpha-helices and the presence of an extended coil. Interaction studies were preformed between RodZn domain and MreB from which no significant results could be extrapolated. Since HTH motif is frequently associated with DNA interaction, the involvement of RodZn in DNA organization is being investigated. At the same time, RodZc domain, which structure has never been reported, was subject of study. Bioinformatic, biophysical and biochemical methodologies were employed to study this domain. A model based in a pseudo-ab initio methodology was built, revealing an Ig-like fold. The Ig superfamily is a large group of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. Therefore, RodZ is thought to be a protein that establishes a link between the inner side of the cell membrane and the outer side, promoting spatiotemporal coordination between peptidoglycan synthesis and cell division