The Bio3D packages for structural bioinformatics

Bio3D is a family of R packages for the analysis of biomolecular sequence, structure, and dynamics. Major functionality includes biomolecular database searching and retrieval, sequence and structure conservation analysis, ensemble normal mode analysis, protein structure and correlation network analysis, principal component, and related multivariate analysis methods. Here, we review recent package developments, including a new underlying segregation into separate packages for distinct analysis, and introduce a new method for structure analysis named ensemble difference distance matrix analysis (eDDM). The eDDM approach calculates and compares atomic distance matrices across large sets of homologous atomic structures to help identify the residue wise determinants underlying specific functional processes. An eDDM workflow is detailed along with an example application to a large protein family. As a new member of the Bio3D family, the Bio3D‐eddm package supports both experimental and theoretical simulation‐generated structures, is integrated with other methods for dissecting sequence‐structure–function relationships, and can be used in a highly automated and reproducible manner. Bio3D is distributed as an integrated set of platform independent open source R packages available from: http://thegrantlab.org/bio3d/ .     

High-throughput modeling and analysis of protein structural dynamics

Protein function is a dynamic property closely related to the conformational mechanisms of protein structure in its physiological environment. To understand and control the function of target proteins, it becomes increasingly important to develop methods and tools for predicting collective motions at the molecular level. In this article, we review computational methods for predicting conformational dynamics and discuss software tools for data analysis. In particular, we discuss a high-throughput, web-based system called iGNM for protein structural dynamics. iGNM contains a database of protein motions for more than 20 000 PDB structures and supports online calculations for newly deposited PDB structures or user-modified structures. iGNM allows dynamics analysis of protein structures ranging from enzymes to large complexes and assemblies, and enables the exploration of protein sequence-structure-dynamics-function relations.     

Integrating experimental data with molecular simulations to investigate RNA structural dynamics

Conformational dynamics is crucial for ribonucleic acid (RNA) function. Techniques such as nuclear magnetic resonance, cryo-electron microscopy, small- and wide-angle X-ray scattering, chemical probing, single-molecule Förster resonance energy transfer, or even thermal or mechanical denaturation experiments probe RNA dynamics at different time and space resolutions. Their combination with accurate atomistic molecular dynamics (MD) simulations paves the way for quantitative and detailed studies of RNA dynamics. First, experiments provide a quantitative validation tool for MD simulations. Second, available data can be used to refine simulated structural ensembles to match experiments. Finally, comparison with experiments allows for improving MD force fields that are transferable to new systems for which data is not available. Here we review the recent literature and provide our perspective on this field.     

Insights into enzyme structure and dynamics elucidated by amide H/D exchange mass spectrometry

Conformational changes and protein dynamics play an important role in the catalytic efficiency of enzymes. Amide H/D exchange mass spectrometry (H/D exchange MS) is emerging as an efficient technique to study the local and global changes in protein structure and dynamics due to ligand binding, protein activation-inactivation by modification, and protein-protein interactions. By monitoring the selective exchange of hydrogen for deuterium along a peptide backbone, this sensitive technique probes protein motions and structural elements that may be relevant to allostery and function. In this report, several applications of H/D exchange MS are presented which demonstrate the unique capability of amide hydrogen/deuterium exchange mass spectrometry for examining dynamic and structural changes associated with enzyme catalysis.     

Contrasting roles of dynamics in protein allostery: NMR and structural studies of CheY and the third PDZ domain from PSD-95

Allosteric regulation is a ubiquitous phenomenon exploited in biological processes to control cells in a myriad of ways. It is also of emerging interest in the design of functional proteins and therapeutics. Even though allostery was proposed over 50 years ago and has been studied intensively from a structural perspective, many key details of allosteric mechanisms remain mysterious. Over the last decade significant attention has been paid to the “dynamic component” of allostery, as opposed to the analysis of rigid structures. Nuclear magnetic resonance spectroscopy and its ability to detect conformationally dynamic processes at atomic resolution have played an important role in expanding our understanding of allosteric mechanisms and opening up new questions. This article focuses on work that highlights how protein dynamics can factor into allosteric processes in distinct ways. Two cases are contrasted. The first considers the “traditionally allosteric” protein CheY, which undergoes a conformational change as a key element of its allostery. The second considers the more rarely observed “dynamic allostery” in a PDZ domain, in which allosteric behavior arises from changes in internal structural dynamics. Interestingly, the dynamic processes in these two contrasting examples occur on different timescales. In the case of the PDZ domain, subsequent experimental and computational work is reviewed to reveal a more complete picture of this interesting case of allostery.     

