skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED

Abstract

Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined.We showwith four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can alsomore » be determined by direct methods.« less

Authors:
 [1];  [1];  [1];  [1];  [2];  [2]; ORCiD logo [2];  [2];  [1]
  1. Univ. of California, Los Angeles, CA (United States)
  2. Howard Hughes Medical Inst., Ashburn, VA (United States)
Publication Date:
Research Org.:
Univ. of California, Los Angeles, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1425979
Grant/Contract Number:  
FC02-02ER63421
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 113; Journal Issue: 40; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; phasing; prion; electron; diffraction; MicroED; nanocrystal

Citation Formats

Sawaya, Michael R., Rodriguez, Jose, Cascio, Duilio, Collazo, Michael J., Shi, Dan, Reyes, Francis E., Hattne, Johan, Gonen, Tamir, and Eisenberg, David S. Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED. United States: N. p., 2016. Web. doi:10.1073/pnas.1606287113.
Sawaya, Michael R., Rodriguez, Jose, Cascio, Duilio, Collazo, Michael J., Shi, Dan, Reyes, Francis E., Hattne, Johan, Gonen, Tamir, & Eisenberg, David S. Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED. United States. doi:10.1073/pnas.1606287113.
Sawaya, Michael R., Rodriguez, Jose, Cascio, Duilio, Collazo, Michael J., Shi, Dan, Reyes, Francis E., Hattne, Johan, Gonen, Tamir, and Eisenberg, David S. Mon . "Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED". United States. doi:10.1073/pnas.1606287113. https://www.osti.gov/servlets/purl/1425979.
@article{osti_1425979,
title = {Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED},
author = {Sawaya, Michael R. and Rodriguez, Jose and Cascio, Duilio and Collazo, Michael J. and Shi, Dan and Reyes, Francis E. and Hattne, Johan and Gonen, Tamir and Eisenberg, David S.},
abstractNote = {Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined.We showwith four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods.},
doi = {10.1073/pnas.1606287113},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 40,
volume = 113,
place = {United States},
year = {2016},
month = {9}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 19 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

High-voltage electron diffraction from bacteriorhodopsin (purple membrane) is measurably dynamical
journal, September 1989

  • Glaeser, R. M.; Ceska, T. A.
  • Acta Crystallographica Section A Foundations of Crystallography, Vol. 45, Issue 9
  • DOI: 10.1107/S0108767389004599

Combined crystal structure prediction and high-pressure crystallization in rational pharmaceutical polymorph screening
journal, July 2015

  • Neumann, M. A.; van de Streek, J.; Fabbiani, F. P. A.
  • Nature Communications, Vol. 6, Issue 1
  • DOI: 10.1038/ncomms8793

Direct phase determination for quasi-kinematical electron diffraction intensity data from organic microcrystals
journal, January 1976


Cross-beta Order and Diversity in Nanocrystals of an Amyloid-forming Peptide
journal, July 2003


Isomorphous replacement and phase determination in non-centrosymmetric space groups
journal, November 1956


A short history of SHELX
journal, December 2007

  • Sheldrick, George M.
  • Acta Crystallographica Section A Foundations of Crystallography, Vol. 64, Issue 1, p. 112-122
  • DOI: 10.1107/S0108767307043930

Advances in direct methods for protein crystallography
journal, October 1999


Indexing amyloid peptide diffraction from serial femtosecond crystallography: new algorithms for sparse patterns
journal, January 2015

  • Brewster, Aaron S.; Sawaya, Michael R.; Rodriguez, Jose
  • Acta Crystallographica Section D Biological Crystallography, Vol. 71, Issue 2
  • DOI: 10.1107/S1399004714026145

The collection of MicroED data for macromolecular crystallography
journal, April 2016

  • Shi, Dan; Nannenga, Brent L.; de la Cruz, M. Jason
  • Nature Protocols, Vol. 11, Issue 5
  • DOI: 10.1038/nprot.2016.046

Structure of the toxic core of α-synuclein from invisible crystals
journal, September 2015

  • Rodriguez, Jose A.; Ivanova, Magdalena I.; Sawaya, Michael R.
  • Nature, Vol. 525, Issue 7570
  • DOI: 10.1038/nature15368

Hydrogen Tunneling Links Protein Dynamics to Enzyme Catalysis
journal, June 2013


The detection of sub-units within the crystallographic asymmetric unit
journal, January 1962


The Born Approximation in Electron Diffraction
journal, August 1952

  • Schomaker, Verner; Glauber, Roy
  • Nature, Vol. 170, Issue 4320
  • DOI: 10.1038/170290b0

Ab initio structure determination of nanocrystals of organic pharmaceutical compounds by electron diffraction at room temperature using a Timepix quantum area direct electron detector
journal, February 2016

  • van Genderen, E.; Clabbers, M. T. B.; Das, P. P.
  • Acta Crystallographica Section A Foundations and Advances, Vol. 72, Issue 2
  • DOI: 10.1107/S2053273315022500

