Cryo-EM structure and B-factor refinement with ensemble representation.

Cryo-EM experiments produce images of macromolecular assemblies that are combined to produce three-dimensional density maps. Typically, atomic models of the constituent molecules are fitted into these maps, followed by a density-guided refinement. We introduce TEMPy-ReFF, a method for atomic structure refinement in cryo-EM density maps. Our method represents atomic positions as components of a Gaussian mixture model, utilising their variances as B-factors, which are used to derive an ensemble description. Extensively tested on a substantial dataset of 229 cryo-EM maps from EMDB ranging in resolution from 2.1-4.9 Å with corresponding PDB and CERES atomic models, our results demonstrate that TEMPy-ReFF ensembles provide a superior representation of cryo-EM maps. On a single-model basis, it performs similarly to the CERES re-refinement protocol, although there are cases where it provides a better fit to the map. Furthermore, our method enables the creation of composite maps free of boundary artefacts. TEMPy-ReFF is useful for better interpretation of flexible structures, such as those involving RNA, DNA or ligands.

RIBFIND2: Identifying rigid bodies in protein and nucleic acid structures.

Molecular structures are often fitted into cryo-EM maps by flexible fitting. When this requires large conformational changes, identifying rigid bodies can help optimize the model-map fit. Tools for identifying rigid bodies in protein structures exist, however an equivalent for nucleic acid structures is lacking. With the increase in cryo-EM maps containing RNA and progress in RNA structure prediction, there is a need for such tools. We previously developed RIBFIND, a program for clustering protein secondary structures into rigid bodies. In RIBFIND2, this approach is extended to nucleic acid structures. RIBFIND2 can identify biologically relevant rigid bodies in important groups of complex RNA structures, capturing a wide range of dynamics, including large rigid-body movements. The usefulness of RIBFIND2-assigned rigid bodies in cryo-EM model refinement was demonstrated on three examples, with two conformations each: Group II Intron complexed IEP, Internal Ribosome Entry Site and the Processome, using cryo-EM maps at 2.7-5 Å resolution. A hierarchical refinement approach, performed on progressively smaller sets of RIBFIND2 rigid bodies, was clearly shown to have an advantage over classical all-atom refinement. RIBFIND2 is available via a web server with structure visualization and as a standalone tool.

Linking Gas-Phase and Solution-Phase Protein Unfolding via Mobile Proton Simulations.

Native mass spectrometry coupled to ion mobility (IM-MS) combined with collisional activation (CA) of ions in the gas phase (in vacuo) is an important method for the study of protein unfolding. It has advantages over classical biophysical and structural techniques as it can be used to analyze small volumes of low-concentration heterogeneous mixtures while maintaining solution-like behavior and does not require labeling with fluorescent or other probes. It is unclear, however, whether the unfolding observed during collision activation experiments mirrors solution-phase unfolding. To bridge the gap between in vacuo and in-solution behavior, we use unbiased molecular dynamics (MD) to create in silico models of in vacuo unfolding of a well-studied protein, the N-terminal domain of ribosomal L9 (NTL9) protein. We utilize a mobile proton algorithm (MPA) to create 100 thermally unfolded and coulombically unfolded in silico models for observed charge states of NTL9. The unfolding behavior in silico replicates the behavior in-solution and is in line with the in vacuo observations; however, the theoretical collision cross section (CCS) of the in silico models was lower compared to that of the in vacuo data, which may reflect reduced sampling.

Evaluation of acquisition modes for semi-quantitative analysis by targeted and untargeted mass spectrometry.

