Analysis of global and site-specific radiation damage in cryo-EM

Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.

[1]  Kay Diederichs,et al.  Some aspects of quantitative analysis and correction of radiation damage. , 2006, Acta crystallographica. Section D, Biological crystallography.

[2]  R. Henderson,et al.  Molecular Structure Determination by Electron Microscopy of Unstained Crystalline Specimens , 1989 .

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

[4]  T. Gonen,et al.  Structure of catalase determined by MicroED , 2014, eLife.

[5]  David Eisenberg,et al.  Atomic resolution structures from fragmented protein crystals by the cryoEM method MicroED , 2017, Nature Methods.

[6]  John L Rubinstein,et al.  The resolution dependence of optimal exposures in liquid nitrogen temperature electron cryomicroscopy of catalase crystals. , 2010, Journal of structural biology.

[7]  T. Gonen,et al.  The collection of MicroED data for macromolecular crystallography , 2016, Nature Protocols.

[8]  Sriram Subramaniam,et al.  Structure of β-galactosidase at 3.2-Å resolution obtained by cryo-electron microscopy , 2014, Proceedings of the National Academy of Sciences.

[9]  Tamir Gonen,et al.  MicroED data collection and processing , 2015, Acta crystallographica. Section A, Foundations and advances.

[10]  Mark A Hill,et al.  Will reduced radiation damage occur with very small crystals? , 2005, Journal of synchrotron radiation.

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

[12]  Wolfgang Kabsch,et al.  Integration, scaling, space-group assignment and post-refinement , 2010, Acta crystallographica. Section D, Biological crystallography.

[13]  Holger Stark,et al.  Electron radiation damage to protein crystals of bacteriorhodopsin at different temperatures , 1996 .

[14]  J. Kirz,et al.  An assessment of the resolution limitation due to radiation-damage in x-ray diffraction microscopy. , 2005, Journal of Electron Spectroscopy and Related Phenomena.

[15]  James M. Holton,et al.  A beginner’s guide to radiation damage , 2009, Journal of synchrotron radiation.

[16]  R. Glaeser,et al.  Radiation damage of purple membrane at low temperature. , 1979, Ultramicroscopy.

[17]  Jesse B. Hopkins,et al.  Global radiation damage: temperature dependence, time dependence and how to outrun it. , 2013, Journal of synchrotron radiation.

[18]  R. Ravelli,et al.  Radiation damage to nucleoprotein complexes in macromolecular crystallography , 2015, Journal of synchrotron radiation.

[19]  Philip R. Evans,et al.  How good are my data and what is the resolution? , 2013, Acta crystallographica. Section D, Biological crystallography.

[20]  W. Chiu,et al.  Quantitative assessment of radiation damage in a thin protein crystal , 1984, Journal of microscopy.

[21]  Alexandre Urzhumtsev,et al.  On effective and optical resolutions of diffraction data sets. , 2013, Acta crystallographica. Section D, Biological crystallography.

[22]  G. Sheldrick.  A short history of SHELX. , 2008, Acta crystallographica. Section A, Foundations of crystallography.

[23]  Ilme Schlichting,et al.  Serial femtosecond crystallography: the first five years , 2015, IUCrJ.

[24]  P. Howell,et al.  Identification of heavy‐atom derivatives by normal probability methods , 1992 .

[25]  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.

[26]  Elspeth F. Garman,et al.  Radiation damage in macromolecular crystallography: what is it and why should we care? , 2010, Acta crystallographica. Section D, Biological crystallography.

[27]  J. Helliwell.  Protein crystal perfection and the nature of radiation damage , 1988 .

[28]  J L Sussman,et al.  Specific chemical and structural damage to proteins produced by synchrotron radiation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Randy J. Read,et al.  Overview of the CCP4 suite and current developments , 2011, Acta crystallographica. Section D, Biological crystallography.

[30]  Tamir Gonen,et al.  High-resolution structure determination by continuous rotation data collection in MicroED , 2014, Nature Methods.

[31]  Owen Johnson,et al.  iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM , 2011, Acta crystallographica. Section D, Biological crystallography.

[32]  José A. Rodríguez,et al.  MicroED Structures from Micrometer Thick Protein Crystals , 2017, bioRxiv.

[33]  Andrew G. W. Leslie,et al.  Processing diffraction data with mosflm , 2007 .

[34]  Tamir Gonen,et al.  Three-dimensional electron crystallography of protein microcrystals , 2013, eLife.

[35]  Stephen Corcoran,et al.  Radiation damage in protein crystals is reduced with a micron-sized X-ray beam , 2011, Proceedings of the National Academy of Sciences.

[36]  Naji S Husseini,et al.  Quantifying X-ray radiation damage in protein crystals at cryogenic temperatures. , 2006, Acta crystallographica. Section D, Biological crystallography.

[37]  Travis E. Oliphant,et al.  Python for Scientific Computing , 2007, Computing in Science & Engineering.

[38]  M. Caffrey,et al.  Unit-cell volume change as a metric of radiation damage in crystals of macromolecules. , 2002, Journal of synchrotron radiation.

[39]  K. Ishizuka,et al.  Crystal structure of Ag · TCNQ , 1980, Nature.

[40]  A. Vagin,et al.  MOLREP: an Automated Program for Molecular Replacement , 1997 .

[41]  T. Gonen,et al.  Modeling truncated pixel values of faint reflections in MicroED images1 , 2016, Journal of applied crystallography.

[42]  N. Pannu,et al.  REFMAC5 for the refinement of macromolecular crystal structures , 2011, Acta crystallographica. Section D, Biological crystallography.

[43]  R. Henderson,et al.  Three-dimensional model of purple membrane obtained by electron microscopy , 1975, Nature.

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

[45]  Z. Dauter,et al.  Radiation decay of thaumatin crystals at three X-ray energies. , 2015, Acta crystallographica. Section D, Biological crystallography.

[46]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[47]  M. Symons,et al.  Structure and mobility of electron gain and loss centres in proteins , 1987, Nature.

[48]  E. Garman,et al.  Investigation of possible free-radical scavengers and metrics for radiation damage in protein cryocrystallography. , 2002, Journal of synchrotron radiation.