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.

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 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 Molecular recognition of M1-linked ubiquitin chains by native and phosphorylated UBAN domains

2019 ◽  
Author(s):  
Lina Herhaus ◽  
Henry van den Bedem ◽  
Sean Tang ◽  
Soichi Wakatsuki ◽  
Ivan Dikic ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Binding Sites ◽  
Coiled Coil ◽  
Domain Specificity ◽  
Binding Properties ◽  
Post Translational Modifications ◽  
Binding Characteristics ◽  
Binding Stoichiometry ◽  
Coil Structure ◽  
Concentration Dependency

AbstractAlthough the Ub-binding domain in ABIN proteins and NEMO (UBAN) is highly conserved, UBAN-containing proteins exhibit different Ub-binding properties, resulting in their diverse biological roles. Post-translational modifications further control UBAN domain specificity for poly-Ub chains. However, precisely, how the UBAN domain structurally confers such functional diversity remains poorly understood. Here we report crystal structures of ABIN-1 alone and in complex with one or two M1-linked di-Ub chains. ABIN-1 UBAN forms a homo-dimer that provides two symmetrical Ub-binding sites on either side of the coiled-coil structure. Moreover, crystal structures of ABIN1 UBAN in complex with di-Ub chains reveal a concentration-dependency of UBAN/di-Ub binding stoichiometry. Analysis of UBAN/M1-linked di-Ub binding characteristics indicates that phosphorylated S473 in OPTN and its corresponding phospho-mimetic residue in ABIN-1 (E484) are essential for high affinity interactions with M1-linked Ub chains. Also, a phospho-mimetic mutation of A303 in NEMO, corresponding to S473 of OPTN, increases binding affinity for M1-linked Ub chains. These findings are in line with the diverse physiological roles of UBAN domains, as phosphorylation of OPTN UBAN is required to enhance its binding to Ub during mitophagy.

About the efficiency of the early FOMs inab initioprotein phasing

2004 ◽  
Vol 37 (5) ◽  
pp. 791-801 ◽  
Author(s):  
Maria C. Burla ◽  
Rocco Caliandro ◽  
Benedetta Carrozzini ◽  
Giovanni L. Cascarano ◽  
Liberato De Caro ◽  
...  
Keyword(s):  
Ab Initio ◽  
Crystal Structures ◽  
Phase Error ◽  
Protein Crystal ◽  
Correct Solution ◽  
Large Set ◽  
Figures Of Merit ◽  
Selection Of

Allab initiotechniques for solving protein crystal structures use multisolution approaches. Several figures of merit that are found in the literature are efficient in the last steps of the phasing process, when some trials converge to the correct solution with a relatively small average phase error. Early figures of merit are much more critical; they should be able to recognize useful trials when the phase error is still large, and their efficiency determines the efficiency of the program. In the present work, a wide variety of known figures of merit at atomic and quasi-atomic (∼1.4 Å) resolution have been tested; new figures have also been devised and tested. Their application to a large set of test structures allows the study of their properties at different data resolutions and the selection of the most efficient figures within theSIR2003-Nframework.

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 Crystallographic molecular replacement using an in silico-generated search model of SARS-CoV-2 ORF8

2021 ◽  
Author(s):  
Thomas G. Flower ◽  
James H. Hurley
Keyword(s):  
Ab Initio ◽  
Structural Parameters ◽  
Search Model ◽  
Problem Solution ◽  
Molecular Replacement ◽  
Phase Problem ◽  
Search Models ◽  
Sad Phasing ◽  
Predicted Model ◽  
Β Sheet