RATIONALE: Analyte quantitation by mass spectrometry underpins a diverse range of scientific endeavors. The fast-growing field of mass spectrometer development has resulted in several targeted and untargeted acquisition modes suitable for these applications. By characterizing the acquisition methods available on an ion mobility (IM)-enabled orthogonal acceleration time-of-flight (oa-ToF) instrument, the optimum modes for analyte semi-quantitation can be deduced. METHODS: Serial dilutions of commercial metabolite, peptide, or cross-linked peptide analytes were prepared in matrices of human urine or Escherichia coli digest. Each analyte dilution was introduced into an IM separation-enabled oa-ToF mass spectrometer by reversed-phase liquid chromatography and electrospray ionization. Data were acquired for each sample in duplicate using nine different acquisition modes, including four IM-enabled acquisitions modes, available on the mass spectrometer. RESULTS: Five (metabolite) or seven (peptide/cross-linked peptide) point calibration curves were prepared for analytes across each of the acquisition modes. A nonlinear response was observed at high concentrations for some modes, attributed to saturation effects. Two correction methods, one MS1 isotope-correction and one MS2 ion intensity-correction, were applied to address this observation, resulting in an up to twofold increase in dynamic range. By averaging the semi-quantitative results across analyte classes, two parameters, linear dynamic range (LDR) and lower limit of quantification (LLOQ), were determined to evaluate each mode. CONCLUSION: A comparison of the acquisition modes revealed that data-independent acquisition and parallel reaction monitoring methods are most robust for semi-quantitation when considering achievable LDR and LLOQ. IM-enabled modes exhibited sensitivity increases, but a simultaneous reduction in dynamic range required correction methods to recover. These findings will assist users in identifying the optimum acquisition mode for their analyte quantitation needs, supporting a diverse range of applications and providing guidance for future acquisition mode developments.

Integration of Mass Spectrometry Data for Structural Biology.

Mass spectrometry (MS) is increasingly being used to probe the structure and dynamics of proteins and the complexes they form with other macromolecules. There are now several specialized MS methods, each with unique sample preparation, data acquisition, and data processing protocols. Collectively, these methods are referred to as structural MS and include cross-linking, hydrogen-deuterium exchange, hydroxyl radical footprinting, native, ion mobility, and top-down MS. Each of these provides a unique type of structural information, ranging from composition and stoichiometry through to residue level proximity and solvent accessibility. Structural MS has proved particularly beneficial in studying protein classes for which analysis by classic structural biology techniques proves challenging such as glycosylated or intrinsically disordered proteins. To capture the structural details for a particular system, especially larger multiprotein complexes, more than one structural MS method with other structural and biophysical techniques is often required. Key to integrating these diverse data are computational strategies and software solutions to facilitate this process. We provide a background to the structural MS methods and briefly summarize other structural methods and how these are combined with MS. We then describe current state of the art approaches for the integration of structural MS data for structural biology. We quantify how often these methods are used together and provide examples where such combinations have been fruitful. To illustrate the power of integrative approaches, we discuss progress in solving the structures of the proteasome and the nuclear pore complex. We also discuss how information from structural MS, particularly pertaining to protein dynamics, is not currently utilized in integrative workflows and how such information can provide a more accurate picture of the systems studied. We conclude by discussing new developments in the MS and computational fields that will further enable in-cell structural studies.

Cryo-EM targets in CASP14.

Structures of seven CASP14 targets were determined using cryo-electron microscopy (cryo-EM) technique with resolution between 2.1 and 3.8 Å. We provide an evaluation of the submitted models versus the experimental data (cryo-EM density maps) and experimental reference structures built into the maps. The accuracy of models is measured in terms of coordinate-to-density and coordinate-to-coordinate fit. A-posteriori refinement of the most accurate models in their corresponding cryo-EM density resulted in structures that are close to the reference structure, including some regions with better fit to the density. Regions that were found to be less "refineable" correlate well with regions of high diversity between the CASP models and low goodness-of-fit to density in the reference structure.

Cyclic Ion Mobility-Collision Activation Experiments Elucidate Protein Behavior in the Gas Phase.

Ion mobility coupled to mass spectrometry (IM-MS) is widely used to study protein dynamics and structure in the gas phase. Increasing the energy with which the protein ions are introduced to the IM cell can induce them to unfold, providing information on the comparative energetics of unfolding between different proteoforms. Recently, a high-resolution cyclic IM-mass spectrometer (cIM-MS) was introduced, allowing multiple, consecutive tandem IM experiments (IMn) to be carried out. We describe a tandem IM technique for defining detailed protein unfolding pathways and the dynamics of disordered proteins. The method involves multiple rounds of IM separation and collision activation (CA): IM-CA-IM and CA-IM-CA-IM. Here, we explore its application to studies of a model protein, cytochrome C, and dimeric human islet amyloid polypeptide (hIAPP), a cytotoxic and amyloidogenic peptide involved in type II diabetes. In agreement with prior work using single stage IM-MS, several unfolding events are observed for cytochrome C. IMn-MS experiments also show evidence of interconversion between compact and extended structures. IMn-MS data for hIAPP shows interconversion prior to dissociation, suggesting that the certain conformations have low energy barriers between them and transition between compact and extended forms.

