MicroED – Three dimensional electron crystallography

We demonstrated that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1 – 1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9Å resolution (Figure 9). This proof of principle paves the way for the implementation of a new technique, which we name “MicroED”, that may have wide applicability in structural biology.

In 2014 we further inmproved the MicroED method. Firstly, we developed an improved data collection protocol for MicroED called Continuous rotation. Microcrystals are continuously rotated during data collection yielding improved data, and allowing data processing with the crystallographic software tool MOSFLM, resulting in improved resolution for the model protein lysozyme to 2.5Å resolution. These improvements pave the way for the broad implementation and application of MicroED in structural biology. Current efforts include new phasing methods, automation and program development.

Secondly, we used the improved MicroED protocols for data collection and analysis to determine the structure of catalase. Bovine liver catalase crystals that were only ~160nm thick were used for the structure analysis. A single crystal yielded data to 3.2Å resolution enabling structure determination rapidly.

In 2015 we published the first two previously unknown structures determined by MicroED. The structures of two peptides from the toxic core of a-synuclein of Parkinsons’ Disease. The structures were determined from vanishingly small crystals, only ~200nm thick and wide, and yielded 1.4Å resolution. These structures, which are currently the highest resolution structures determined to date by any cryo EM method, show new and important structural information that could aid in the development of pharmaceuticals against this devastating neurological disease. The study, which was published by Nature also show a number of protons for the very first time.

Raw datasets available for download:

  • Lysozyme: DOI 10.15785/SBGRID/185 Download here.
  • Catalase: DOI 10.15785/SBGRID/186 Download here.
  • a-synuclein G11A: DOI 10.15785/SBGRID/193 Download here.

Hardware

Stage rotation controller developed at Janelia. Full description can be found here.

Software downloads

All software that we developed for MicroED data processing can be found here.

Follow us on Twitter @gonenlab #cryoem #gonenlab #MicroED

Relevant papers

Hattne, Johan; Shi, Dan; de la Cruz, Jason M; Reyes, Francis E; Gonen, Tamir

Modeling truncated pixel values of faint reflections in MicroED images Journal Article

J Appl Crystallogr, 49 (Pt 3), pp. 1029–1034, 2016.

Abstract | Links

Shi, Dan; Nannenga, Brent L; de la Cruz, Jason M; Liu, Jinyang; Sawtelle, Steven; Calero, Guillermo; Reyes, Francis E; Hattne, Johan; Gonen, Tamir

The collection of MicroED data for macromolecular crystallography Journal Article

Nat Protoc, 11 (5), pp. 895–904, 2016.

Abstract | Links

Rodriguez, Jose A; Ivanova, Magdalena I; Sawaya, Michael R; Cascio, Duilio; Reyes, Francis E; Shi, Dan; Sangwan, Smriti; Guenther, Elizabeth L; Johnson, Lisa M; Zhang, Meng; Jiang, Lin; Arbing, Mark A; Nannenga, Brent L; Hattne, Johan; Whitelegge, Julian; Brewster, Aaron S; Messerschmidt, Marc; Boutet, Sébastien; Sauter, Nicholas K; Gonen, Tamir; Eisenberg, David S

Structure of the toxic core of α-synuclein from invisible crystals Journal Article

Nature, 525 (7570), pp. 486–490, 2015.

Abstract | Links

Hattne, Johan; Reyes, Francis E; Nannenga, Brent L; Shi, Dan; de la Cruz, Jason M; Leslie, Andrew G W; Gonen, Tamir

MicroED data collection and processing Journal Article

Acta Crystallogr A Found Adv, 71 (Pt 4), pp. 353–360, 2015.

Abstract | Links

Nannenga, Brent L; Shi, Dan; Hattne, Johan; Reyes, Francis E; Gonen, Tamir

Structure of catalase determined by MicroED Journal Article

Elife, 3 , pp. e03600, 2014.

Abstract | Links

Nannenga, Brent L; Shi, Dan; Leslie, Andrew G W; Gonen, Tamir

High-resolution structure determination by continuous-rotation data collection in MicroED Journal Article

Nat. Methods, 11 (9), pp. 927–930, 2014.

Abstract | Links

Iadanza, Matthew G; Gonen, Tamir

A suite of software for processing MicroED data of extremely small protein crystals Journal Article

J Appl Crystallogr, 47 (Pt 3), pp. 1140–1145, 2014.

Abstract | Links

Shi, Dan; Nannenga, Brent L; Iadanza, Matthew G; Gonen, Tamir

Three-dimensional electron crystallography of protein microcrystals Journal Article

Elife, 2 , pp. e01345, 2013.

Abstract | Links

Wisedchaisri, Goragot; Gonen, Tamir

Fragment-Based Phase Extension for Three-Dimensional Structure Determination of Membrane Proteins by Electron Crystallography Journal Article

Structure, 19 (7), pp. 976–987, 2011.

Abstract | Links

Relevant Reviews and Book Chapters:

Nannenga, Brent L; Gonen, Tamir

Protein structure determination by MicroED Journal Article

Curr. Opin. Struct. Biol., 27 , pp. 24–31, 2014.

Abstract | Links

Nannenga, Brent L; Iadanza, Matthew G; Vollmar, Breanna S; Gonen, Tamir

Overview of Electron Crystallography of Membrane Proteins: Crystallization and Screening Strategies Using Negative Stain Electron Microscopy Journal Article

Curr Protoc Protein Sci, 72 (1), pp. 17.15.1–17.15.11, 2013.

Abstract | Links

Wisedchaisri, Goragot; Gonen, Tamir

Phasing Electron Diffraction Data by Molecular Replacement: Strategy for Structure Determination and Refinement Book Chapter

Electron Crystallography of Soluble and Membrane Proteins, 955 , Chapter 14, pp. 243–272, 2012.

Abstract | Links

Gonen, Tamir

The Collection of High-Resolution Electron Diffraction Data Book Chapter

Electron Crystallography of Soluble and Membrane Proteins, 955 , Chapter 9, pp. 153–169, 2012.

Abstract | Links

Stokes, David L; Ubarretxena-Belandia, Iban; Gonen, Tamir; Engel, Andreas

High-Throughput Methods for Electron Crystallography Book Chapter

Electron Crystallography of Soluble and Membrane Proteins, 955 , Chapter 15, pp. 273–296, 2012.

Abstract | Links

Wisedchaisri, Goragot; Reichow, Steve L; Gonen, Tamir

Advances in Structural and Functional Analysis of Membrane Proteins by Electron Crystallography Journal Article

Structure, 19 (10), pp. 1381–1393, 2011.

Abstract | Links