A microtubule RELION-based pipeline for cryo-EM image processing
Ontology highlight
ABSTRACT:
SUBMITTER: Joseph Michael Atherton
PROVIDER: EMPIAR-10796 | biostudies-other |
REPOSITORIES: biostudies-other
Publications
A microtubule RELION-based pipeline for cryo-EM image processing.
Cook Alexander D AD Manka Szymon W SW Wang Su S Moores Carolyn A CA Atherton Joseph J
Journal of structural biology 20191011 1
Microtubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and β-tubulin. The de ...[more]
Similar Datasets
Project description:Microtubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and β-tubulin. The determination of the αβ-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins.
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Project description: Not available
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Project description:The use of cryoEM and three-dimensional image reconstruction is becoming increasingly common. Our vision for this technique is to provide a straightforward manner in which users can proceed from raw data to a reliable 3D reconstruction through a pipeline that both facilitates management of the processing steps and makes the results at each step more transparent. Tightly integrated with a relational SQL database, Appion is a modular and transparent pipeline that extends existing software applications and procedures. The user manages and controls the software modules via web-based forms, and all results are similarly available using web-based viewers directly linked to the underlying database, enabling even naive users to quickly deduce the quality of their results. The Appion API was designed with the principle that applications should be compatible with a broad range of specimens and that libraries and routines are modular and extensible. Presented here is a description of the design and architecture of the working Appion pipeline prototype and some results of its use.
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Project description:Macromolecular structural determination by Electron Microscopy under cryogenic conditions is revolutionizing the field of structural biology, interesting a large community of potential users. Still, the path from raw images to density maps is complex, and sophisticated image processing suites are required in this process, often demanding the installation and understanding of different software packages. Here, we present Scipion Web Tools, a web-based set of tools/workflows derived from the Scipion image processing framework, specially tailored to nonexpert users in need of very precise answers at several key stages of the structural elucidation process.
| S-EPMC5734268 | biostudies-literature
Project description:SummaryThe Electron Microscopy Hole Punch (EMHP) is a streamlined suite of tools for quick assessment, sorting and hole masking of electron micrographs. With recent advances in single-particle electron cryo-microscopy (cryo-EM) data processing allowing for the rapid determination of protein structures using a smaller computational footprint, we saw the need for a fast and simple tool for data pre-processing that could run independent of existing high-performance computing (HPC) infrastructures. EMHP provides a data preprocessing platform in a small package that requires minimal python dependencies to function.Availability and implementationhttps://www.bitbucket.org/chazbot/emhp Apache 2.0 License.Contactbowman@scripps.edu.Supplementary informationSupplementary data are available at Bioinformatics online.
| S-EPMC5860320 | biostudies-literature
Project description:Segmentation is one of the most important stages in the 3D reconstruction of macromolecule structures in cryo-electron microscopy. Due to the variability of macromolecules and the low signal-to-noise ratio of the structures present, there is no generally satisfactory solution to this process. This work proposes a new unsupervised particle picking and segmentation algorithm based on the composition of two well-known image filters: Anisotropic (Perona-Malik) diffusion and non-negative matrix factorization. This study focused on keyhole limpet hemocyanin (KLH) macromolecules which offer both a top view and a side view. Our proposal was able to detect both types of views and separate them automatically. In our experiments, we used 30 images from the KLH dataset of 680 positive classified regions. The true positive rate was 95.1% for top views and 77.8% for side views. The false negative rate was 14.3%. Although the false positive rate was high at 21.8%, it can be lowered with a supervised classification technique.
