High-throughput cryo-EM enabled by user-free preprocessing routines

The growth of single-particle cryo-EM into a mainstream structural biology tool has allowed for many important biological discoveries. Continued developments in data collection strategies alongside new sample preparation devices heralds a future where users will collect multiple datasets per microscope session. To make cryo-EM data processing more automatic and user-friendly, we have developed an automatic pipeline for cryo-EM data preprocessing and assessment using a combination of deep learning and image analysis tools. We have verified the performance of this pipeline on a number of datasets and extended its scope to include sample screening by the user-free assessment of the qualities of a series of datasets under different conditions. We propose that our workflow provides a decision-free solution for cryo-EM, making data preprocessing more generalized and robust in the high-throughput era as well as more convenient for users from a range of backgrounds.

[1]  Yanan Zhu,et al.  A deep convolutional neural network approach to single-particle recognition in cryo-electron microscopy , 2016, BMC Bioinformatics.

[2]  E. Zeitler,et al.  Cryo electron microscopy. , 1982, Ultramicroscopy.

[3]  B. Carragher,et al.  Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM. , 2012, Journal of structural biology.

[4]  Christopher Irving,et al.  Appion: an integrated, database-driven pipeline to facilitate EM image processing. , 2009, Journal of structural biology.

[5]  Filiz Bunyak,et al.  DRPnet - Automated Particle Picking in Cryo-Electron Micrographs using Deep Regression , 2019 .

[6]  M. Darrow,et al.  Chameleon: Next Generation Sample Preparation for CryoEM based on Spotiton , 2019, Microscopy and Microanalysis.

[7]  David J. Fleet,et al.  cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination , 2017, Nature Methods.

[8]  William J. Rice,et al.  High Resolution Single Particle Cryo-Electron Microscopy using Beam-Image Shift , 2018, bioRxiv.

[9]  Renmin Han,et al.  PIXER: an automated particle-selection method based on segmentation using a deep neural network , 2019, BMC Bioinformatics.

[10]  Liqing Zhang,et al.  Saliency Detection: A Spectral Residual Approach , 2007, 2007 IEEE Conference on Computer Vision and Pattern Recognition.

[11]  Clinton S Potter,et al.  Big data in cryoEM: automated collection, processing and accessibility of EM data. , 2018, Current opinion in microbiology.

[12]  Bonnie Berger,et al.  Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs , 2018, Nature Methods.

[13]  Guangwen Yang,et al.  A fast method for particle picking in cryo-electron micrographs based on fast R-CNN , 2017 .

[14]  W. Kühlbrandt The Resolution Revolution , 2014, Science.

[15]  Soumith Chintala,et al.  Unsupervised Representation Learning with Deep Convolutional Generative Adversarial Networks , 2015, ICLR.

[16]  Markus Stabrin,et al.  High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE , 2017, Journal of visualized experiments : JoVE.

[17]  Wah Chiu,et al.  Comparing cryo-EM structures. , 2018, Journal of structural biology.

[18]  Mona Wong-Barnum,et al.  COSMIC2: A Science Gateway for Cryo-Electron Microscopy Structure Determination , 2017, PEARC.

[19]  Sjors H. W. Scheres,et al.  Unravelling biological macromolecules with cryo-electron microscopy , 2016, Nature.

[20]  Dmitry Lyumkis,et al.  Challenges and opportunities in cryo-EM single-particle analysis , 2019, The Journal of Biological Chemistry.

[21]  Andres E Leschziner,et al.  Low cost, high performance processing of single particle cryo-electron microscopy data in the cloud , 2015, bioRxiv.

[22]  R. Ravelli,et al.  Automated cryo-EM sample preparation by pin-printing and jet vitrification , 2019, bioRxiv.

[23]  Tian Xia,et al.  DeepPicker: a Deep Learning Approach for Fully Automated Particle Picking in Cryo-EM , 2016, Journal of structural biology.

[24]  Dimitry Tegunov,et al.  Real-time cryo–EM data pre-processing with Warp , 2019, Nature Methods.

[25]  Jian Sun,et al.  Deep Residual Learning for Image Recognition , 2015, 2016 IEEE Conference on Computer Vision and Pattern Recognition (CVPR).

[26]  William Graf,et al.  Deep learning for cellular image analysis , 2019, Nature Methods.

[27]  N. Grigorieff,et al.  CTFFIND4: Fast and accurate defocus estimation from electron micrographs , 2015, bioRxiv.

[28]  A. Cheng,et al.  2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy , 2015, eLife.

[29]  Erik Lindahl,et al.  New tools for automated high-resolution cryo-EM structure determination in RELION-3 , 2018, eLife.

[30]  Sjors H.W. Scheres,et al.  RELION: Implementation of a Bayesian approach to cryo-EM structure determination , 2012, Journal of structural biology.

[31]  F. Young Biochemistry , 1955, The Indian Medical Gazette.

[32]  Thorsten Wagner,et al.  SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM , 2019, Communications Biology.

[33]  Sjors H.W. Scheres,et al.  A pipeline approach to single-particle processing in RELION , 2016 .

[34]  Yong Zi Tan,et al.  Reducing effects of particle adsorption to the air-water interface in cryoEM , 2018, Nature Methods.

[35]  Anchi Cheng,et al.  Automated molecular microscopy: the new Leginon system. , 2005, Journal of structural biology.

[36]  Rafael Fernandez-Leiro,et al.  A pipeline approach to single-particle processing in RELION , 2016, bioRxiv.

[37]  Michael A. Cianfrocco,et al.  Cryo–electron microscopy structure and analysis of the P-Rex1–Gβγ signaling scaffold , 2019, Science Advances.

[38]  Jianlin Cheng,et al.  DeepCryoPicker: fully automated deep neural network for single protein particle picking in cryo-EM , 2019, bioRxiv.

[39]  Andrej Bieri,et al.  Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts. , 2017, Journal of structural biology.