61 research outputs found
Integrative modelling of cellular assemblies
A wide variety of experimental techniques can be used for understanding the precise molecular mechanisms underlying the activities of cellular assemblies. The inherent limitations of a single experimental technique often requires integration of data from complementary approaches to gain sufficient insights into the assembly structure and function. Here, we review popular computational approaches for integrative modelling of cellular assemblies, including protein complexes and genomic assemblies. We provide recent examples of integrative models generated for such assemblies by different experimental techniques, especially including data from 3D electron microscopy (3D-EM) and chromosome conformation capture experiments, respectively. We highlight general concepts in integrative modelling and discuss the need for careful formulation and merging of different types of information
Analysis of protein chameleon sequence characteristics
Conversion of local structural state of a protein from an α-helix to a β-strand is usually associated with a major change in the
tertiary structure. Similar changes were observed during the self assembly of amyloidogenic proteins to form fibrils, which
are implicated in severe diseases conditions, e.g., Alzheimer disease. Studies have emphasized that certain protein sequence
fragments known as chameleon sequences do not have a strong preference for either helical or the extended conformations.
Surprisingly, the information on the local sequence neighborhood can be used to predict their secondary at a high accuracy
level. Here we report a large scale-analysis of chameleon sequences to estimate their propensities to be associated with
different local structural states such as α -helices, β-strands and coils. With the help of the propensity information derived
from the amino acid composition, we underline their complexity, as more than one quarter of them prefers coil state over to
the regular secondary structures. About half of them show preference for both α-helix and β-sheet conformations and either
of these two states is favored by the rest
HIV reverse transcriptase: Structural interpretation of drug resistant genetic variants from India
The reverse transcriptase (RT) enzyme is the prime target of nucleoside/ nucleotide (NRTI) and non-nucleoside (NNRTI) reverse transcriptase
inhibitors. Here we investigate the structural basis of effects of drug-resistance mutations in clade C RT using three-dimensional structural
modeling. Apropos the expectation was for unique mechanisms in clade C based on interactions with amino acids of p66 subunit in RT molecule.
3-D structures of RT with mutations found in sequences from 2 treatment naïve, 8 failed and one reference clade C have been modeled and
analyzed. Models were generated by computational mutation of available crystal structures of drug bound homologous RT. Energy minimization
of the models and the structural analyses were carried out using standard methods. Mutations at positions 75,101,118,190,230,238 and 318
known to confer drug resistance were investigated. Different mutations produced different effects such as alteration of geometry of the drugbinding
pocket, structural changes at the site of entry of the drug (into the active site), repositioning the template bases or by discriminating the
inhibitors from their natural substrates. For the mutations analyzed, NRTI resistance was mediated mainly by the ability to discriminate between
inhibitors and natural substrate, whereas, NNRTI resistance affected either the drug entry or the geometry of the active site. Our analysis
suggests that different mutations result in different structural effects affecting the ability of a given drug to bind to the RT. Our studies will help
in the development of newer drugs taking into account the presence of these mutations and the structural basis of drug resistance
Refinement of atomic models in high resolution EM reconstructions using Flex-EM and local assessment
As the resolutions of Three Dimensional Electron Microscopic reconstructions of biological macromolecules are being improved, there is a need for better fitting and refinement methods at high resolutions and robust approaches for model assessment. Flex-EM/MODELLER has been used for flexible fitting of atomic models in intermediate-to-low resolution density maps of different biological systems. Here, we demonstrate the suitability of the method to successfully refine structures at higher resolutions (2.5–4.5 Å) using both simulated and experimental data, including a newly processed map of Apo-GroEL. A hierarchical refinement protocol was adopted where the rigid body definitions are relaxed and atom displacement steps are reduced progressively at successive stages of refinement. For the assessment of local fit, we used the SMOC (segment-based Manders’ overlap coefficient) score, while the model quality was checked using the Qmean score. Comparison of SMOC profiles at different stages of refinement helped in detecting regions that are poorly fitted. We also show how initial model errors can have significant impact on the goodness-of-fit. Finally, we discuss the implementation of Flex-EM in the CCP-EM software suite
PBxplore: a tool to analyze local protein structure and deformability with Protein Blocks
This paper describes the development and application of a suite of tools, called PBxplore, to analyze the dynamics and deformability of protein structures using Protein Blocks (PBs). Proteins are highly dynamic macromolecules, and a classical way to analyze their inherent flexibility is to perform molecular dynamics simulations. The advantage of using small structural prototypes such as PBs is to give a good approximation of the local structure of the protein backbone. More importantly, by reducing the conformational complexity of protein structures, PBs allow analysis of local protein deformability which cannot be done with other methods and had been used efficiently in different applications. PBxplore is able to process large amounts of data such as those produced by molecular dynamics simulations. It produces frequencies, entropy and information logo outputs as text and graphics. PBxplore is available at https://github.com/pierrepo/PBxplore and is released under the open-source MIT license