一种快速非比对的蛋白质序列相似性与进化分析方法

本文提出了一种新的快速非比对的蛋白质序列相似性与进化分析方法。在刻画蛋白质序列特征时,首先将氨基酸的10种理化性质通过主成分分析浓缩为6个主成分,并且将每条蛋白质序列里的氨基酸数目作为权重对主成分得分值进行加权平均,然后再融合氨基酸的位置信息构成一个26维的蛋白质序列特征向量,最后利用欧式距离度量蛋白质序列间的相似性及进化关系。通过对3个蛋白质序列数据集的测试表明,本文提出的方法能将每条蛋白质序列准确聚类,并且简便快捷,说明了该方法的有效性。     

1H and 15N NMR assignment and solution structure of the SH3 domain of spectrin: Comparison of unrefined and refined structure sets with the crystal structure

The assignment of the ¹H and ¹⁵Nnuclear magnetic resonance spectra of the Src-homology region 3 domain ofchicken brain -spectrin has been obtained. A set of solutionstructures has been determined from distance and dihedral angle restraints,which provide a reasonable representation of the protein structure insolution, as evaluated by a principal component analysis of the globalpairwise root-mean-square deviation (rmsd) in a large set of structuresconsisting of the refined and unrefined solution structures and the crystalstructure. The solution structure is well defined, with a lower degree ofconvergence between the structures in the loop regions than in the secondarystructure elements. The average pairwise rmsd between the 15 refinedsolution structures is 0.71 ± 0.13 Å for the backbone atoms and1.43 ± 0.14 Å for all heavy atoms. The solution structure isbasically the same as the crystal structure. The average rmsd between the 15refined solution structures and the crystal structure is 0.76 Å forthe backbone atoms and 1.45 ± 0.09 Å for all heavy atoms. Thereare, however, small differences probably caused by intermolecular contactsin the crystal structure.     

Protein flexibility in psychrophilic and mesophilic trypsins. Evidence of evolutionary conservation of protein dynamics in trypsin-like serine-proteases

To shed light on the molecular features related to cold-adaptation in serine-proteases, we have carried out molecular dynamics simulations of homologous mesophilic and psychrophilic trypsins, with particular attention to evaluation of intramolecular interactions and flexibility. Psychrophilic trypsins present fewer interdomain interactions and enhanced localized flexibility in regions close to the catalytic site. Notably, these regions fit well with the pattern of protein flexibility previously reported for psychrophilic elastases. Our results indicate that specific sites within the serine-protease fold can be considered hot spots of cold-adaptation and that psychrophilic trypsins and elastases have independently discovered similar molecular strategies to optimize flexibility at low temperatures.     

Structure from NMR and molecular dynamics: Distance restraining inhibits motion in the essential subspace

Summary We address the question how well proteins can be modelled on the basis of NMR data, when these data are incorporated into the protein model using distance restraints in a molecular dynamics simulation. We found, using HPr as a model protein, that distance restraining freezes the essential motion of proteins, as defined by Amadei et al. [Amadei, A., Linssen, A.B.M. and Berendsen, H.J.C. (1993) Protein Struct. Funct. Genet., 17, 412–425]. We discuss how modelling protocols can be improved in order to solve this problem.     

