scholarly journals Extending the scope of coiled-coil crystal structure solution by AMPLE through improved ab initio modelling

2020 ◽  
Vol 76 (3) ◽  
pp. 272-284 ◽  
Author(s):  
Jens M. H. Thomas ◽  
Ronan M. Keegan ◽  
Daniel J. Rigden ◽  
Owen R. Davies
Keyword(s):  
Ab Initio ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Molecular Replacement ◽  
Helical Structures ◽  
Major Barrier ◽  
X Ray Crystallography ◽  
Structure Solution ◽  
Coil Structure ◽  
Experimental Phasing

The phase problem remains a major barrier to overcome in protein structure solution by X-ray crystallography. In recent years, new molecular-replacement approaches using ab initio models and ideal secondary-structure components have greatly contributed to the solution of novel structures in the absence of clear homologues in the PDB or experimental phasing information. This has been particularly successful for highly α-helical structures, and especially coiled-coils, in which the relatively rigid α-helices provide very useful molecular-replacement fragments. This has been seen within the program AMPLE, which uses clustered and truncated ensembles of numerous ab initio models in structure solution, and is already accomplished for α-helical and coiled-coil structures. Here, an expansion in the scope of coiled-coil structure solution by AMPLE is reported, which has been achieved through general improvements in the pipeline, the removal of tNCS correction in molecular replacement and two improved methods for ab initio modelling. Of the latter improvements, enforcing the modelling of elongated helices overcame the bias towards globular folds and provided a rapid method (equivalent to the time requirements of the existing modelling procedures in AMPLE) for enhanced solution. Further, the modelling of two-, three- and four-helical oligomeric coiled-coils, and the use of full/partial oligomers in molecular replacement, provided additional success in difficult and lower resolution cases. Together, these approaches have enabled the solution of a number of parallel/antiparallel dimeric, trimeric and tetrameric coiled-coils at resolutions as low as 3.3 Å, and have thus overcome previous limitations in AMPLE and provided a new functionality in coiled-coil structure solution at lower resolutions. These new approaches have been incorporated into a new release of AMPLE in which automated elongated monomer and oligomer modelling may be activated by selecting `coiled-coil' mode.

scholarly journals Rapid molecular replacement of coiled-coil and transmembrane proteins with AMPLE

2014 ◽  
Vol 70 (a1) ◽  
pp. C347-C347
Author(s):  
Jens Thomas ◽  
Ronan Keegan ◽  
Jaclyn Bibby ◽  
Martyn Winn ◽  
Olga Mayans ◽  
...  
Keyword(s):  
Ab Initio ◽  
Protein Structures ◽  
Coiled Coil ◽  
Molecular Replacement ◽  
Helical Structures ◽  
Desktop Computer ◽  
Search Models ◽  
Nmr Structures ◽  
Structure Solution ◽  
Helical Geometry

Molecular Replacement (MR) is an increasingly popular route to protein structure solution. AMPLE[1] is a software pipeline that uses either cheaply obtained ab inito protein models, or NMR structures to extend the scope of MR, allowing it to solve entirely novel protein structures in a completely automated pipeline on a standard desktop computer. AMPLE employs a cluster-and-truncate approach, combined with multiple modes of side chain treatment, to analyse the candidate models and extract the consensual features most likely to solve the structure. The search models generated in this way are screened by MrBump using Phaser and Molrep and correct solutions are detected using main chain tracing and phase modification with Shelxe. AMPLE proved capable of processing rapidly obtained ab initio structure predictions into successful search models and more recently proved effective in assembling NMR structures for MR[2]. Coiled-coil proteins are a distinct class of protein fold whose structure solution by MR is not typically straightforward. We show here that AMPLE can quickly and routinely solve most coiled-coil structures using ab initio predictions from Rosetta. The predictions are generally not globally accurate, but by encompassing different degrees of truncation of clustered models, AMPLE succeeds by sampling across a range of search models. These sometimes succeed through capturing locally well-modelled conformations, but often simply contain small helical units. Remarkably, the latter regularly succeed despite out-of-register placement and poor MR statistics. We demonstrate that single structures derived from successful ensembles perform less well, and comparable ideal helices solve few targets. Thus, both modelling of distortions from ideal helical geometry and the ensemble nature of the search models contribute to success. AMPLE is a framework applicable to any set of input structures in which variability is correlated with inaccuracy. We also present preliminary data demonstrating structure solution of transmembrane helical structures using Rosetta modelling. We finally consider future sources of starting models which offer the hope that MR with AMPLE, in the absence of close homology between a known structure and the target, may soon be possible with larger proteins.

