MonoRes: Automatic and Accurate Estimation of Local Resolution for Electron Microscopy Maps

Vilas, José Luis; Gómez-Blanco, J.; Conesa, Pablo; Melero, Roberto; Rosa-Trevín, J.M. de la; Otón, Joaquı́n; Cuenca, J.; Marabini, Roberto; Carazo, José Marı́a; Vargas, Javier; Sorzano, Carlos Óscar S.

doi:10.1016/j.str.2017.12.018

Cited by 194 publications

(175 citation statements)

References 30 publications

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“…The twin problems of the resolution at which a reconstruction remains interpretable, and of variable local "resolution" or SNR, have been heavily investigated within the single particle analysis field. Independent halfset refinement, in which over-fitting is mitigated against during the refinement process (Grigorieff, 2000;Scheres & Chen, 2012;Henderson et al, 2012), local resolution measurement (Cardone et al, 2013, Kucukelbir et al, 2014 and local resolution filtering (Cardone et al, 2013;Vilas et al, 2018a), pursued after the refinement process, have been widely adopted to avoid over-interpretation of reconstructed densities.…”

Section: Discussionmentioning

confidence: 99%

“…where signal becomes indistinguishable from noise (Stewart & Grigorieff, 2004). Several methods for evaluation and treatment of local resolution in cryo-EM reconstruction have previously been proposed (Stewart & Grigorieff, 2004;Cardone et al, 2013;Chen et al, 2013;Kucukelbir et al, 2014;Vilas et al, 2018a;Ramírez-Aportela et al, 2018), however none provide a way to minimise over-fitting during the reconstruction process.…”

Section: Globally Filtered Reconstruction Methods Must Be Expected Tomentioning

confidence: 99%

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Mitigating Local Over-fitting During Single Particle Reconstruction with SIDESPLITTER

Ramlaul¹,

Palmer

Aylett

2019

Preprint

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Abbreviations2D/3D -2/3-Dimensional; Cryo-EM -Electron Cryo-Microscopy; EM -Electron microscopy; FSC -Fourier shell correlation; LAFTER -Local Agreement Filter for Transmission EM Reconstructions; SNR -Signal to noise ratio. AbstractSingle particle analysis of cryo-EM images enables macromolecular structure determination at resolutions approaching the atomic scale. Experimental images are extremely noisy, however, and during iterative refinement it is possible to stably incorporate noise into the reconstructed density. Such "over-fitting" can lead to misinterpretation of the structure, and thereby flawed biological results. Several strategies are routinely used to prevent the spurious incorporation of noise within reconstructed volumes, the most common being independent refinement of two sides of a split dataset.In this study, we show that over-fitting remains an issue within regions of low local signal-to-noise in reconstructed volumes refined using the half-set strategy. We propose a modified filtering process during refinement through the application of a local signal-to-noise filter, SIDESPLITTER, which we show to be capable of reducing over-fitting in both idealised and experimental settings, while maintaining independence between the two sides of a split refinement. SIDESPLITTER can also improve the final resolution in refinements of structures prone to severe over-fitting, such as membrane proteins in detergent micelles.

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Section: Discussionmentioning

confidence: 99%

Section: Globally Filtered Reconstruction Methods Must Be Expected Tomentioning

confidence: 99%

Mitigating Local Over-fitting During Single Particle Reconstruction with SIDESPLITTER

Ramlaul¹,

Palmer

Aylett

2019

Preprint

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“…CCV particles were picked using FindEM Template picking (44), resulting in a total of 80k CCV particle images (894-pixel box size). FSC was generated using even and odd masked half maps, and the local resolution for the mini-coat vertex was calculated using monores (45).…”

Section: Tryptic Peptides Separation and Detectionmentioning

confidence: 99%

The Structures of Natively Assembled Clathrin Coated Vesicles

Paraan

Mendez

Sharum

et al. 2020

Preprint

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Vesicle trafficking by clathrin coated vesicles (CCVs) is one of the major mechanisms by which proteins and nutrients are absorbed by the cell and transported between organelles. The individual proteins comprising the coated vesicles include clathrin heavy chain, clathrin light chain, and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2appendages crosslink adjacent CHC β-propellers and that the appendage densities reside almost exclusively in CCV hexagonal faces. We resolve how AP2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.

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“…We processed four classes (155,192,315, and 240 particles) using a 50 pixel box size and 15 iterations. The limited number of particles available in cryo-ET experiments did not allow to obtain a high-resolution structure; the resolution estimated using the local resolution algorithm MonoRes (Vilas et al, 2018) is around 3 nm. The method is reference-free; the process was started from a weighted average structure obtained from random orientations of all particles.…”

Section: Subtomogram Averaging and Classificationmentioning

confidence: 99%

“…Due to the large box size, part of the plate associated with decorative nucleosomes is also included in the subtomogram average. The limited number of particles available in cryo-ET experiments did not allow to obtain a high-resolution structure; the resolution estimated using the local resolution algorithm MonoRes (Vilas et al, 2018) is around 3 nm.…”

Section: Subtomogram Averaging and Classificationmentioning

confidence: 99%

Frozen‐hydrated chromatin from metaphase chromosomes has an interdigitated multilayer structure

et al. 2019

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Cryo‐electron tomography and small‐angle X‐ray scattering were used to investigate the chromatin folding in metaphase chromosomes. The tomographic 3D reconstructions show that frozen‐hydrated chromatin emanated from chromosomes is planar and forms multilayered plates. The layer thickness was measured accounting for the contrast transfer function fringes at the plate edges, yielding a width of ~ 7.5 nm, which is compatible with the dimensions of a monolayer of nucleosomes slightly tilted with respect to the layer surface. Individual nucleosomes are visible decorating distorted plates, but typical plates are very dense and nucleosomes are not identifiable as individual units, indicating that they are tightly packed. Two layers in contact are ~ 13 nm thick, which is thinner than the sum of two independent layers, suggesting that nucleosomes in the layers interdigitate. X‐ray scattering of whole chromosomes shows a main scattering peak at ~ 6 nm, which can be correlated with the distance between layers and between interdigitating nucleosomes interacting through their faces. These observations support a model where compact chromosomes are composed of many chromatin layers stacked along the chromosome axis.

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MonoRes: Automatic and Accurate Estimation of Local Resolution for Electron Microscopy Maps

Cited by 194 publications

References 30 publications

Mitigating Local Over-fitting During Single Particle Reconstruction with SIDESPLITTER

Mitigating Local Over-fitting During Single Particle Reconstruction with SIDESPLITTER

The Structures of Natively Assembled Clathrin Coated Vesicles

Frozen‐hydrated chromatin from metaphase chromosomes has an interdigitated multilayer structure

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