Interview with Tamir Gonen about MicroED

We demonstrated that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (cryo-EM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å 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 Å2. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. 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 improved 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 2.5 Å 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.

Example of MicroED data: lysozyme
Example of MicroED data: catalase

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 ~160 nm thick were used for the structure analysis. A single crystal yielded data to 3.2 Å resolution enabling rapid structure determination.

In 2015 we published the first two previously unknown structures determined by MicroED. The structures of two peptides from the toxic core of α-synuclein of Parkinson’s disease. The structures were determined from vanishingly small crystals, only ~200 nm thick and wide, and yielded 1.4 Å 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.

2021: Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion-beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous rotation data were collected using an ultra-low exposure rate on a Falcon 4 direct electron detector in electron-counting mode. For the first sample, triclinic lysozyme extending to 0.87 Å resolution, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to 1.5 Å resolution. These results demonstrate that macromolecules can be determined to sub-Ångström resolution by MicroED and that ab initio phasing can be successfully applied to counting data collected on a direct electron detector.

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Raw datasets available for download

Hardware

The MicroED Stage Controller page contains a full description of the stage rotation controller developed at Janelia Research Campus. This device is not needed on Thermo Fisher systems from 2015 or later.

Software downloads

All software that we developed for MicroED data processing can be found on the downloads pages.

See also

MicroED Imaging Center at UCLA

Selected publications

Martynowycz, Michael W; Clabbers, Max T B; Unge, Johan; Hattne, Johan; Gonen, Tamir

Benchmarking the ideal sample thickness in cryo-EM Journal Article

In: Proc Natl Acad Sci U S A, 118 (49), 2021, ISSN: 1091-6490.

Abstract | Links

Zhu, Lan; Bu, Guanhong; Jing, Liang; Shi, Dan; Lee, Ming-Yue; Gonen, Tamir; Liu, Wei; Nannenga, Brent L.

Structure Determination from Lipidic Cubic Phase Embedded Microcrystals by MicroED Journal Article

In: Structure, 28 (10), pp. 1149–1159.e4, 2020.

Abstract | Links

Jones, Christopher G; Martynowycz, Michael W; Hattne, Johan; Fulton, Tyler J; Stoltz, Brian M; Rodriguez, Jose A; Nelson, Hosea M; Gonen, Tamir

The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination Journal Article

In: ACS Cent Sci, 4 (11), pp. 1587–1592, 2018.

Abstract | Links

Liu, Shian; Gonen, Tamir

MicroED structure of the NaK ion channel reveals a Na⁺ partition process into the selectivity filter Journal Article

In: Commun Biol, 1 , pp. 38, 2018.

Abstract | Links

Hattne, Johan; Shi, Dan; Glynn, Calina; Zee, Chih-Te; Gallagher-Jones, Marcus; Martynowycz, Michael W; Rodriguez, Jose A; Gonen, Tamir

Analysis of Global and Site-Specific Radiation Damage in Cryo-EM Journal Article

In: Structure, 26 (5), pp. 759–766, 2018.

Abstract | Links

de la Cruz, Jason M; Hattne, Johan; Shi, Dan; Seidler, Paul; Rodriguez, Jose; Reyes, Francis E; Sawaya, Michael R; Cascio, Duilio; Weiss, Simon C; Kim, Sun Kyung; Hinck, Cynthia S; Hinck, Andrew P; Calero, Guillermo; Eisenberg, David; Gonen, Tamir

Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED Journal Article

In: Nat. Methods, 14 (4), pp. 399–402, 2017.

Abstract | Links

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

In: 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

In: 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

In: 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

In: 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

In: 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

In: 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

In: 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

In: 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

In: Structure, 19 (7), pp. 976–987, 2011.

Abstract | Links

Relevant reviews and book chapters

Clark, Lisa J.; Bu, Guanhong; Nannenga, Brent L.; Gonen, Tamir

MicroED for the study of protein–ligand interactions and the potential for drug discovery Journal Article

In: Nat Rev Chem, 5 , pp. 853–858, 2021.

Abstract | Links

Mu, Xuelang; Gillman, Cody; Nguyen, Chi; Gonen, Tamir

An Overview of Microcrystal Electron Diffraction (MicroED) Journal Article

In: Annu Rev Biochem, 90 , pp. 431–450, 2021, ISSN: 1545-4509.

Abstract | Links

Danelius, E; Gonen, T

Protein and Small Molecule Structure Determination by the Cryo-EM Method MicroED Book Chapter

In: Owens, Raymond J. (Ed.): 2305 , pp. 323–342, 2021.

Abstract | Links

Nannenga, Brent L; Gonen, Tamir

The cryo-EM method microcrystal electron diffraction (MicroED) Journal Article

In: Nat. Methods, 16 (5), pp. 369–379, 2019.

Abstract | Links

Martynowycz, Michael W; Gonen, Tamir

From electron crystallography of 2D crystals to MicroED of 3D crystals Journal Article

In: Curr Opin Colloid Interface Sci, 34 , pp. 9–16, 2018.

Abstract | Links

Nannenga, Brent L; Gonen, Tamir

Protein structure determination by MicroED Journal Article

In: 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

In: 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

In: 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

In: 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

In: 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

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

Abstract | Links