TEMPy2: a Python library with improved 3D electron microscopy density-fitting and validation workflows.

Structural determination of molecular complexes by cryo-EM requires large, often complex processing of the image data that are initially obtained. Here, TEMPy2, an update of the TEMPy package to process, optimize and assess cryo-EM maps and the structures fitted to them, is described. New optimization routines, comprehensive automated checks and workflows to perform these tasks are described.

Automated Modeling and Validation of Protein Complexes in Cryo-EM Maps.

The resolving power of cryo-EM experiments has dramatically improved in recent years. However, many cryo-EM maps may still not achieve a resolution that is sufficiently high to allow model building directly from the map. Instead, it is common practice to fit an initial atomic model to the map and refine this model. Depending on the resolution and whether the structure suffers from inherent flexibility or experimental limitations, different methods can be applied, to obtain high-quality, well-fitted atomic model of the macromolecular assembly represented by the map, and to assess its properties. In this review, we describe some of these methods, with the main focus on those that have been developed in our group over the last decade.

Cryo-electron microscopy targets in CASP13: Overview and evaluation of results.

Structures of seven CASP13 targets were determined using cryo-electron microscopy (cryo-EM) technique with resolution between 3.0 and 4.0 Å. We provide an overview of the experimentally derived structures and describe results of the numerical evaluation of the submitted models. The evaluation is carried out by comparing coordinates of models to those of reference structures (CASP-style evaluation), as well as checking goodness-of-fit of modeled structures to the cryo-EM density maps. The performance of contributing research groups in the CASP-style evaluation is measured in terms of backbone accuracy, all-atom local geometry and similarity of inter-subunit interfaces. The results on the cryo-EM targets are compared with those on the whole set of eighty CASP13 targets. A posteriori refinement of the best models in their corresponding cryo-EM density maps resulted in structures that are very close to the reference structure, including some regions with better fit to the density.

A Polymer Physics Framework for the Entropy of Arbitrary Pseudoknots.

The accurate prediction of RNA secondary structure from primary sequence has had enormous impact on research from the past 40 years. Although many algorithms are available to make these predictions, the inclusion of non-nested loops, termed pseudoknots, still poses challenges arising from two main factors: 1) no physical model exists to estimate the loop entropies of complex intramolecular pseudoknots, and 2) their NP-complete enumeration has impeded their study. Here, we address both challenges. First, we develop a polymer physics model that can address arbitrarily complex pseudoknots using only two parameters corresponding to concrete physical quantities-over an order of magnitude fewer than the sparsest state-of-the-art phenomenological methods. Second, by coupling this model to exhaustive enumeration of the set of possible structures, we compute the entire free energy landscape of secondary structures resulting from a primary RNA sequence. We demonstrate that for RNA structures of ∼80 nucleotides, with minimal heuristics, the complete enumeration of possible secondary structures can be accomplished quickly despite the NP-complete nature of the problem. We further show that despite our loop entropy model's parametric sparsity, it performs better than or on par with previously published methods in predicting both pseudoknotted and non-pseudoknotted structures on a benchmark data set of RNA structures of ≤80 nucleotides. We suggest ways in which the accuracy of the model can be further improved.

Multifunctional energy landscape for a DNA G-quadruplex: An evolved molecular switch.

We explore the energy landscape for a four-fold telomere repeat, obtaining interconversion pathways between six experimentally characterised G-quadruplex topologies. The results reveal a multi-funnel system, with a variety of intermediate configurations and misfolded states. This organisation is identified with the intrinsically multi-functional nature of the system, suggesting a new paradigm for the classification of such biomolecules and clarifying issues regarding apparently conflicting experimental results.

The crystal structure of the 5΄ functional domain of the transcription riboregulator 7SK.

