Automation in Single-Particle Electron Microscopy

Lyumkis, Dmitry; Moeller, Arne; Cheng, Anchi; Herold, Amber; Hou, Eric; Irving, Christopher; Jacovetty, Erica L.; Lau, Pick Wei; Mulder, Anke M.; Pulokas, James; Quispe, Joel; Voss, Neil R.; Potter, Clinton S.; Carragher, Bridget

doi:10.1016/s0076-6879(10)83015-0

Cited by 23 publications

(19 citation statements)

References 171 publications

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“…Much effort has been invested in simplifying and automating the collection of EM images and the use of image processing software (reviewed in Lyumkis et al, 2010). The problematic issue with single-particle EM, however, is that there is still no objective quality criterion that is simple and easy to use, such as the R-free value in X-ray crystallography, that would allow one to assess whether the determined density map is accurate or not.…”

Section: Introductionmentioning

confidence: 99%

A Primer to Single-Particle Cryo-Electron Microscopy

Cheng

Grigorieff

Penczek

et al. 2015

Cell

521

411

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Summary Cryo-electron microscopy (cryo-EM) of single-particle specimens is used to determine the structure of proteins and macromolecular complexes without the need for crystals. Recent advances in detector technology and software algorithms now allow images of unprecedented quality to be recorded and structures to be determined at near-atomic resolution. However, compared with X-ray crystallography, cryo-EM is a young technique with distinct challenges. This primer explains the different steps and considerations involved in structure determination by single-particle cryo-EM to provide an overview for scientists wishing to understand more about this technique and the interpretation of data obtained with it, as well as a starting guide for new practitioners.

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Section: Introductionmentioning

confidence: 99%

A Primer to Single-Particle Cryo-Electron Microscopy

Cheng

Grigorieff

Penczek

et al. 2015

Cell

521

411

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“…Nanodiscs when viewed edgewise have a distinct high-contrast shape that allows the use of automated particle selection procedures to potentially generate the large data sets of particles necessary for high resolution analysis. [8][9][10] We have used a simple preparative procedure that preferentially displays the nanodiscs in edge-view, most useful for their identification and inherent angular variability. We have faced the problem of low contrast of the PA in these images, limiting the initial analysis to a modest (16 Å ) nominal resolution.…”

Section: Introductionmentioning

confidence: 99%

Three dimensional structure of the anthrax toxin translocon–lethal factor complex by cryo‐electron microscopy

et al. 2013

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We have visualized by cryo-electron microscopy (cryo-EM) the complex of the anthrax protective antigen (PA) translocon and the N-terminal domain of anthrax lethal factor (LF N ) inserted into a nanodisc model lipid bilayer. We have determined the structure of this complex at a nominal resolution of 16 Å by single-particle analysis and three-dimensional reconstruction. Consistent with our previous analysis of negatively stained unliganded PA, the translocon comprises a globular structure (cap) separated from the nanodisc bilayer by a narrow stalk that terminates in a transmembrane channel (incompletely distinguished in this reconstruction). The globular cap is larger than the unliganded PA pore, probably due to distortions introduced in the previous negatively stained structures. The cap exhibits larger, more distinct radial protrusions, previously identified with PA domain three, fitted by elements of the NMFF PA prepore crystal structure. The presence of LF N , though not distinguished due to the seven-fold averaging used in the reconstruction, contributes to the distinct protrusions on the cap rim volume distal to the membrane. Furthermore, the lumen of the cap region is less resolved than the unliganded negatively stained PA, due to the low contrast obtained in our images of this specimen. Presence of the LF N extended helix and N terminal unstructured regions may also contribute to this additional internal density within the interior of the cap. Initial NMFF fitting of the cryoEM-defined PA pore cap region positions the Phe clamp region of the PA pore translocon directly above an internal vestibule, consistent with its role in toxin translocation.

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“…Automation is more than a means to save time and more than the mere elimination of a tedious repetitive task. It is a strategy that allows to gather data sets of unprecedented size and quality (Lyumkis et al, 2010).…”

Section: Discussionmentioning

confidence: 99%