Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge.

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.

Small molecules enhance the potency of natural antimicrobial peptides.

The skin-associated microbiome plays an important role in general well-being and in a variety of treatable skin conditions. In this regard, endogenous antimicrobial peptides have both a direct and indirect role in determining the composition of the microbiota. We demonstrate here that certain small molecular species can amplify the antimicrobial potency of naturally occurring antimicrobial peptides. In this study, we have used niacinamide, a form of vitamin B3 naturally found in foods and widely used in cosmetic skincare products, and two of its structural analogs, to investigate their cooperativity with the human antimicrobial peptide LL37 on the bacterium Staphylococcus aureus. We observed a clear synergistic effect of niacinamide and, to some extent, N-methylnicotinamide, whereas isonicotinamide showed no significant cooperativity with LL37. Adaptively biased molecular dynamics simulations using simplified model membrane substrates and single peptides revealed that these molecules partition into the headgroup region of an anionic bilayer used to mimic the bacterial membrane. The simulated effects on the physical properties of the simulated model membrane are well correlated with experimental activity observed in real biological assays despite the simplicity of the model. In contrast, these molecules have little effect on zwitterionic bilayers that mimic a mammalian membrane. We conclude that niacinamide and N-methylnicotinamide can therefore potentiate the activity of host peptides by modulating the physical properties of the bacterial membrane, and to a lesser extent through direct interactions with the peptide. The level of cooperativity is strongly dependent on the detailed chemistry of the additive, suggesting an opportunity to fine-tune the behavior of host peptides.

Overview and applications of map and model validation tools in the CCP-EM software suite.

Cryogenic electron microscopy (cryo-EM) has recently been established as a powerful technique for solving macromolecular structures. Although the best resolutions achievable are improving, a significant majority of data are still resolved at resolutions worse than 3 Å, where it is non-trivial to build or fit atomic models. The map reconstructions and atomic models derived from the maps are also prone to errors accumulated through the different stages of data processing. Here, we highlight the need to evaluate both model geometry and fit to data at different resolutions. Assessment of cryo-EM structures from SARS-CoV-2 highlights a bias towards optimising the model geometry to agree with the most common conformations, compared to the agreement with data. We present the CoVal web service which provides multiple validation metrics to reflect the quality of atomic models derived from cryo-EM data of structures from SARS-CoV-2. We demonstrate that further refinement can lead to improvement of the agreement with data without the loss of geometric quality. We also discuss the recent CCP-EM developments aimed at addressing some of the current shortcomings.

Comparing Cryo-EM Reconstructions and Validating Atomic Model Fit Using Difference Maps.

Cryogenic electron microscopy (cryo-EM) is a powerful technique for determining structures of multiple conformational or compositional states of macromolecular assemblies involved in cellular processes. Recent technological developments have led to a leap in the resolution of many cryo-EM data sets, making atomic model building more common for data interpretation. We present a method for calculating differences between two cryo-EM maps or a map and a fitted atomic model. The proposed approach works by scaling the maps using amplitude matching in resolution shells. To account for variability in local resolution of cryo-EM data, we include a procedure for local amplitude scaling that enables appropriate scaling of local map contrast. The approach is implemented as a user-friendly tool in the CCP-EM software package. To obtain clean and interpretable differences, we propose a protocol involving steps to process the input maps and output differences. We demonstrate the utility of the method for identifying conformational and compositional differences including ligands. We also highlight the use of difference maps for evaluating atomic model fit in cryo-EM maps.

Indexed variation graphs for efficient and accurate resistome profiling.

Motivation: Antimicrobial resistance (AMR) remains a major threat to global health. Profiling the collective AMR genes within a metagenome (the 'resistome') facilitates greater understanding of AMR gene diversity and dynamics. In turn, this can allow for gene surveillance, individualized treatment of bacterial infections and more sustainable use of antimicrobials. However, resistome profiling can be complicated by high similarity between reference genes, as well as the sheer volume of sequencing data and the complexity of analysis workflows. We have developed an efficient and accurate method for resistome profiling that addresses these complications and improves upon currently available tools. Results: Our method combines a variation graph representation of gene sets with a locality-sensitive hashing Forest indexing scheme to allow for fast classification of metagenomic sequence reads using similarity-search queries. Subsequent hierarchical local alignment of classified reads against graph traversals enables accurate reconstruction of full-length gene sequences using a scoring scheme. We provide our implementation, graphing Resistance Out Of meTagenomes (GROOT), and show it to be both faster and more accurate than a current reference-dependent tool for resistome profiling. GROOT runs on a laptop and can process a typical 2 gigabyte metagenome in 2 min using a single CPU. Our method is not restricted to resistome profiling and has the potential to improve current metagenomic workflows. Availability and implementation: GROOT is written in Go and is available at https://github.com/will-rowe/groot (MIT license). Supplementary information: Supplementary data are available at Bioinformatics online.

Modulation of Antimicrobial Peptide Potency in Stressed Lipid Bilayers.

