RELION-3: new tools for automated high-resolution cryo-EM structure determination

Here, we describe the third major release of relion. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations. Reference-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2D-classification, de novo model generation and 3D-classification. Perparticle refinement of CTF parameters and correction of estimated beam tilt provides higher-resolution reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resolution for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beam-induced motion correction it leads to resolution improvements of 0.2-0.7 Å compared to previous relion versions.

[1]  Jasenko Zivanov,et al.  A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis , 2018, bioRxiv.

[2]  S. Scheres,et al.  Advances in Single-Particle Electron Cryomicroscopy Structure Determination applied to Sub-tomogram Averaging , 2015, Structure.

[3]  S. Harrison,et al.  Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction , 2008, Proceedings of the National Academy of Sciences.

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

[5]  Wen Jiang,et al.  EMAN2: an extensible image processing suite for electron microscopy. , 2007, Journal of structural biology.

[6]  F. Zemlin,et al.  Coma-free alignment of high-resolution electron microscopes with the aid of optical diffractograms , 1978 .

[7]  Xueming Li,et al.  Alignment of direct detection device micrographs using a robust Optical Flow approach. , 2015, Journal of structural biology.

[8]  J. V. Van Etten,et al.  Pushing the resolution limit by correcting the Ewald sphere effect in single-particle Cryo-EM reconstructions , 2018, Nature Communications.

[9]  A. Bartesaghi,et al.  2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor , 2015, Science.

[10]  Sjors H.W. Scheres,et al.  A Bayesian View on Cryo-EM Structure Determination , 2012, 2012 9th IEEE International Symposium on Biomedical Imaging (ISBI).

[11]  G. Ferraro,et al.  Cisplatin Binding Sites in Human H-Chain Ferritin. , 2017, Inorganic chemistry.

[12]  R. Henderson,et al.  Detective quantum efficiency of electron area detectors in electron microscopy , 2009, Ultramicroscopy.

[13]  David N Mastronarde,et al.  Automated electron microscope tomography using robust prediction of specimen movements. , 2005, Journal of structural biology.

[14]  C O S Sorzano,et al.  Scipion: A software framework toward integration, reproducibility and validation in 3D electron microscopy. , 2016, Journal of structural biology.

[15]  John Steel,et al.  Cross-neutralization of influenza A viruses mediated by a single antibody loop , 2012, Nature.

[16]  Shaoxia Chen,et al.  Prevention of overfitting in cryo-EM structure determination , 2012, Nature Methods.

[17]  Robert M Glaeser,et al.  Precise beam-tilt alignment and collimation are required to minimize the phase error associated with coma in high-resolution cryo-EM. , 2011, Journal of structural biology.

[18]  M Radermacher,et al.  DoG Picker and TiltPicker: software tools to facilitate particle selection in single particle electron microscopy. , 2009, Journal of structural biology.

[19]  Alwyn Eades,et al.  Obtaining TEM images with a uniform deviation parameter. , 2006, Ultramicroscopy.

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

[21]  Henning Stahlberg,et al.  Focus: The interface between data collection and data processing in cryo-EM. , 2017, Journal of structural biology.

[22]  R. Crichton,et al.  Structural analysis of haemin demetallation by L-chain apoferritins. , 2012, Journal of inorganic biochemistry.

[23]  Kai Zhang,et al.  Gctf: Real-time CTF determination and correction , 2015, bioRxiv.

[24]  Sjors H. W. Scheres,et al.  An atomic structure of human γ-secretase , 2015, Nature.

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

[26]  Pierre Stadelmann,et al.  Effect of three-fold astigmatism on high resolution electron micrographs , 1995 .

[27]  C. Russo,et al.  Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy , 2017, Nature Communications.

[28]  D. Agard,et al.  Electron counting and beam-induced motion correction enable near atomic resolution single particle cryoEM , 2013, Nature Methods.

[29]  D. Agard,et al.  MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy , 2017, Nature Methods.

[30]  Mindy I. Davis,et al.  Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery , 2016, Cell.

[31]  J. Kowal,et al.  Robust image alignment for cryogenic transmission electron microscopy. , 2017, Journal of structural biology.