scholarly journals Combinatorial use of disulfide bridges and native sulfur-SAD phasing for rapid structure determination of coiled-coils

2018 ◽  
Vol 38 (5) ◽  
Author(s):  
Sebastian H.W. Kraatz ◽  
Sarah Bianchi ◽  
Michel O. Steinmetz
Keyword(s):  
Coiled Coil ◽  
Coiled Coils ◽  
Molecular Replacement ◽  
Disulfide Bridges ◽  
X Ray ◽  
X Ray Crystallography ◽  
Protein Protein Interaction ◽  
Eukaryotic Proteins ◽  
Sad Phasing

Coiled-coils are ubiquitous protein–protein interaction motifs found in many eukaryotic proteins. The elongated, flexible and often irregular nature of coiled-coils together with their tendency to form fibrous arrangements in crystals imposes challenges on solving the phase problem by molecular replacement. Here, we report the successful combinatorial use of native and rational engineered disulfide bridges together with sulfur-SAD phasing as a powerful tool to stabilize and solve the structure of coiled-coil domains in a straightforward manner. Our study is a key example of how modern sulfur SAD combined with mutagenesis can help to advance and simplify the structural study of challenging coiled-coil domains by X-ray crystallography.

scholarly journals Routine phasing of coiled-coil protein crystal structures withAMPLE

IUCrJ ◽  
2015 ◽  
Vol 2 (2) ◽  
pp. 198-206 ◽  
Author(s):  
Jens M. H. Thomas ◽  
Ronan M. Keegan ◽  
Jaclyn Bibby ◽  
Martyn D. Winn ◽  
Olga Mayans ◽  
...  
Keyword(s):  
Ab Initio ◽  
Crystal Structures ◽  
Coiled Coil ◽  
Protein Crystal ◽  
Molecular Replacement ◽  
Search Models ◽  
Homologous Protein ◽  
Coil Structure ◽  
Convenient Route ◽  
Chain Lengths

Coiled-coil protein folds are among the most abundant in nature. These folds consist of long wound α-helices and are architecturally simple, but paradoxically their crystallographic structures are notoriously difficult to solve with molecular-replacement techniques. The programAMPLEcan solve crystal structures by molecular replacement usingab initiosearch models in the absence of an existent homologous protein structure.AMPLEhas been benchmarked on a large and diverse test set of coiled-coil crystal structures and has been found to solve 80% of all cases. Successes included structures with chain lengths of up to 253 residues and resolutions down to 2.9 Å, considerably extending the limits on size and resolution that are typically tractable byab initiomethodologies. The structures of two macromolecular complexes, one including DNA, were also successfully solved using their coiled-coil components. It is demonstrated that both theab initiomodelling and the use of ensemble search models contribute to the success ofAMPLEby comparison with phasing attempts using single structures or ideal polyalanine helices. These successes suggest that molecular replacement withAMPLEshould be the method of choice for the crystallographic elucidation of a coiled-coil structure. Furthermore,AMPLEmay be able to exploit the presence of a coiled coil in a complex to provide a convenient route for phasing.