It is shown that the tendency of an archetypal antimicrobial peptide to insert into and perforate a simple lipid bilayer is strongly modulated by tensile stress in the membrane. The results, obtained through molecular dynamics simulations, have been demonstrated with several lipid compositions and appear to be general, although quantitative details differ. The findings imply that the potency of antimicrobial peptides may not be a purely intrinsic chemical property and, instead, depends on the mechanical state of the target membrane.

Accelerating molecular discovery through data and physical sciences: Applications to peptide-membrane interactions.

Simulation and data analysis have evolved into powerful methods for discovering and understanding molecular modes of action and designing new compounds to exploit these modes. The combination provides a strong impetus to create and exploit new tools and techniques at the interfaces between physics, biology, and data science as a pathway to new scientific insight and accelerated discovery. In this context, we explore the rational design of novel antimicrobial peptides (short protein sequences exhibiting broad activity against multiple species of bacteria). We show how datasets can be harvested to reveal features which inform new design concepts. We introduce new analysis and visualization tools: a graphical representation of the k-mer spectrum as a fundamental property encoded in antimicrobial peptide databases and a data-driven representation to illustrate membrane binding and permeation of helical peptides.

K-mer clustering algorithm using a MapReduce framework: application to the parallelization of the Inchworm module of Trinity.

BACKGROUND: De novo transcriptome assembly is an important technique for understanding gene expression in non-model organisms. Many de novo assemblers using the de Bruijn graph of a set of the RNA sequences rely on in-memory representation of this graph. However, current methods analyse the complete set of read-derived k-mer sequence at once, resulting in the need for computer hardware with large shared memory. RESULTS: We introduce a novel approach that clusters k-mers as the first step. The clusters correspond to small sets of gene products, which can be processed quickly to give candidate transcripts. We implement the clustering step using the MapReduce approach for parallelising the analysis of large datasets, which enables the use of compute clusters. The computational task is distributed across the compute system using the industry-standard MPI protocol, and no specialised hardware is required. Using this approach, we have re-implemented the Inchworm module from the widely used Trinity pipeline, and tested the method in the context of the full Trinity pipeline. Validation tests on a range of real datasets show large reductions in the runtime and per-node memory requirements, when making use of a compute cluster. CONCLUSIONS: Our study shows that MapReduce-based clustering has great potential for distributing challenging sequencing problems, without loss of accuracy. Although we have focussed on the Trinity package, we propose that such clustering is a useful initial step for other assembly pipelines.

Improved metrics for comparing structures of macromolecular assemblies determined by 3D electron-microscopy.

Recent developments in 3-dimensional electron microcopy (3D-EM) techniques and a concomitant drive to look at complex molecular structures, have led to a rapid increase in the amount of volume data available for biomolecules. This creates a demand for better methods to analyse the data, including improved scores for comparison, classification and integration of data at different resolutions. To this end, we developed and evaluated a set of scoring functions that compare 3D-EM volumes. To test our scores we used a benchmark set of volume alignments derived from the Electron Microscopy Data Bank. We find that the performance of different scores vary with the map-type, resolution and the extent of overlap between volumes. Importantly, adding the overlap information to the local scoring functions can significantly improve their precision and accuracy in a range of resolutions. A combined score involving the local mutual information and overlap (LMI_OV) performs best overall, irrespective of the map category, resolution or the extent of overlap, and we recommend this score for general use. The local mutual information score itself is found to be more discriminatory than cross-correlation coefficient for intermediate-to-low resolution maps or when the map size and density distribution differ significantly. For comparing map surfaces, we implemented two filters to detect the surface points, including one based on the 'extent of surface exposure'. We show that scores that compare surfaces are useful at low resolutions and for maps with evident surface features. All the scores discussed are implemented in TEMPy (http://tempy.ismb.lon.ac.uk/).

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 (2.5-4.5Å) 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.

Effect of phosphorylation on EGFR dimer stability probed by single-molecule dynamics and FRET/FLIM.

Deregulation of epidermal growth factor receptor (EGFR) signaling has been correlated with the development of a variety of human carcinomas. EGF-induced receptor dimerization and consequent trans- auto-phosphorylation are among the earliest events in signal transduction. Binding of EGF is thought to induce a conformational change that consequently unfolds an ectodomain loop required for dimerization indirectly. It may also induce important allosteric changes in the cytoplasmic domain. Despite extensive knowledge on the physiological activation of EGFR, the effect of targeted therapies on receptor conformation is not known and this particular aspect of receptor function, which can potentially be influenced by drug treatment, may in part explain the heterogeneous clinical response among cancer patients. Here, we used Förster resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM) combined with two-color single-molecule tracking to study the effect of ATP-competitive small molecule tyrosine kinase inhibitors (TKIs) and phosphatase-based manipulation of EGFR phosphorylation on live cells. The distribution of dimer on-times was fitted to a monoexponential to extract dimer off-rates (koff). Our data show that pretreatment with gefitinib (active conformation binder) stabilizes the EGFR ligand-bound homodimer. Overexpression of EGFR-specific DEP-1 phosphatase was also found to have a stabilizing effect on the homodimer. No significant difference in the koff of the dimer could be detected when an anti-EGFR antibody (425 Snap single-chain variable fragment) that allows for dimerization of ligand-bound receptors, but not phosphorylation, was used. These results suggest that both the conformation of the extracellular domain and phosphorylation status of the receptor are involved in modulating the stability of the dimer. The relative fractions of these two EGFR subpopulations (interacting versus free) were obtained by a fractional-intensity analysis of ensemble FRET/FLIM images. Our combined imaging approach showed that both the fraction and affinity (surrogate of conformation at a single-molecule level) increased after gefitinib pretreatment or DEP-1 phosphatase overexpression. Using an EGFR mutation (I706Q, V948R) that perturbs the ability of EGFR to dimerize intracellularly, we showed that a modest drug-induced increase in the fraction/stability of the EGFR homodimer may have a significant biological impact on the tumor cell's proliferation potential.