Three-dimensional model of purple membrane obtained by electron microscopy
journal, September 1975

  • Henderson, R.; Unwin, P. N. T.
  • Nature, Vol. 257, Issue 5521
  • DOI: 10.1038/257028a0

Modeling truncated pixel values of faint reflections in MicroED images
journal, May 2016

  • Hattne, Johan; Shi, Dan; de la Cruz, M. Jason
  • Journal of Applied Crystallography, Vol. 49, Issue 3
  • DOI: 10.1107/S1600576716007196

REFMAC 5 for the refinement of macromolecular crystal structures
journal, March 2011

  • Murshudov, Garib N.; Skubák, Pavol; Lebedev, Andrey A.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
  • DOI: 10.1107/S0907444911001314

Cryo-electron tomography: The challenge of doing structural biology in situ
journal, August 2013

  • Lučić, Vladan; Rigort, Alexander; Baumeister, Wolfgang
  • The Journal of Cell Biology, Vol. 202, Issue 3
  • DOI: 10.1083/jcb.201304193

Protein structure determination by MicroED
journal, August 2014


Atomic structures of amyloid cross-β spines reveal varied steric zippers
journal, April 2007

  • Sawaya, Michael R.; Sambashivan, Shilpa; Nelson, Rebecca
  • Nature, Vol. 447, Issue 7143
  • DOI: 10.1038/nature05695

A Primer to Single-Particle Cryo-Electron Microscopy
journal, April 2015


PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

  • Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
  • DOI: 10.1107/S0907444909052925

A Fourier Series Method for the Determination of the Components of Interatomic Distances in Crystals
journal, September 1934


Solving protein nanocrystals by cryo-EM diffraction: Multiple scattering artifacts
journal, January 2015


MicroED data collection and processing
journal, July 2015

  • Hattne, Johan; Reyes, Francis E.; Nannenga, Brent L.
  • Acta Crystallographica Section A Foundations and Advances, Vol. 71, Issue 4
  • DOI: 10.1107/S2053273315010669

An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated  -sheet structure for amyloid
journal, February 2001

  • Balbirnie, M.; Grothe, R.; Eisenberg, D. S.
  • Proceedings of the National Academy of Sciences, Vol. 98, Issue 5
  • DOI: 10.1073/pnas.041617698

Phase annealing in SHELX-90: direct methods for larger structures
journal, June 1990

  • Sheldrick, G. M.
  • Acta Crystallographica Section A Foundations of Crystallography, Vol. 46, Issue 6, p. 467-473
  • DOI: 10.1107/S0108767390000277

Structure of catalase determined by MicroED
journal, October 2014


Dynamical theory for electron scattering from crystal defects and disorder
journal, January 1979


High-resolution electron crystallography of protein molecules
journal, December 1993


Three-dimensional electron crystallography of protein microcrystals
journal, November 2013


Peptoid nanosheets exhibit a new secondary-structure motif
journal, October 2015

  • Mannige, Ranjan V.; Haxton, Thomas K.; Proulx, Caroline
  • Nature, Vol. 526, Issue 7573
  • DOI: 10.1038/nature15363

XDS
journal, January 2010

  • Kabsch, Wolfgang
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2
  • DOI: 10.1107/S0907444909047337

Using RosettaLigand for Small Molecule Docking into Comparative Models
journal, December 2012


Phasing methods for protein crystallography
journal, October 1997


Imaging protein three-dimensional nanocrystals with cryo-EM
journal, April 2013

  • Nederlof, Igor; Li, Yao Wang; van Heel, Marin
  • Acta Crystallographica Section D Biological Crystallography, Vol. 69, Issue 5
  • DOI: 10.1107/S0907444913002734

Comparison of calculated and observed dynamical diffraction from purple membrane: implications
journal, September 1996


The Direct Methods of X-ray Crystallography
journal, July 1986


Structure of the cross-β spine of amyloid-like fibrils
journal, June 2005

  • Nelson, Rebecca; Sawaya, Michael R.; Balbirnie, Melinda
  • Nature, Vol. 435, Issue 7043
  • DOI: 10.1038/nature03680

Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography
journal, December 2014

  • Wan, Qun; Bennett, Brad C.; Wilson, Mark A.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 51
  • DOI: 10.1073/pnas.1415856111

Relativistic Hartree–Fock X-ray and electron scattering factors
journal, May 1968


    Works referencing / citing this record:

    Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED
    journal, February 2017

    • de la Cruz, M. Jason; Hattne, Johan; Shi, Dan
    • Nature Methods, Vol. 14, Issue 4
    • DOI: 10.1038/nmeth.4178

    Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED
    journal, February 2017

    • de la Cruz, M. Jason; Hattne, Johan; Shi, Dan
    • Nature Methods, Vol. 14, Issue 4
    • DOI: 10.1038/nmeth.4178