Automatedde novophasing and model building of coiled-coil proteins

2015 ◽  
Vol 71 (3) ◽  
pp. 606-614 ◽  
Author(s):  
Sebastian Rämisch ◽  
Robert Lizatović ◽  
Ingemar André
Keyword(s):  
Structure Prediction ◽  
Initial Phase ◽  
Model Building ◽  
De Novo ◽  
Protein Complexes ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Phase Estimation ◽  
Molecular Replacement ◽  
Experimental Phasing

Models generated byde novostructure prediction can be very useful starting points for molecular replacement for systems where suitable structural homologues cannot be readily identified. Protein–protein complexes andde novo-designed proteins are examples of systems that can be challenging to phase. In this study, the potential ofde novomodels of protein complexes for use as starting points for molecular replacement is investigated. The approach is demonstrated using homomeric coiled-coil proteins, which are excellent model systems for oligomeric systems. Despite the stereotypical fold of coiled coils, initial phase estimation can be difficult and many structures have to be solved with experimental phasing. A method was developed for automatic structure determination of homomeric coiled coils from X-ray diffraction data. In a benchmark set of 24 coiled coils, ranging from dimers to pentamers with resolutions down to 2.5 Å, 22 systems were automatically solved, 11 of which had previously been solved by experimental phasing. The generated models contained 71–103% of the residues present in the deposited structures, had the correct sequence and had freeRvalues that deviated on average by 0.01 from those of the respective reference structures. The electron-density maps were of sufficient quality that only minor manual editing was necessary to produce final structures. The method, namedCCsolve, combines methods forde novostructure prediction, initial phase estimation and automated model building into one pipeline.CCsolveis robust against errors in the initial models and can readily be modified to make use of alternative crystallographic software. The results demonstrate the feasibility ofde novophasing of protein–protein complexes, an approach that could also be employed for other small systems beyond coiled coils.

scholarly journals ARCIMBOLDOon coiled coils

2018 ◽  
Vol 74 (3) ◽  
pp. 194-204 ◽  
Author(s):  
Iracema Caballero ◽  
Massimo Sammito ◽  
Claudia Millán ◽  
Andrey Lebedev ◽  
Nicolas Soler ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Coiled Coils ◽  
Translational Symmetry ◽  
Command Line ◽  
Phase Problem ◽  
Final Solution ◽  
Resolution Limit ◽  
Helical Structures ◽  
Small Model ◽  
Test Pool

ARCIMBOLDOsolves the phase problem by combining the location of small model fragments usingPhaserwith density modification and autotracing usingSHELXE. Mainly helical structures constitute favourable cases, which can be solved using polyalanine helical fragments as search models. Nevertheless, the solution of coiled-coil structures is often complicated by their anisotropic diffraction and apparent translational noncrystallographic symmetry. Long, straight helices have internal translational symmetry and their alignment in preferential directions gives rise to systematic overlap of Patterson vectors. This situation has to be differentiated from the translational symmetry relating different monomers.ARCIMBOLDO_LITEhas been run on single workstations on a test pool of 150 coiled-coil structures with 15–635 amino acids per asymmetric unit and with diffraction data resolutions of between 0.9 and 3.0 Å. The results have been used to identify and address specific issues when solving this class of structures usingARCIMBOLDO. Features fromPhaserv.2.7 onwards are essential to correct anisotropy and produce translation solutions that will pass the packing filters. As the resolution becomes worse than 2.3 Å, the helix direction may be reversed in the placed fragments. Differentiation between true solutions and pseudo-solutions, in which helix fragments were correctly positioned but in a reverse orientation, was found to be problematic at resolutions worse than 2.3 Å. Therefore, after every new fragment-placement round, complete or sparse combinations of helices in alternative directions are generated and evaluated. The final solution is once again probed by helix reversal, refinement and extension. To conclude, density modification andSHELXEautotracing incorporating helical constraints is also exploited to extend the resolution limit in the case of coiled coils and to enhance the identification of correct solutions. This study resulted in a specialized mode withinARCIMBOLDOfor the solution of coiled-coil structures, which overrides the resolution limit and can be invoked from the command line (keyword coiled_coil) orARCIMBOLDO_LITEtask interface inCCP4i.