Study on the mechanism of the BtuF periplasmic-binding protein for vitamin B12

BtuF is the periplasmic binding protein (PBP) that binds vitamin B₁₂ and delivers it to the periplasmic surface of the ABC transporter BtuCD. PBPs generally exhibit considerable conformational changes during ligand binding process, however, BtuF belongs to a subclass of PBPs that, doesn't show such behavior on the basis of the crystal structures. Employing steered molecular dynamics on the B₁₂-bound BtuF, we investigated the energetics and mechanism of BtuF. A potential of mean force along the postulated vitamin B₁₂ unbinding pathway was constructed through Jarzynski's equality. The large free energy differences of the postulated B₁₂ unbinding process suggests the B₁₂-bound structure is in a stable closed state and some conformation changes may be necessary to the B₁₂ unbinding. From the result of the principal component analysis, we found the BtuF-B₁₂ complex shows clear opening-closing and twisting motion tendencies which may facilitate the unbinding of B₁₂ from the binding pocket. The intrinsic flexibility of BtuF was also explored, and it's suggested the Trp44-Gln45 pair, which is situated at the mouth of the B₁₂ binding pocket, may act as a gate in the B₁₂ binding and unbinding process.     

Evolutionary conservation of protein vibrational dynamics

The aim of the present work is to study the evolutionary divergence of vibrational protein dynamics. To this end, we used the Gaussian Network Model to perform a systematic analysis of normal mode conservation on a large dataset of proteins classified into homologous sets of family pairs and superfamily pairs. We found that the lowest most collective normal modes are the most conserved ones. More precisely, there is, on average, a linear correlation between normal mode conservation and mode collectivity. These results imply that the previously observed conservation of backbone flexibility (B-factor) profiles is due to the conservation of the most collective modes, which contribute the most to such profiles. We discuss the possible roles of normal mode robustness and natural selection in the determination of the observed behavior. Finally, we draw some practical implications for dynamics-based protein alignment and classification and discuss possible caveats of the present approach.     

Simulating nanoscale functional motions of biomolecules

We are describing efficient dynamics simulation methods for the characterization of functional motion of biomolecules on the nanometer scale. Multivariate statistical methods are widely used to extract and enhance functional collective motions from molecular dynamics (MD) simulations. A dimension reduction in MD is often realized through a principal component analysis (PCA) or a singular value decomposition (SVD) of the trajectory. Normal mode analysis (NMA) is a related collective coordinate space approach, which involves the decomposition of the motion into vibration modes based on an elastic model. Using the myosin motor protein as an example we describe a hybrid technique termed amplified collective motions (ACM) that enhances sampling of conformational space through a combination of normal modes with atomic level MD. Unfortunately, the forced orthogonalization of modes in collective coordinate space leads to complex dependencies that are not necessarily consistent with the symmetry of biological macromolecules and assemblies. In many biological molecules, such as HIV-1 protease, reflective or rotational symmetries are present that are broken using standard orthogonal basis functions. We present a method to compute the plane of reflective symmetry or the axis of rotational symmetry from the trajectory frames. Moreover, we develop an SVD that best approximates the given trajectory while respecting the symmetry. Finally, we describe a local feature analysis (LFA) to construct a topographic representation of functional dynamics in terms of local features. The LFA representations are low-dimensional, and provide a reduced basis set for collective motions, but unlike global collective modes they are sparsely distributed and spatially localized. This yields a more reliable assignment of essential dynamics modes across different MD time windows.     

Structure-based protein NMR assignments using native structural ensembles

An important step in NMR protein structure determination is the assignment of resonances and NOEs to corresponding nuclei. Structure-based assignment (SBA) uses a model structure ("template") for the target protein to expedite this process. Nuclear vector replacement (NVR) is an SBA framework that combines multiple sources of NMR data (chemical shifts, RDCs, sparse NOEs, amide exchange rates, TOCSY) and has high accuracy when the template is close to the target protein's structure (less than 2 A backbone RMSD). However, a close template may not always be available. We extend the circle of convergence of NVR for distant templates by using an ensemble of structures. This ensemble corresponds to the low-frequency perturbations of the given template and is obtained using normal mode analysis (NMA). Our algorithm assigns resonances and sparse NOEs using each of the structures in the ensemble separately, and aggregates the results using a voting scheme based on maximum bipartite matching. Experimental results on human ubiquitin, using four distant template structures show an increase in the assignment accuracy. Our algorithm also improves the robustness of NVR with respect to structural noise. We provide a confidence measure for each assignment using the percentage of the structures that agree on that assignment. We use this measure to assign a subset of the peaks with even higher accuracy. We further validate our algorithm on data for two additional proteins with NVR. We then show the general applicability of our approach by applying our NMA ensemble-based voting scheme to another SBA tool, MARS. For three test proteins with corresponding templates, including the 370-residue maltose binding protein, we increase the number of reliable assignments made by MARS. Finally, we show that our voting scheme is sound and optimal, by proving that it is a maximum likelihood estimator of the correct assignments.     