| S-EPMC6995569 | biostudies-literature
Project description:The existence of structural intermediates in the processes of microtubule assembly and disassembly, and their relationship with the nucleotide state of tubulin, have been the subject of significant study and recent controversy. The first part of this chapter describes experiments and methods designed to characterize, using cryo-electron microscopy (cryo-EM) and image analysis, the structure of stabilized tubulin assemblies that we propose mimic the growth and shortening states at microtubule ends. We further put forward the idea that these intermediates have important biological functions, especially during cellular processes where the dynamic character of microtubules is essential. One such process is the attachment of spindle microtubules to kinetochores in eukaryotic cell division. The second part of this chapter is consequently dedicated to studies of the yeast Dam 1 kinetochore complex and its interaction with microtubules. This complex is essential for accurate chromosome segregation and is an important target of the Aurora B spindle check-point kinase. The Dam 1 complex self-assembles in a microtubule-dependent manner into rings and spirals. The rings are able to track microtubule-depolymerizing ends against a load and in a highly processive manner, an essential property for their function in vivo. We describe the experimental in vitro protocols to produce biologically relevant self-assembled structures of Dam 1 around microtubules and their structural characterization by cryo-EM.
| S-EPMC4167756 | biostudies-literature
Project description:To compare cryo-EM images and 3D reconstructions with atomic structures in a quantitative way it is essential to model the electron scattering by solvent (water or ice) that surrounds protein assemblies. The most rigorous method for determining the density of solvating water atoms for this purpose has been to perform molecular-dynamics (MD) simulations of the protein-water system. In this paper we adapt the ideas of bulk-water modeling that are used in the refinement of X-ray crystal structures to the cryo-EM solvent-modeling problem. We present a continuum model for solvent density which matches MD-based results to within sampling errors. However, we also find that the simple binary-mask model of Jiang and Brünger (1994) performs nearly as well as the new model. We conclude that several methods are now available for rapid and accurate modeling of cryo-EM images and maps of solvated proteins.
| S-EPMC3577369 | biostudies-literature
Project description:The development of cryo-electron microscopy (cryo-EM) allowed microtubules to be captured in their solution-like state, enabling decades of insight into their dynamic mechanisms and interactions with binding partners. Cryo-EM micrographs provide 2D visualization of microtubules, and these 2D images can also be used to reconstruct the 3D structure of the polymer and any associated binding partners. In this way, the binding sites for numerous components of the microtubule cytoskeleton-including motor domains from many kinesin motors, and the microtubule-binding domains of dynein motors and an expanding collection of microtubule associated proteins-have been determined. The effects of various microtubule-binding drugs have also been studied. High-resolution cryo-EM structures have also been used to probe the molecular basis of microtubule dynamic instability, driven by the GTPase activity of ?-tubulin. These studies have shown the conformational changes in lattice-confined tubulin dimers in response to steps in the tubulin GTPase cycle, most notably lattice compaction at the longitudinal inter-dimer interface. Although work is ongoing to define a complete structural model of dynamic instability, attention has focused on the role of gradual destabilization of lateral contacts between tubulin protofilaments, particularly at the microtubule seam. Furthermore, lower resolution cryo-electron tomography 3D structures are shedding light on the heterogeneity of microtubule ends and how their 3D organization contributes to dynamic instability. The snapshots of these polymers captured using cryo-EM will continue to provide critical insights into their dynamics, interactions with cellular components, and the way microtubules contribute to cellular functions in diverse physiological contexts.
| S-EPMC6281474 | biostudies-literature
Project description:Successful sample preparation is the foundation to any structural biology technique. Membrane proteins are of particular interest as these are important targets for drug design, but also notoriously difficult to work with. For electron cryo-microscopy (cryo-EM), the biophysical characterization of sample purity, homogeneity, and integrity as well as biochemical activity is the prerequisite for the preparation of good quality cryo-EM grids as these factors impact the result of the computational reconstruction. Here, we present a quality control pipeline prior to single particle cryo-EM grid preparation using a combination of biophysical techniques to address the integrity, purity, and oligomeric states of membrane proteins and its complexes to enable reproducible conditions for sample vitrification. Differential scanning fluorimetry following the intrinsic protein fluorescence (nDSF) is used for optimizing buffer and detergent conditions, whereas mass photometry and dynamic light scattering are used to assess aggregation behavior, reconstitution efficiency, and oligomerization. The data collected on nDSF and mass photometry instruments can be analyzed with web servers publicly available at spc.embl-hamburg.de. Case studies to optimize conditions prior to cryo-EM sample preparation of membrane proteins present an example quality assessment to corroborate the usefulness of our pipeline.
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