scholarly journals Structural Insights into the Molecular Mechanisms of Cauliflower Mosaic Virus Transmission by Its Insect Vector

2010 ◽  
Vol 84 (9) ◽  
pp. 4706-4713 ◽  
Author(s):  
François Hoh ◽  
Marilyne Uzest ◽  
Martin Drucker ◽  
Célia Plisson-Chastang ◽  
Patrick Bron ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Molecular Mechanisms ◽  
Structural Model ◽  
Coiled Coil ◽  
Virus Transmission ◽  
Cauliflower Mosaic Virus ◽  
Insect Vector ◽  
Virus Surface ◽  
X Ray Crystallography ◽  
Coil Structure

ABSTRACT Cauliflower mosaic virus (CaMV) is transmitted from plant to plant through a seemingly simple interaction with insect vectors. This process involves an aphid receptor and two viral proteins, P2 and P3. P2 binds to both the aphid receptor and P3, itself tightly associated with the virus particle, with the ensemble forming a transmissible viral complex. Here, we describe the conformations of both unliganded CaMV P3 protein and its virion-associated form. X-ray crystallography revealed that the N-terminal domain of unliganded P3 is a tetrameric parallel coiled coil with a unique organization showing two successive four-stranded subdomains with opposite supercoiling handedness stabilized by a ring of interchain disulfide bridges. A structural model of virus-liganded P3 proteins, folding as an antiparallel coiled-coil network coating the virus surface, was derived from molecular modeling. Our results highlight the structural and biological versatility of this coiled-coil structure and provide new insights into the molecular mechanisms involved in CaMV acquisition and transmission by the insect vector.

scholarly journals Molecular symmetry-constrained systematic search approach to structure solution of the coiled-coil SRGAP2 F-BARx domain

2016 ◽  
Vol 72 (12) ◽  
pp. 1241-1253 ◽  
Author(s):  
Michael Sporny ◽  
Julia Guez-Haddad ◽  
David G. Waterman ◽  
Michail N. Isupov ◽  
Yarden Opatowsky
Keyword(s):  
Diffraction Data ◽  
Coiled Coil ◽  
Systematic Search ◽  
Molecular Symmetry ◽  
Structure Solution ◽  
Experimental Phasing ◽  
Membrane Protrusions ◽  
Group A ◽  
Search Approach ◽  
Statistical Criteria

SRGAP2 (Slit–Robo GTPase-activating protein 2) is a cytoplasmic protein found to be involved in neuronal branching, restriction of neuronal migration and restriction of the length and density of dendritic postsynaptic spines. The extended F-BAR (F-BARx) domain of SRGAP2 generates membrane protrusions when expressed in COS-7 cells, while most F-BARs induce the opposite effect: membrane invaginations. As a first step to understand this discrepancy, the F-BARx domain of SRGAP2 was isolated and crystallized after co-expression with the carboxy domains of the protein. Diffraction data were collected from two significantly non-isomorphous crystals in the same monoclinicC2 space group. A correct molecular-replacment solution was obtained by applying a molecular symmetry-constrained systematic search approach that took advantage of the conserved biological symmetry of the F-BAR domains. It is shown that similar approaches can solve other F-BAR structures that were previously determined by experimental phasing. Diffraction data were reprocessed with a high-resolution cutoff of 2.2 Å, chosen using less strict statistical criteria. This has improved the outcome of multi-crystal averaging and other density-modification procedures.

scholarly journals Exploring the speed and performance of molecular replacement withAMPLEusingQUARK ab initioprotein models

2015 ◽  
Vol 71 (2) ◽  
pp. 338-343 ◽  
Author(s):  
Ronan M. Keegan ◽  
Jaclyn Bibby ◽  
Jens Thomas ◽  
Dong Xu ◽  
Yang Zhang ◽  
...  
Keyword(s):  
Protein Structure ◽  
Ab Initio ◽  
Molecular Replacement ◽  
Test Set ◽  
Search Models ◽  
Structure Solution ◽  
And Performance ◽  
Almost All