TopoStats – a program for automated tracing of biomolecules from AFM images
We present TopoStats, a Python toolkit for automated editing and analysis of Atomic Force Microscopy images. The program automates identification and tracing of individual molecules in circular and linear conformations without user input. TopoStats was able to identify and trace a range of molecules within AFM images, finding, on average, ~90% of all individual molecules and molecular assemblies within a wide field of view, and without the need for prior processing. DNA minicircles of varying size, DNA origami rings and pore forming proteins were identified and accurately traced with contour lengths of traces typically within 10 nm of the predicted contour length. TopoStats was also able to reliably identify and trace linear and enclosed circular molecules within a mixed population. The program is freely available via GitHub (https://github.com/afm-spm/TopoStats) and is intended to be modified and adapted for use if required
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
Structural basis of human kinesin-8 function and inhibition
Kinesin motors play diverse roles in mitosis and are targets for anti-mitotic drugs. The clinical significance of these motors emphasizes the importance of understanding the molecular basis of their function. Equally, investigations into the modes of inhibition of these motors provide crucial information about their molecular mechanisms. Kif18A regulates spindle microtubules through its dual functionality – microtubule-based stepping and regulation of microtubule dynamics. We investigated the mechanism of Kif18A and its inhibition by the small molecule BTB-1. The Kif18A motor domain drives ATP-dependent plus-end microtubule gliding, and undergoes conformational changes consistent with canonical mechanisms of plus-end directed motility. The Kif18A motor domain also depolymerises microtubule plus and minus ends. BTB-1 inhibits both microtubule-based Kif18A activities. A reconstruction of BTB-1-bound, microtubule-bound Kif18A, in combination with computational modelling, identified an allosteric BTB-1 binding site near loop5, where it blocks the ATP-dependent conformational changes we characterised. Strikingly, BTB-1 binding is close to that of well-characterised Kif11 inhibitors that block tight microtubule binding, whereas BTB-1 traps Kif18A on the microtubule. Our work highlights a general mechanism of kinesin inhibition in which small molecule binding near loop5 prevents a range of conformational changes, blocking motor function
Assignment of PolyProline II Conformation and Analysis of Sequence – Structure Relationship
International audienceBACKGROUND: Secondary structures are elements of great importance in structural biology, biochemistry and bioinformatics. They are broadly composed of two repetitive structures namely α-helices and β-sheets, apart from turns, and the rest is associated to coil. These repetitive secondary structures have specific and conserved biophysical and geometric properties. PolyProline II (PPII) helix is yet another interesting repetitive structure which is less frequent and not usually associated with stabilizing interactions. Recent studies have shown that PPII frequency is higher than expected, and they could have an important role in protein - protein interactions. METHODOLOGY/PRINCIPAL FINDINGS: A major factor that limits the study of PPII is that its assignment cannot be carried out with the most commonly used secondary structure assignment methods (SSAMs). The purpose of this work is to propose a PPII assignment methodology that can be defined in the frame of DSSP secondary structure assignment. Considering the ambiguity in PPII assignments by different methods, a consensus assignment strategy was utilized. To define the most consensual rule of PPII assignment, three SSAMs that can assign PPII, were compared and analyzed. The assignment rule was defined to have a maximum coverage of all assignments made by these SSAMs. Not many constraints were added to the assignment and only PPII helices of at least 2 residues length are defined. CONCLUSIONS/SIGNIFICANCE: The simple rules designed in this study for characterizing PPII conformation, lead to the assignment of 5% of all amino as PPII. Sequence - structure relationships associated with PPII, defined by the different SSAMs, underline few striking differences. A specific study of amino acid preferences in their N and C-cap regions was carried out as their solvent accessibility and contact patterns. Thus the assignment of PPII can be coupled with DSSP and thus opens a simple way for further analysis in this field
TEMPy: a Python library for assessment of 3D electron microscopy density fits
Three-dimensional electron microscopy (3D EM) is currently one of the most promising techniques used to study macromolecular assemblies. Rigid and flexible fitting of atomic models into density maps is often essential to gain further insights into the assemblies they represent. Currently, tools that facilitate the assessment of fitted atomic models and maps are needed. TEMPy – Template and EM comparison using Python – is a toolkit designed for this purpose. The library includes a set of methods to assess density fits in intermediate-to-low resolution maps, both globally and locally. It also provides procedures for single fit assessment, ensemble generation of fits, clustering, multiple and consensus scoring, as well as plots and output files for visualisation purposes to help the user in analysing rigid and flexible fits. The modular nature of TEMPy helps the integration of scoring and assessment of fits into large pipelines, making it a tool for both novice and expert structural biologists
- …