In vertebrates, the 7SK RNA forms the scaffold of a complex, which regulates transcription pausing of RNA-polymerase II. By binding to the HEXIM protein, the complex comprising proteins LARP7 and MePCE captures the positive transcription elongation factor P-TEFb and prevents phosphorylation of pausing factors. The HEXIM-binding site embedded in the 5΄-hairpin of 7SK (HP1) encompasses a short signature sequence, a GAUC repeat framed by single-stranded uridines. The present crystal structure of HP1 shows a remarkably straight helical stack involving several unexpected triples formed at a central region. Surprisingly, two uridines of the signature sequence make triple interactions in the major groove of the (GAUC)2. The third uridine is turned outwards or inward, wedging between the other uridines, thus filling the major groove. A molecular dynamics simulation indicates that these two conformations of the signature sequence represent stable alternatives. Analyses of the interaction with the HEXIM protein confirm the importance of the triple interactions at the signature sequence. Altogether, the present structural analysis of 7SK HP1 highlights an original mechanism of swapping bases, which could represent a possible '7SK signature' and provides new insight into the functional importance of the plasticity of RNA.

Coarse-Grained Simulations Complemented by Atomistic Molecular Dynamics Provide New Insights into Folding and Unfolding of Human Telomeric G-Quadruplexes.

G-quadruplexes are the most important noncanonical DNA architectures. Many quadruplex-forming sequences, including the human telomeric sequence d(GGGTTA)n, have been investigated due to their implications in cancer and other diseases, and because of their potential in DNA-based nanotechnology. Despite the availability of atomistic structural studies of folded G-quadruplexes, their folding pathways remain mysterious, and mutually contradictory models of folding coexist in the literature. Recent experiments convincingly demonstrated that G-quadruplex folding often takes days to reach thermodynamic equilibrium. Based on atomistic simulations of diverse classes of intermediates in G-quadruplex folding, we have suggested that the folding is an extremely multipathway process combining a kinetic partitioning mechanism with conformational diffusion. However, complete G-quadruplex folding is far beyond the time scale of atomistic simulations. Here we use high-resolution coarse-grained simulations to investigate potential unfolding intermediates, whose structural dynamics are then further explored with all-atom simulations. This multiscale approach indicates how various pathways are interconnected in a complex network. Spontaneous conversions between different folds are observed. We demonstrate the inability of simple order parameters, such as radius of gyration or the number of native H-bonds, to describe the folding landscape of the G-quadruplexes. Our study also provides information relevant to further development of the coarse-grained force field.

Coarse-Grained HiRE-RNA Model for ab Initio RNA Folding beyond Simple Molecules, Including Noncanonical and Multiple Base Pairings.

HiRE-RNA is a coarse-grained model for RNA structure prediction and the dynamical study of RNA folding. Using a reduced set of particles and detailed interactions accounting for base-pairing and stacking, we show that noncanonical and multiple base interactions are necessary to capture the full physical behavior of complex RNAs. In this paper, we give a full account of the model and present results on the folding, stability, and free energy surfaces of 16 systems with 12 to 76 nucleotides of increasingly complex architectures, ranging from monomers to dimers, using a total of 850 µs of simulation time.

Ab initio RNA folding.

RNA molecules are essential cellular machines performing a wide variety of functions for which a specific three-dimensional structure is required. Over the last several years, the experimental determination of RNA structures through x-ray crystallography and NMR seems to have reached a plateau in the number of structures resolved each year, but as more and more RNA sequences are being discovered, the need for structure prediction tools to complement experimental data is strong. Theoretical approaches to RNA folding have been developed since the late nineties, when the first algorithms for secondary structure prediction appeared. Over the last 10 years a number of prediction methods for 3D structures have been developed, first based on bioinformatics and data-mining, and more recently based on a coarse-grained physical representation of the systems. In this review we are going to present the challenges of RNA structure prediction and the main ideas behind bioinformatic approaches and physics-based approaches. We will focus on the description of the more recent physics-based phenomenological models and on how they are built to include the specificity of the interactions of RNA bases, whose role is critical in folding. Through examples from different models, we will point out the strengths of physics-based approaches, which are able not only to predict equilibrium structures, but also to investigate dynamical and thermodynamical behavior, and the open challenges to include more key interactions ruling RNA folding.