AMPLEclusters and truncatesab initioprotein structure predictions, producing search models for molecular replacement. Here, an interesting degree of complementarity is shown between targets solved using the differentab initiomodelling programsQUARKandROSETTA. Search models derived from either program collectively solve almost all of the all-helical targets in the test set. Initial solutions produced byPhaserafter only 5 min perform surprisingly well, improving the prospects forin situstructure solution byAMPLEduring synchrotron visits. Taken together, the results show the potential forAMPLEto run more quickly and successfully solve more targets than previously suspected.

scholarly journals Phasertng: directed acyclic graphs for crystallographic phasing

2021 ◽  
Vol 77 (1) ◽  
pp. 1-10
Author(s):  
Airlie J. McCoy ◽  
Duncan H. Stockwell ◽  
Massimo D. Sammito ◽  
Robert D. Oeffner ◽  
Kaushik S. Hatti ◽  
...  
Keyword(s):  
Directed Acyclic Graph ◽  
Directed Acyclic Graphs ◽  
Molecular Replacement ◽  
Molecular Fragments ◽  
Graph Data ◽  
Acyclic Graphs ◽  
Structure Solution ◽  
Experimental Phasing ◽  
Graph Management ◽  
Extract Phase

Crystallographic phasing strategies increasingly require the exploration and ranking of many hypotheses about the number, types and positions of atoms, molecules and/or molecular fragments in the unit cell, each with only a small chance of being correct. Accelerating this move has been improvements in phasing methods, which are now able to extract phase information from the placement of very small fragments of structure, from weak experimental phasing signal or from combinations of molecular replacement and experimental phasing information. Describing phasing in terms of a directed acyclic graph allows graph-management software to track and manage the path to structure solution. The crystallographic software supporting the graph data structure must be strictly modular so that nodes in the graph are efficiently generated by the encapsulated functionality. To this end, the development of new software, Phasertng, which uses directed acyclic graphs natively for input/output, has been initiated. In Phasertng, the codebase of Phaser has been rebuilt, with an emphasis on modularity, on scripting, on speed and on continuing algorithm development. As a first application of phasertng, its advantages are demonstrated in the context of phasertng.xtricorder, a tool to analyse and triage merged data in preparation for molecular replacement or experimental phasing. The description of the phasing strategy with directed acyclic graphs is a generalization that extends beyond the functionality of Phasertng, as it can incorporate results from bioinformatics and other crystallographic tools, and will facilitate multifaceted search strategies, dynamic ranking of alternative search pathways and the exploitation of machine learning to further improve phasing strategies.

scholarly journals MrParse: Finding homologues in the PDB and the EBI AlphaFold database for Molecular Replacement and more

2021 ◽  
Author(s):  
Adam J Simpkin ◽  
Jens M H Thomas ◽  
Ronan M Keegan ◽  
Daniel J Rigden
Keyword(s):  
Secondary Structure ◽  
Decision Process ◽  
Computational Modelling ◽  
Coiled Coil ◽  
Search Model ◽  
Molecular Replacement ◽  
Regular Secondary Structure ◽  
Reflection Data ◽  
Structure Solution ◽  
Transmembrane Regions

Crystallographers have an array of search model options for structure solution by Molecular Replacement (MR). Well-established options of homologous experimental structures and regular secondary structure elements or motifs are increasingly supplemented by computational modelling. Such modelling may be carried out locally or use pre-calculated predictions retrieved from databases such as the EBI AlphaFold database. MrParse is a new pipeline to help streamline the decision process in MR by consolidating bioinformatic predictions in one place. When reflection data are provided, MrParse can rank any homologues found using eLLG which indicates the likelihood that a given search model will work in MR. In-built displays of predicted secondary structure, coiled-coil and transmembrane regions further inform the choice of MR protocol. MrParse can also identify and rank homologues in the EBI AlphaFold database, a function that will also interest other structural biologists and bioinformaticians.

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