SPHIRE-crYOLO: A fast and accurate fully automated particle picker for cryo-EM

Wagner, Tobias; Merino, Felipe; Stabrin, Markus; Moriya, Toshio; Antoni, Claudia; Apelbaum, Amir; Hagel, Philine; Sitsel, Oleg; Raisch, Tobias; Prumbaum, Daniel; Quentin, Dennis; Roderer, Daniel; Tacke, Sebastian; Siebolds, Birte; Schubert, Evelyn; Shaikh, Tanvir R.; Lill, Pascal; Gatsogiannis, Christos; Raunser, Stefan

doi:10.1101/356584

Cited by 266 publications

(315 citation statements)

References 33 publications

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“…Additionally, early frames can be removed to reduce blurring that results from initial exposure of the sample, and later frames can be removed to reduce the impact of radiation damage that accrues during data acquisition (Scheres 2014). Improvements to image processing software have kept pace (Punjani et al 2017;Zivanov et al 2018;Wagner et al 2019), and with more processing power and pipelined approaches, it is now faster and easier to generate 3D models. Taken together, these innovations have improved the resolution of cryoEM reconstructions to the nearatomic range and allowed the analysis of increasingly smaller proteins or protein complexes.…”

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confidence: 99%

Structure and mechanism of the RNA polymerase II transcription machinery

2020

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RNA polymerase II (Pol II) transcribes all protein-coding genes and many noncoding RNAs in eukaryotic genomes. Although Pol II is a complex, 12-subunit enzyme, it lacks the ability to initiate transcription and cannot consistently transcribe through long DNA sequences. To execute these essential functions, an array of proteins and protein complexes interact with Pol II to regulate its activity. In this review, we detail the structure and mechanism of over a dozen factors that govern Pol II initiation (e.g., TFIID, TFIIH, and Mediator), pausing, and elongation (e.g., DSIF, NELF, PAF, and P-TEFb). The structural basis for Pol II transcription regulation has advanced rapidly in the past decade, largely due to technological innovations in cryoelectron microscopy. Here, we summarize a wealth of structural and functional data that have enabled a deeper understanding of Pol II transcription mechanisms; we also highlight mechanistic questions that remain unanswered or controversial.

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confidence: 99%

Structure and mechanism of the RNA polymerase II transcription machinery

2020

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“…CrYOLO is a particle-picking procedure based on the YOLO framework (Redmon & Farhadi, 2017). For a technical description of crYOLO, we refer the reader to our original publication (Wagner et al, 2019).…”

Section: Cryolomentioning

confidence: 99%

“…The training of crYOLO works as described for singleparticle projects (Wagner et al, 2019). The network is trained on the manually labeled micrographs, while a small subset of those micrographs is used for validation.…”

Section: Cryolo Filament Modementioning

confidence: 99%

“…The determination of protein structures using singleparticle cryo-EM requires the selection of thousands of particles within micrographs. For single particles, various methods have been developed to automate this task (Voss et al, 2009;Scheres, 2015;Huang & Penczek, 2004;Nicholson & Glaeser, 2001;Wagner et al, 2019;Bepler et al, 2019;Tegunov & Cramer, 2019;Wang et al, 2016;Zhu et al, 2017). In particular, the introduction of deep-learning-based procedures have dramatically reduced the false-positive rates of picking and have made the automatic picking of particles the standard in single-particle cryo-EM (Wagner et al, However, the picking of filaments is more challenging because of the line-like structure of the specimens.…”

Section: Introductionmentioning

confidence: 99%

“…Here, we present a new deep-learning filament-picking procedure implemented in our single-particle selection tool SPHIRE-crYOLO (Wagner et al, 2019). CrYOLO is based on a convolutional neural network (CNN) and the 'you only look once' (YOLO) approach (Redmon & Farhadi, 2017).…”

Section: Introductionmentioning

confidence: 99%

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Two particle-picking procedures for filamentous proteins: SPHIRE-crYOLO filament mode and SPHIRE-STRIPER

Wagner¹,

Lusnig²,

Pospich³

et al. 2020

Acta Cryst Sect D Struct Biol

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Structure determination of filamentous molecular complexes involves the selection of filaments from cryo-EM micrographs. The automatic selection of helical specimens is particularly difficult, and thus many challenging samples with issues such as contamination or aggregation are still manually picked. Here, two approaches for selecting filamentous complexes are presented: one uses a trained deep neural network to identify the filaments and is integrated in SPHIRE-crYOLO, while the other, called SPHIRE-STRIPER, is based on a classical line-detection approach. The advantage of the crYOLO-based procedure is that it performs accurately on very challenging data sets and selects filaments with high accuracy. Although STRIPER is less precise, the user benefits from less intervention, since in contrast to crYOLO, STRIPER does not require training. The performance of both procedures on Tobacco mosaic virus and filamentous F-actin data sets is described to demonstrate the robustness of each method.

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Cryo‐Electron Microscopy in Drug Discovery and Development: Opportunities and Challenges

Gomez‐Llorente

Hong

Scapin

2021

Burger's Medicinal Chemistry and Drug Discovery

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While utilization of structural information has been a staple technology in drug discovery for many years, electron microscopy creates opportunities that have been thus far out of reach for the conventional techniques, like the visualization of very large molecular complexes in solution or obtaining high‐resolution structural data for small molecules from microcrystals. These opportunities come at a cost: hardware is extremely expensive, software is ever‐changing, and constantly challenged by new developments in the hardware and requests by the users, and IT departments are faced with significant requirements for storage and computational power. Nevertheless, the establishment of centralized facilities, commercial consortia, and cooperative research centers is well positioned to provide access to Cryo‐EM to an expanded base of researchers, from academia as well as from industry. Understanding of the requirements, advantages, and limitations of this technique becomes then necessary to ensure correct decision‐making processes and resource allocation when undertaking a structure‐guided drug design project.

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SPHIRE-crYOLO: A fast and accurate fully automated particle picker for cryo-EM

Cited by 266 publications

References 33 publications

Structure and mechanism of the RNA polymerase II transcription machinery

Structure and mechanism of the RNA polymerase II transcription machinery

Two particle-picking procedures for filamentous proteins: SPHIRE-crYOLO filament mode and SPHIRE-STRIPER

Cryo‐Electron Microscopy in Drug Discovery and Development: Opportunities and Challenges

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