Network analysis of protein dynamics

The network paradigm is increasingly used to describe the topology and dynamics of complex systems. Here, we review the results of the topological analysis of protein structures as molecular networks describing their small-world character, and the role of hubs and central network elements in governing enzyme activity, allosteric regulation, protein motor function, signal transduction and protein stability. We summarize available data how central network elements are enriched in active centers and ligand binding sites directing the dynamics of the entire protein. We assess the feasibility of conformational and energy networks to simplify the vast complexity of rugged energy landscapes and to predict protein folding and dynamics. Finally, we suggest that modular analysis, novel centrality measures, hierarchical representation of networks and the analysis of network dynamics will soon lead to an expansion of this field.     

Exploring the energy landscape of protein folding using replica-exchange and conventional molecular dynamics simulations

Two independent replica-exchange molecular dynamics (REMD) simulations with an explicit water model were performed of the Trp-cage mini-protein. In the first REMD simulation, the replicas started from the native conformation, while in the second they started from a nonnative conformation. Initially, the first simulation yielded results qualitatively similar to those of two previously published REMD simulations: the protein appeared to be over-stabilized, with the predicted melting temperature 50-150K higher than the experimental value of 315K. However, as the first REMD simulation progressed, the protein unfolded at all temperatures. In our second REMD simulation, which starts from a nonnative conformation, there was no evidence of significant folding. Transitions from the unfolded to the folded state did not occur on the timescale of these simulations, despite the expected improvement in sampling of REMD over conventional molecular dynamics (MD) simulations. The combined 1.42 micros of simulation time was insufficient for REMD simulations with different starting structures to converge. Conventional MD simulations at a range of temperatures were also performed. In contrast to REMD, the conventional MD simulations provide an estimate of Tm in good agreement with experiment. Furthermore, the conventional MD is a fraction of the cost of REMD and continuous, realistic pathways of the unfolding process at atomic resolution are obtained.     

Cryo-electron microscopy for structural analysis of dynamic biological macromolecules

Background

Since the introduction of what became today's standard for cryo-embedding of biological macromolecules at native conditions more than 30 years ago, techniques and equipment have been drastically improved and the structure of biomolecules can now be studied at near atomic resolution by cryo-electron microscopy (cryo-EM) while capturing multiple dynamic states. Here we review the recent progress in cryo-EM for structural studies of dynamic biological macromolecules.

Conformational analysis and dynamics of mannobiosides and mannotriosides using Monte Carlo/stochastic dynamics simulations

The conformation and dynamics of alpha-(1-->2)-mannobioside, alpha-(1-->6)-mannobioside, and of the trisaccharide alpha-Man-(1-->2)-alpha-Man-(1-->6)-alpha- Man-OMe were studied using Monte Carlo/stochastic dynamics (MC/SD) simulations, the AMBER* force field, and the GB/SA implicit water solvation model. The results are in agreement with available experimental data.     

Acyl carrier protein structural classification and normal mode analysis

All acyl carrier protein primary and tertiary structures were gathered into the ThYme database. They are classified into 16 families by amino acid sequence similarity, with members of the different families having sequences with statistically highly significant differences. These classifications are supported by tertiary structure superposition analysis. Tertiary structures from a number of families are very similar, suggesting that these families may come from a single distant ancestor. Normal vibrational mode analysis was conducted on experimentally determined freestanding structures, showing greater fluctuations at chain termini and loops than in most helices. Their modes overlap more so within families than between different families. The tertiary structures of three acyl carrier protein families that lacked any known structures were predicted as well.