Achieving better than 3 Å resolution by single particle cryo-EM at 200 keV

Nearly all single-particle cryo-EM structures resolved to better than 4-Å resolution have been determined using 300-keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain reconstructions of macromolecular complexes of different sizes to better than 3-Å resolution using a 200-keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules.

[1]  J. Dubochet,et al.  Cryo-electron microscopy of vitrified specimens , 1988, Quarterly Reviews of Biophysics.

[2]  R. Henderson The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules , 1995, Quarterly Reviews of Biophysics.

[3]  Toshihiko Ogura,et al.  Topology representing network enables highly accurate classification of protein images taken by cryo electron-microscope without masking. , 2003, Journal of structural biology.

[4]  Cathy H. Wu,et al.  UniProt: the Universal Protein knowledgebase , 2004, Nucleic Acids Res..

[5]  A M Roseman,et al.  FindEM--a fast, efficient program for automatic selection of particles from electron micrographs. , 2004, Journal of structural biology.

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

[7]  Joachim Frank,et al.  Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes , 2008, Nature Protocols.

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

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

[10]  Vincent B. Chen,et al.  Correspondence e-mail: , 2000 .

[11]  Randy J. Read,et al.  Acta Crystallographica Section D Biological , 2003 .

[12]  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.

[13]  A. Cheng,et al.  Movies of ice-embedded particles enhance resolution in electron cryo-microscopy. , 2012, Structure.

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

[15]  M. Baker,et al.  Outcome of the First Electron Microscopy Validation Task Force Meeting , 2012, Structure.

[16]  R. Henderson,et al.  High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy☆ , 2013, Ultramicroscopy.

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

[18]  A. Steven,et al.  One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions. , 2013, Journal of structural biology.

[19]  S. Scheres Beam-induced motion correction for sub-megadalton cryo-EM particles , 2014, eLife.

[20]  R. Egerton Choice of operating voltage for a transmission electron microscope. , 2014, Ultramicroscopy.

[21]  John E. Johnson,et al.  Near-atomic resolution reconstructions using a mid-range electron microscope operated at 200 kV. , 2014, Journal of structural biology.

[22]  Lori A. Passmore,et al.  Ultrastable gold substrates for electron cryomicroscopy , 2014, Science.

[23]  Nikolaus Grigorieff,et al.  Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6 , 2015, eLife.

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

[25]  Nathaniel Echols,et al.  EMRinger: Side-chain-directed model and map validation for 3D Electron Cryomicroscopy , 2015, Nature Methods.

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

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

[28]  P. Penczek,et al.  A Primer to Single-Particle Cryo-Electron Microscopy , 2015, Cell.

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

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

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

[32]  M. Janeček,et al.  Modern Electron Microscopy in Physical and Life Sciences , 2016 .

[33]  Frank DiMaio,et al.  Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta , 2016, bioRxiv.

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

[35]  G. Lander,et al.  Setting up the Talos Arctica electron microscope and Gatan K2 direct detector for high-resolution cryogenic single-particle data acquisition , 2017 .

[36]  W. Baumeister,et al.  Cryo-EM structure of haemoglobin at 3.2 Å determined with the Volta phase plate , 2016, Nature Communications.

[37]  Lingpeng Cheng,et al.  Near-Atomic Resolution Structure Determination of a Cypovirus Capsid and Polymerase Complex Using Cryo-EM at 200kV. , 2017, Journal of molecular biology.

[38]  Wolfgang Baumeister,et al.  Using the Volta phase plate with defocus for cryo-EM single particle analysis , 2016, bioRxiv.

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

[40]  Gabriel C. Lander,et al.  A multi-model approach to assessing local and global cryo-EM map quality , 2017, bioRxiv.