AbstractThe majority of crystal structures are determined by the method of molecular replacement (MR). The range of application of MR is limited mainly by the need for an accurate search model. In most cases, pre-existing experimentally determined structures are used as search models. In favorable cases, ab initio predicted structures have yielded search models adequate for molecular replacement. The ORF8 protein of SARS-CoV-2 represents a challenging case for MR using an ab initio prediction because ORF8 has an all β-sheet fold and few orthologs. We previously determined experimentally the structure of ORF8 using the single anomalous dispersion (SAD) phasing method, having been unable to find an MR solution to the crystallographic phase problem. Following a report of an accurate prediction of the ORF8 structure, we assessed whether the predicted model would have succeeded as an MR search model. A phase problem solution was found, and the resulting structure was refined, yielding structural parameters equivalent to the original experimental solution.

scholarly journals Ab initio solution of macromolecular crystal structures without direct methods

2017 ◽  
Vol 114 (14) ◽  
pp. 3637-3641 ◽  
Author(s):  
Airlie J. McCoy ◽  
Robert D. Oeffner ◽  
Antoni G. Wrobel ◽  
Juha R. M. Ojala ◽  
Karl Tryggvason ◽  
...  
Keyword(s):  
Ab Initio ◽  
Crystal Structures ◽  
Signal To Noise Ratio ◽  
Direct Methods ◽  
Molecular Replacement ◽  
Signal To Noise ◽  
Model Quality ◽  
Theoretical Understanding ◽  
Unknown Structure ◽  
Noise Ratio

The majority of macromolecular crystal structures are determined using the method of molecular replacement, in which known related structures are rotated and translated to provide an initial atomic model for the new structure. A theoretical understanding of the signal-to-noise ratio in likelihood-based molecular replacement searches has been developed to account for the influence of model quality and completeness, as well as the resolution of the diffraction data. Here we show that, contrary to current belief, molecular replacement need not be restricted to the use of models comprising a substantial fraction of the unknown structure. Instead, likelihood-based methods allow a continuum of applications depending predictably on the quality of the model and the resolution of the data. Unexpectedly, our understanding of the signal-to-noise ratio in molecular replacement leads to the finding that, with data to sufficiently high resolution, fragments as small as single atoms of elements usually found in proteins can yield ab initio solutions of macromolecular structures, including some that elude traditional direct methods.

scholarly journals Application of theAMPLEcluster-and-truncate approach to NMR structures for molecular replacement

2013 ◽  
Vol 69 (11) ◽  
pp. 2194-2201 ◽  
Author(s):  
Jaclyn Bibby ◽  
Ronan M. Keegan ◽  
Olga Mayans ◽  
Martyn D. Winn ◽  
Daniel J. Rigden
Keyword(s):  
Protein Structure ◽  
Ab Initio ◽  
Target Protein ◽  
Molecular Replacement ◽  
Search Models ◽  
Sequence Identity ◽  
Nmr Structures ◽  
Core Cluster ◽  
Structural Divergence ◽  
Improved Performance

AMPLEis a program developed for clustering and truncatingab initioprotein structure predictions into search models for molecular replacement. Here, it is shown that its core cluster-and-truncate methods also work well for processing NMR ensembles into search models.Rosettaremodelling helps to extend success to NMR structures bearing low sequence identity or high structural divergence from the target protein. Potential future routes to improved performance are considered and practical, general guidelines on usingAMPLEare provided.

scholarly journals Unsupervised determination of protein crystal structures

2019 ◽  
Vol 116 (22) ◽  
pp. 10813-10818 ◽  
Author(s):  
Ivan S. Ufimtsev ◽  
Michael Levitt
Keyword(s):  
Crystal Structures ◽  
Protein Crystal ◽  
Search Model ◽  
Molecular Replacement ◽  
Initial Model ◽  
Large Pool

We present a method for automatic solution of protein crystal structures. The method proceeds with a single initial model obtained, for instance, by molecular replacement (MR). If a good-quality search model is not available, as often is the case with MR of distant homologs, our method first can automatically screen a large pool of poorly placed models and single out promising candidates for further processing if there are any. We demonstrate its utility by solving a set of synthetic cases in the 2.9- to 3.45-Å resolution.

Sign in / Sign up

Export Citation Format

Share Document