[32]  Hemant D. Tagare,et al.  The Local Resolution of Cryo-EM Density Maps , 2013, Nature Methods.

[33]  Nikolaus Grigorieff,et al.  FREALIGN: high-resolution refinement of single particle structures. , 2007, Journal of structural biology.

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

[35]  N. Grigorieff,et al.  Accurate determination of local defocus and specimen tilt in electron microscopy. , 2003, Journal of structural biology.

[36]  R. Henderson,et al.  Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. , 2003, Journal of molecular biology.

[37]  Yong Zi Tan,et al.  Sub-2 Å Ewald Curvature Corrected Single-Particle Cryo-EM , 2018, bioRxiv.

[38]  Jasenko Zivanov,et al.  A Bayesian Approach to Beam-Induced Motion Correction in Cryo-EM Single-Particle Analysis , 2018 .

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

[40]  Alan Brown,et al.  Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions , 2015, Acta crystallographica. Section D, Biological crystallography.

[41]  Ben Hankamer,et al.  The Laplacian of Gaussian and arbitrary z-crossings approach applied to automated single particle reconstruction. , 2007, Journal of structural biology.

[42]  Guillermo Sapiro,et al.  Atomic Resolution Cryo-EM Structure of β-Galactosidase. , 2018, Structure.

[43]  Alexis Rohou,et al.  cisTEM: user-friendly software for single-particle image processing , 2017 .

[44]  M van Heel,et al.  A new generation of the IMAGIC image processing system. , 1996, Journal of structural biology.

[45]  John A. Nelder,et al.  A Simplex Method for Function Minimization , 1965, Comput. J..

[46]  Matthew L. Baker,et al.  Backbone structure of the infectious Epsilon15 virus capsid revealed by electron cryomicroscopy , 2008 .

[47]  Ardan Patwardhan,et al.  EMPIAR: a public archive for raw electron microscopy image data , 2016, Nature Methods.

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

[49]  E. Lindahl,et al.  Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION , 2018, bioRxiv.

[50]  W. O. Saxton.  Observation of lens aberrations for very high‐resolution electron microscopy. I. Theory , 1995 .

[51]  Paul D. Adams,et al.  Accurate model annotation of a near-atomic resolution cryo-EM map , 2017, Proceedings of the National Academy of Sciences.

[52]  R. Henderson,et al.  Ewald sphere correction using a single side-band image processing algorithm , 2018, Ultramicroscopy.

[53]  Matthew L. Baker,et al.  Backbone structure of the infectious ε15 virus capsid revealed by electron cryomicroscopy , 2008, Nature.

[54]  Marcus A. Brubaker,et al.  Alignment of cryo-EM movies of individual particles by optimization of image translations. , 2014, Journal of structural biology.

[55]  Sjors H.W. Scheres,et al.  Helical reconstruction in RELION , 2016, bioRxiv.

[56]  E. Lindahl,et al.  Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2 , 2016, bioRxiv.

[57]  Joseph H. Davis,et al.  Addressing preferred specimen orientation in single-particle cryo-EM through tilting , 2017, Nature Methods.

[58]  Ardan Patwardhan,et al.  Trends in the Electron Microscopy Data Bank (EMDB) , 2017, Acta crystallographica. Section D, Structural biology.

[59]  Z. Zhou,et al.  3.88 Å structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy , 2008, Nature.

[60]  Guanghui Yang,et al.  Sampling the conformational space of the catalytic subunit of human γ-secretase , 2015, bioRxiv.

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

[62]  Sjors H.W. Scheres,et al.  Semi-automated selection of cryo-EM particles in RELION-1.3 , 2015, Journal of structural biology.

[63]  Dimitry Tegunov,et al.  Real-time cryo–EM data pre-processing with Warp , 2018, Nature Methods.

[64]  Alexis Rohou,et al.  cisTEM: User-friendly software for single-particle image processing , 2017, bioRxiv.

[65]  Sydney R. Hall,et al.  The STAR file: a new format for electronic data transfer and archiving , 1991, J. Chem. Inf. Comput. Sci..