MRC2014: Extensions to the MRC format header for electron cryo-microscopy and tomography.

The MRC binary file format is widely used in the three-dimensional electron microscopy field for storing image and volume data. Files contain a header which describes the kind of data held, together with other important metadata. In response to advances in electron microscopy techniques, a number of variants to the file format have emerged which contain useful additional data, but which limit interoperability between different software packages. Following extensive discussions, the authors, who represent leading software packages in the field, propose a set of extensions to the MRC format standard designed to accommodate these variants, while restoring interoperability. The MRC format is equivalent to the map format used in the CCP4 suite for macromolecular crystallography, and the proposal also maintains interoperability with crystallography software. This Technical Note describes the proposed extensions, and serves as a reference for the standard.

Collaborative computational project for electron cryo-microscopy.

The Collaborative Computational Project for Electron cryo-Microscopy (CCP-EM) has recently been established. The aims of the project are threefold: to build a coherent cryoEM community which will provide support for individual scientists and will act as a focal point for liaising with other communities, to support practising scientists in their use of cryoEM software and finally to support software developers in producing and disseminating robust and user-friendly programs. The project is closely modelled on CCP4 for macromolecular crystallography, and areas of common interest such as model fitting, underlying software libraries and tools for building program packages are being exploited. Nevertheless, cryoEM includes a number of techniques covering a large range of resolutions and a distinct project is required. In this article, progress so far is reported and future plans are discussed.

Exploring the speed and performance of molecular replacement with AMPLE using QUARK ab initio protein models.

AMPLE clusters and truncates ab initio protein structure predictions, producing search models for molecular replacement. Here, an interesting degree of complementarity is shown between targets solved using the different ab initio modelling programs QUARK and ROSETTA. Search models derived from either program collectively solve almost all of the all-helical targets in the test set. Initial solutions produced by Phaser after only 5 min perform surprisingly well, improving the prospects for in situ structure solution by AMPLE during synchrotron visits. Taken together, the results show the potential for AMPLE to run more quickly and successfully solve more targets than previously suspected.

Conversion of a disulfide bond into a thioacetal group during echinomycin biosynthesis.

Echinomycin is a nonribosomal depsipeptide natural product with a range of interesting bioactivities that make it an important target for drug discovery and development. It contains a thioacetal bridge, a unique chemical motif derived from the disulfide bond of its precursor antibiotic triostin A by the action of an S-adenosyl-L-methionine-dependent methyltransferase, Ecm18. The crystal structure of Ecm18 in complex with its reaction products S-adenosyl-L-homocysteine and echinomycin was determined at 1.50 Å resolution. Phasing was achieved using a new molecular replacement package called AMPLE, which automatically derives search models from structure predictions based on ab initio protein modelling. Structural analysis indicates that a combination of proximity effects, medium effects, and catalysis by strain drives the unique transformation of the disulfide bond into the thioacetal linkage.

Community recommendations on cryoEM data archiving and validation: Outcomes of a wwPDB/EMDB workshop on cryoEM data management, deposition and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and consensus recommendations resulting from the workshop. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Community recommendations on cryoEM data archiving and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Ligand binding and dynamics of the monomeric epidermal growth factor receptor ectodomain.

The ectodomain of the human epidermal growth factor receptor (hEGFR) controls input to several cell signalling networks via binding with extracellular growth factors. To gain insight into the dynamics and ligand binding of the ectodomain, the hEGFR monomer was subjected to molecular dynamics simulation. The monomer was found to be substantially more flexible than the ectodomain dimer studied previously. Simulations where the endogeneous ligand EGF binds to either Subdomain I or Subdomain III, or where hEGFR is unbound, show significant differences in dynamics. The molecular mechanics Poisson-Boltzmann surface area method has been used to derive relative free energies of ligand binding, and we find that the ligand is capable of binding either subdomain with a slight preference for III. Alanine-scanning calculations for the effect of selected ligand mutants on binding reproduce the trends of affinity measurements. Taken together, these results emphasize the possible role of the ectodomain monomer in the initial step of ligand binding, and add details to the static picture obtained from crystal structures.

Application of the AMPLE cluster-and-truncate approach to NMR structures for molecular replacement.

AMPLE is a program developed for clustering and truncating ab initio protein 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. Rosetta remodelling 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 using AMPLE are provided.