| 1. |
- Luis Vilas, Jose, et al.
(författare)
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MonoRes : Automatic and Accurate Estimation of Local Resolution for Electron Microscopy Maps
- 2018
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 26:2, s. 337-344
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Tidskriftsartikel (refereegranskat)abstract
- Since the beginning of electron microscopy, resolution has been a critical parameter. In this article, we propose a fully automatic, accurate method for determining the local resolution of a 3D map (MonoRes). The foundation of this algorithm is an extension of the concept of analytic signal, termed monogenic signal. The map is filtered at different frequencies and the amplitude of the monogenic signal is calculated, after which a criterion is applied to determine the resolution at each voxel. MonoRes is fully automatic without compulsory user parameters, with great accuracy in all tests, and is computationally more rapid than existing methods in the field. In addition, MonoRes offers the option of local filtering of the original map based on the calculated local resolution.
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| 2. |
- Abrishami, Vahid, et al.
(författare)
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Localized reconstruction in Scipion expedites the analysis of symmetry mismatches in cryo-EM data
- 2021
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Ingår i: Progress in Biophysics and Molecular Biology. - : Elsevier BV. - 0079-6107 .- 1873-1732. ; 160, s. 43-52
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Forskningsöversikt (refereegranskat)abstract
- Technological advances in transmission electron microscopes and detectors have turned cryogenic electron microscopy (cryo-EM) into an essential tool for structural biology. A commonly used cryo-EM data analysis method, single particle analysis, averages hundreds of thousands of low-dose images of individual macromolecular complexes to determine a density map of the complex. The presence of symmetry in the complex is beneficial since each projection image can be assigned to multiple views of the complex. However, data processing that applies symmetry can average out asymmetric features and consequently data analysis methods are required to resolve asymmetric structural features. Scipion is a cryo-EM image processing framework that integrates functions from different image processing packages as plugins. To extend its functionality for handling symmetry mismatches, we present here a Scipion plugin termed LocalRec implementing the localized reconstruction method. When tested on an adenovirus data set, the plugin enables resolving the symmetry-mismatched trimeric fibre bound to the five-fold vertices of the capsid. Furthermore, it improves the structure determination of the icosahedral capsid by dealing with the defocus gradient across the particle. LocalRec is expected to be widely applicable in a range of cryo-EM investigations of flexible and symmetry mismatched complexes.
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| 3. |
- Cheng, Kimberley, et al.
(författare)
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tmRNA-SmpB complex mimics native aminoacyl-tRNAs in the A site of stalled ribosomes
- 2010
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Ingår i: Journal of Structural Biology. - : Elsevier BV. - 1047-8477 .- 1095-8657. ; 169:3, s. 342-348
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial ribosomes stalled on faulty, often truncated, mRNAs lacking stop codons are rescued by trans-translation. It relies on an RNA molecule (tmRNA) capable of replacing the faulty mRNA with its own open reading frame (ORF). Translation of tmRNA ORF results in the tagging of faulty protein for degradation and its release from the ribosome. We used single-particle cryo-electron microscopy to visualize tmRNA together with its helper protein SmpB on the 70S Escherichia coli ribosome in states subsequent to GTP hydrolysis on elongation factor Tu (EF-Tu). Three-dimensional reconstruction and heterogeneity analysis resulted in a 15 A resolution structure of the tmRNA-SmpB complex accommodated in the A site of the ribosome, which shows that SmpB mimics the anticodon- and D-stem of native tRNAs missing in the tRNA-like domain of tmRNA. We conclude that the tmRNA-SmpB complex accommodates in the ribosomal A site very much like an aminoacyl-tRNA during protein elongation.
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| 4. |
- Elmlund, Hans, 1978-
(författare)
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Protein structure dynamics and interplay : by single-particle electron microscopy
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Single-particle cryo-electron microscopy (cryo-EM) is a method capable of obtaining information about the structural organization and dynamics of large macromolecular assemblies. In the late nineties, the method was suggested to have the potential of generating “atomic resolution” reconstructions of particles above a certain mass. However, visualization of secondary structure elements in cryo-EM reconstructions has so far been achieved mainly for highly symmetrical macromolecular assemblies or by using previously existing X-ray structures to solve the initial alignment problem. A factor that severely limits the resolution for low-symmetry (point group symmetry Cn) particles is the problem of ab initio three-dimensional alignment of cryo-EM projection images of proteins in vitreous ice. A more general problem in the field of molecular biology is the study of heterogeneous structural properties of particles in preparations of purified macromolecular complexes. If not resolved, structural heterogeneity limits the achievable resolution of a cryo-EM reconstruction and makes correct biological interpretation difficult. If resolved, the heterogeneity instead offers a tremendous biological insight into the dynamic behaviour of a structure, and statistical information about partitioning over subpopulations with distinct structural features within the ensemble of particles may be gained. This thesis adds to the existing body of methods in the field of single-particle cryo-EM by addressing the problem of ab initio rotational alignment and the problem of resolving structural heterogeneity without using a priori information about the structural variability within large populations of cryo-EM projections of unstained proteins. The thesis aims at making the single-particle cryo-EM method a generally applicable tool for generating subnanometer resolution reconstructions and perform heterogeneity analysis of biological macromolecules.
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