Beyond blob-ology

Smith, Martin T. J.; Rubinstein, John L.

doi:10.1126/science.1256358

Cited by 47 publications

(25 citation statements)

References 14 publications

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“…Recent advances in single particle cryo-EM instrumentation and data processing have facilitated the calculation of high-resolution 3D-maps of biomolecules as small as 60 kDa (Bai et al, 2015; Smith and Rubinstein, 2014; Khoshouei et al, 2016). Figure 7A–B show cryo-EM density maps of free and RNF169-bound NCPs, determined at 8.1 and 6.6 Å resolution, respectively (Figure 7—figure supplement 1).…”

Section: Resultsmentioning

confidence: 99%

The RNF168 paralog RNF169 defines a new class of ubiquitylated histone reader involved in the response to DNA damage

Kitevski-LeBlanc

Fradet-Turcotte

Kukić

et al. 2017

eLife

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Site-specific histone ubiquitylation plays a central role in orchestrating the response to DNA double-strand breaks (DSBs). DSBs elicit a cascade of events controlled by the ubiquitin ligase RNF168, which promotes the accumulation of repair factors such as 53BP1 and BRCA1 on the chromatin flanking the break site. RNF168 also promotes its own accumulation, and that of its paralog RNF169, but how they recognize ubiquitylated chromatin is unknown. Using methyl-TROSY solution NMR spectroscopy and molecular dynamics simulations, we present an atomic resolution model of human RNF169 binding to a ubiquitylated nucleosome, and validate it by electron cryomicroscopy. We establish that RNF169 binds to ubiquitylated H2A-Lys13/Lys15 in a manner that involves its canonical ubiquitin-binding helix and a pair of arginine-rich motifs that interact with the nucleosome acidic patch. This three-pronged interaction mechanism is distinct from that by which 53BP1 binds to ubiquitylated H2A-Lys15 highlighting the diversity in site-specific recognition of ubiquitylated nucleosomes.DOI: http://dx.doi.org/10.7554/eLife.23872.001

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Section: Resultsmentioning

confidence: 99%

The RNF168 paralog RNF169 defines a new class of ubiquitylated histone reader involved in the response to DNA damage

Kitevski-LeBlanc

Fradet-Turcotte

Kukić

et al. 2017

eLife

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“…Although several models that have attempted to model the G. sulfurreducens pilus structure have questioned whether sufficient pi‐pi stacking of aromatic amino acids for metallic‐like conductivity is possible (Feliciano et al ., ; Boesen and Nielsen, ; Bonanni et al ., ; Reardon and Mueller, ), actual experimental results trump modelling, which can be based on faulty assumptions, and the recent EFM imaging of charge propagation (Malvankar et al ., ) further supports the abundant experimental evidence (Malvankar et al ., ; Vargas et al ., ) supporting metallic‐like conductivity. At this point, structural studies, potentially taking advantage of recent advances in cryo‐electron microscopy (Glaeser, ; Smith and Rubinstein, ), are more likely to advance understanding of the features that confer metallic‐like conductivity than will theoretical modelling.…”

Section: Discussionmentioning

confidence: 99%

Seeing is believing: novel imaging techniques help clarify microbial nanowire structure and function

Lovley

Malvankar

2015

Environmental Microbiology

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SummaryNovel imaging approaches have recently helped to clarify the properties of 'microbial nanowires'. Geobacter sulfurreducens pili are actual wires. They possess metallic-like conductivity, which can be attributed to overlapping pi-pi orbitals of key aromatic amino acids. Electrostatic force microscopy recently confirmed charge propagation along the pili, in a manner similar to carbon nanotubes. The pili are essential for long-range electron transport to insoluble electron acceptors and interspecies electron transfer. Previous claims that Shewanella oneidensis also produce conductive pili have recently been recanted, based on novel live-imaging studies. The putative pili are, in fact, long extensions of the cytochrome-rich outer membrane and periplasm that, when dried, collapse to form filaments with dimensions similar to pili. It has yet to be demonstrated whether the cytochrome-to-cytochrome electron hopping documented in the dried membrane extensions takes place in intact hydrated membrane extensions or whether the membrane extensions enhance electron transport to insoluble electron acceptors such as Fe(III) oxides or electrodes. These findings demonstrate that G. sulfurreducens conductive pili and the outer membrane extensions of S. oneidensis are fundamentally different in composition, mechanism of electron transport and physiological role. New methods for evaluating filament conductivity will facilitate screening the microbial world for nanowires and elucidating their function.

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“…Anisotropic magnification in electron microscope images of 2D crystals was first described more than 30 years ago [1] but more recently has not been detected with most modern electron microscopes used under conditions typical for cryo-EM with film or charge-coupled device (CCD) cameras. Direct detector device (DDDs) cameras have revolutionized single particle electron cryomicroscopy (cryo-EM) [2,3]. Two DDD manufacturers, Gatan and Direct Electron, have produced cameras with pixel sizes between 5.0 and 6.5 µm, which is significantly smaller than is typical with CCD cameras.…”

Section: Introductionmentioning

confidence: 99%

Description and comparison of algorithms for correcting anisotropic magnification in cryo-EM images

Zhao

Brubaker

Rubinstein

2015

Journal of Structural Biology

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Single particle electron cryomicroscopy (cryo-EM) allows for structures of proteins and protein complexes to be determined from images of non-crystalline specimens. Cryo-EM data analysis requires electron microscope images of randomly oriented ice-embedded protein particles to be rotated and translated to allow for coherent averaging when calculating three-dimensional (3D) structures. Rotation of 2D images is usually done with the assumption that the magnification of the electron microscope is the same in all directions. However, due to electron optical aberrations, this condition is not met with some electron microscopes when used with the settings necessary for cryo-EM with a direct detector device (DDD) camera. Correction of images by linear interpolation in real space has allowed high-resolution structures to be calculated from cryo-EM images for symmetric particles. Here we describe and compare a simple real space method, a simple Fourier space method, and a somewhat more sophisticated Fourier space method to correct images for a measured anisotropy in magnification. Further, anisotropic magnification causes contrast transfer function (CTF) parameters estimated from image power spectra to have an apparent systematic astigmatism. To address this problem we develop an approach to adjust CTF parameters measured from distorted images so that they can be used with corrected images. The effect of anisotropic magnification on CTF parameters provides a simple way of detecting magnification anisotropy in cryo-EM datasets.

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Beyond blob-ology

Abstract: Advanced cryo–electron microscopy yields high-resolution structures of proteins

Cited by 47 publications

References 14 publications

The RNF168 paralog RNF169 defines a new class of ubiquitylated histone reader involved in the response to DNA damage

The RNF168 paralog RNF169 defines a new class of ubiquitylated histone reader involved in the response to DNA damage

Seeing is believing: novel imaging techniques help clarify microbial nanowire structure and function

Description and comparison of algorithms for correcting anisotropic magnification in cryo-EM images

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