Epock: rapid analysis of protein pocket dynamics.

SUMMARY: The volume of an internal protein pocket is fundamental to ligand accessibility. Few programs that compute such volumes manage dynamic data from molecular dynamics (MD) simulations. Limited performance often prohibits analysis of large datasets. We present Epock, an efficient command-line tool that calculates pocket volumes from MD trajectories. A plugin for the VMD program provides a graphical user interface to facilitate input creation, run Epock and analyse the results. AVAILABILITY AND IMPLEMENTATION: Epock C++ source code, Python analysis scripts, VMD Tcl plugin, documentation and installation instructions are freely available at http://epock.bitbucket.org. CONTACT: benoist.laurent@gmail.com or baaden@smplinux.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

WEBnm@ v2.0: Web server and services for comparing protein flexibility.

BACKGROUND: Normal mode analysis (NMA) using elastic network models is a reliable and cost-effective computational method to characterise protein flexibility and by extension, their dynamics. Further insight into the dynamics-function relationship can be gained by comparing protein motions between protein homologs and functional classifications. This can be achieved by comparing normal modes obtained from sets of evolutionary related proteins. RESULTS: We have developed an automated tool for comparative NMA of a set of pre-aligned protein structures. The user can submit a sequence alignment in the FASTA format and the corresponding coordinate files in the Protein Data Bank (PDB) format. The computed normalised squared atomic fluctuations and atomic deformation energies of the submitted structures can be easily compared on graphs provided by the web user interface. The web server provides pairwise comparison of the dynamics of all proteins included in the submitted set using two measures: the Root Mean Squared Inner Product and the Bhattacharyya Coefficient. The Comparative Analysis has been implemented on our web server for NMA, WEBnm@, which also provides recently upgraded functionality for NMA of single protein structures. This includes new visualisations of protein motion, visualisation of inter-residue correlations and the analysis of conformational change using the overlap analysis. In addition, programmatic access to WEBnm@ is now available through a SOAP-based web service. Webnm@ is available at http://apps.cbu.uib.no/webnma . CONCLUSION: WEBnm@ v2.0 is an online tool offering unique capability for comparative NMA on multiple protein structures. Along with a convenient web interface, powerful computing resources, and several methods for mode analyses, WEBnm@ facilitates the assessment of protein flexibility within protein families and superfamilies. These analyses can give a good view of how the structures move and how the flexibility is conserved over the different structures.

The OPEP protein model: from single molecules, amyloid formation, crowding and hydrodynamics to DNA/RNA systems.

The OPEP coarse-grained protein model has been applied to a wide range of applications since its first release 15 years ago. The model, which combines energetic and structural accuracy and chemical specificity, allows the study of single protein properties, DNA-RNA complexes, amyloid fibril formation and protein suspensions in a crowded environment. Here we first review the current state of the model and the most exciting applications using advanced conformational sampling methods. We then present the current limitations and a perspective on the ongoing developments.

Coarse-grained simulations of RNA and DNA duplexes.

Although RNAs play many cellular functions, little is known about the dynamics and thermodynamics of these molecules. In principle, all-atom molecular dynamics simulations can investigate these issues, but with current computer facilities, these simulations have been limited to small RNAs and to short times. HiRe-RNA, a recently proposed high-resolution coarse-grained RNA that captures many geometric details such as base pairing and stacking, is able to fold RNA molecules to near-native structures in a short computational time. So far, it had been applied to simple hairpins, and here we present its application to duplexes of a couple dozen nucleotides and show how with replica exchange molecular dynamics (REMD) we can easily predict the correct double helix from a completely random configuration and study the dissociation curve. To show the versatility of our model, we present an application to a double stranded DNA molecule as well. A reconstruction algorithm allows us to obtain full atom structures from the coarse-grained model. Through atomistic molecular dynamics (MD), we can compare the dynamics starting from a representative structure of a low temperature replica or from the experimental structure, and show how the two are statistically identical, highlighting the validity of a coarse-grained approach for structured RNAs and DNAs.