FEI’s direct electron detector developments: Embarking on a revolution in cryo-TEM

Kuijper, Maarten; Hoften, Gerald van; Janssen, Bart; Geurink, Rudolf; Carlo, Sacha De; Vos, Matthijn; Duinen, G. van; Haeringen, Bart van; Storms, M.

doi:10.1016/j.jsb.2015.09.014

Cited by 67 publications

(40 citation statements)

References 16 publications

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“…one electron per 150 pixels). This figure is similar to that for Falcon III in Kuijper et al (2015). regions near the metal grid bars of the EM grid does the temperature fall more slowly due to the thermal inertia of the grid bar, so there is a 3-5 µm wide region near each grid bar where the ice is frequently crystalline.…”

Section: Beam-induced Specimen Movementsupporting

confidence: 71%

Single particle electron cryomicroscopy: trends, issues and future perspective

Vinothkumar

Henderson

2016

Quart. Rev. Biophys.

175

115

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Abstract. There has been enormous progress during the last few years in the determination of three-dimensional biological structures by single particle electron cryomicroscopy (cryoEM), allowing maps to be obtained with higher resolution and from fewer images than required previously. This is due principally to the introduction of a new type of direct electron detector that has 2-to 3-fold higher detective quantum efficiency than available previously, and to the improvement of the computational algorithms for image processing. In spite of the great strides that have been made, quantitative analysis shows that there are still significant gains to be made provided that the problems associated with image degradation can be solved, possibly by minimising beam-induced specimen movement and charge build up during imaging. If this can be achieved, it should be possible to obtain near atomic resolution structures of smaller single particles, using fewer images and resolving more conformational states than at present, thus realising the full potential of the method. The recent popularity of cryoEM for molecular structure determination also highlights the need for lower cost microscopes, so we encourage development of an inexpensive, 100 keV electron cryomicroscope with a high-brightness field emission gun to make the method accessible to individual groups or institutions that cannot afford the investment and running costs of a state-of-the-art 300 keV installation. A key requisite for successful high-resolution structure determination by cryoEM includes interpretation of images and optimising the biochemistry and grid preparation to obtain nicely distributed macromolecules of interest. We thus include in this review a gallery of cryoEM micrographs that shows illustrative examples of single particle images of large and small macromolecular complexes.

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Section: Beam-induced Specimen Movementsupporting

confidence: 71%

Single particle electron cryomicroscopy: trends, issues and future perspective

Vinothkumar

Henderson

2016

Quart. Rev. Biophys.

175

115

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“…S3). They show similar characteristics to the same DEDs on different microscopes (Faruqi and McMullan, 2018; Kuijper et al, 2015; Ruskin et al, 2013) as expected.…”

Section: Resultssupporting

confidence: 80%

Sub-3 Å resolution structure of apoferritin using a multi-purpose TEM with a side-entry cryo-holder

Kayama

Burton‐Smith

Song

et al. 2020

Preprint

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15The structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single 16 particle analysis (SPA) has had great impact as a biophysical method in recent years. Many results 17 of cryo-EM SPA are based on state-of-the-art cryo-electron microscopes customized for SPA. 18These are currently only available in limited locations around the world, where securing machine 19 time is highly competitive. One potential solution for this time-competitive situation is to reuse 20 existing multi-purpose equipment. Here, we used a multi-purpose TEM with a side entry cryo-21 holder at our facility to evaluate the potential of high-resolution SPA. We report a 3 Å resolution 22 map of apoferritin with local resolution extending to 2.6 Å. The map clearly showed two positions 23 of an aromatic side chain. We also verified the optimal imaging conditions depending on different 24 electron microscope and camera combinations. This study demonstrates the possibilities of more 25 widely available and established electron microscopes, and their applications for cryo-EM SPA. 26 27 Keywords 28 apoferritin; benchmarking; cryo-electron microscopy; multi-purpose TEM; single particle 29 analysis; side-entry cryo-holder 30

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“…It only emerges here owing to the 'full-data-set' aspect of the eigenvector analysis, bringing even the weakest of effects to statistical significance. The Falcon II is not a 'super-resolution' single-electron detection camera, as is, for example, the Gatan K2 Summit or the Falcon III camera (Kuijper et al, 2015). Nevertheless, our MSA analysis reveals that significant 'superresolution' information could be recorded.…”

Section: Anisotropic Magnificationmentioning

confidence: 81%

Single-particle cryo-EM using alignment by classification (ABC): the structure ofLumbricus terrestrishaemoglobin

Afanasyev

Seer-Linnemayr

Ravelli

et al. 2017

IUCrJ

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Single-particle cryogenic electron microscopy (cryo-EM) can now yield nearatomic resolution structures of biological complexes. However, the referencebased alignment algorithms commonly used in cryo-EM suffer from reference bias, limiting their applicability (also known as the 'Einstein from random noise' problem). Low-dose cryo-EM therefore requires robust and objective approaches to reveal the structural information contained in the extremely noisy data, especially when dealing with small structures. A reference-free pipeline is presented for obtaining near-atomic resolution three-dimensional reconstructions from heterogeneous ('four-dimensional') cryo-EM data sets. The methodologies integrated in this pipeline include a posteriori camera correction, movie-based full-data-set contrast transfer function determination, movie-alignment algorithms, (Fourier-space) multivariate statistical data compression and unsupervised classification, 'random-startup' three-dimensional reconstructions, four-dimensional structural refinements and Fourier shell correlation criteria for evaluating anisotropic resolution. The procedures exclusively use information emerging from the data set itself, without external 'starting models'. Euler-angle assignments are performed by angular reconstitution rather than by the inherently slower projection-matching approaches. The comprehensive 'ABC-4D' pipeline is based on the two-dimensional referencefree 'alignment by classification' (ABC) approach, where similar images in similar orientations are grouped by unsupervised classification. Some fundamental differences between X-ray crystallography versus single-particle cryo-EM data collection and data processing are discussed. The structure of the giant haemoglobin from Lumbricus terrestris at a global resolution of $3.8 Å is presented as an example of the use of the ABC-4D procedure.

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FEI’s direct electron detector developments: Embarking on a revolution in cryo-TEM

Cited by 67 publications

References 16 publications

Single particle electron cryomicroscopy: trends, issues and future perspective

Single particle electron cryomicroscopy: trends, issues and future perspective

Sub-3 Å resolution structure of apoferritin using a multi-purpose TEM with a side-entry cryo-holder

Single-particle cryo-EM using alignment by classification (ABC): the structure ofLumbricus terrestrishaemoglobin

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