Microcrystal electron diffraction

Microcrystal electron diffraction, or MicroED,[1][2] is a CryoEM method that was developed by the Gonen laboratory in late 2013 at the Janelia Research Campus of the Howard Hughes Medical Institute. MicroED is a form of electron crystallography where thin 3D crystals are used for structure determination by electron diffraction.

The method was developed for structure determination of proteins from nanocrystals that are typically not suitable for X-ray diffraction because of their size. Crystals that are one billionth the size needed for X-ray crystallography can yield high quality data.[3] The samples are frozen hydrated as for all other CryoEM modalities but instead of using the transmission electron microscope (TEM) in imaging mode one uses it in diffraction mode with an extremely low electron exposure (typically < 0.01 e−/Å2/s). The nano crystal is exposed to the diffracting beam and continuously rotated[2] while diffraction is collected on a fast camera as a movie.[2] MicroED data is then processed using traditional software for X-ray crystallography without the need for specialized software for structure analysis and refinement.[4] Importantly, both the hardware and software used in a MicroED experiment are standard and broadly available.

Development

The first successful demonstration of MicroED was reported in 2013 by the Gonen laboratory.[1] The structure of lysozyme, a classic test protein in X-ray crystallography. Earlier in 2013, the Abrahams group independently reported 3D electron diffraction data using a Medipix quantum area detector on lysozyme crystals but were unable to solve the structure due to technical limitations.[5]

Experimental setup

Detailed protocols for setting up the electron microscope and for data collections have been published.[6]

Microscope

MicroED data is collected using transmission electron (cryogenic) microscopy. The microscope must be equipped with a selected area aperture to use selected area diffraction.

Detectors

A variety of detectors have been used to collected electron diffraction data in MicroED experiments. Detectors utilizing charge-coupled device (CCD) and complementary metal–oxide–semiconductor (CMOS) technology have been used. With CMOS detectors, individual electron counts can be interpreted.[7]

Still diffraction

The initial proof of concept publication on MicroED used lysozyme crystals.[1] Up to 90 degrees of data were collected from a single nano crystal, with discrete 1 degree steps between frames. Each diffraction pattern was collected with an ultra-low dose rate of ∼0.01 e−/Å2/s. Data from 3 crystals was merged to yield a 2.9Å resolution structure with good refinement statistics, and represented the first time electron diffraction had been used successfully to determine the structure of a dose-sensitive protein from 3D microcrystals in cryogenic conditions.

Continuous stage rotation

Shortly after the proof of principle paper MicroED was improved by applying continuous rotation during the data collection scheme.[2] Here the crystal is slowly rotated in a single direction while diffraction is recorded on a fast camera as a movie. The methodology is like the rotation method in x-ray crystallography. This led to several improvements in data quality and allowed data processing using standard X-ray crystallographic software.[2] Benefits of continuous rotation MicroED include a decrease in dynamical scattering and improved sampling of reciprocal space. Continuous-rotation is the standard method of MicroED data collection since 2014.

Data processing

Detailed protocols for MicroED data processing have been published.[4] When MicroED data is collected using continuous stage rotation, standard crystallography software can be used.

Differences between MicroED and other electron diffraction methods

Other electron diffraction methods that have been developed for material science of radiation insensitive material like inorganic salts include Automated Diffraction Tomography (ADT)[8] and Rotation Electron Diffraction (RED[9]). These methods significantly differ from MicroED: In ADT discrete steps of goniometer tilt are used to cover reciprocal space in combination with beam precession to fill in the gaps.[8] ADT uses specialized hardware for precession and scanning transmission electron microscopy for crystal tracking.[8] RED is done in TEM but the goniometer is coarsely tilted in discrete steps and beam tilting is used to fill in the gaps.[9] Specialized software is used to process ADT and RED data.[9] Importantly, ADT and RED were developed and tested on radiation insensitive inorganic materials and salts and have not been demonstrated for use with proteins or radiation sensitive organic material studied in a frozen hydrated state.

Milestones

Method scope

MicroED has been used to determine the structures of large globular proteins,[10] small proteins,[2] peptides,[11] membrane proteins,[12] organic molecules,[13][14] and inorganic compounds.[15] In many of these examples hydrogens and charged ions were observed.[11][12]

Novel structures of α-synuclein of Parkinson's disease

The first novel structures solved by MicroED were published in late 2015.[11] These structures were of peptide fragments that form the toxic core of α-synculein, the protein responsible for Parkinson's disease and lead to insight into the aggregation mechanism toxic aggregates. The structures were solved at 1.4 Å resolution.

Novel protein structure of R2lox

The first novel structure of a protein solved by MicroED was published in 2019.[16] The protein is the metalloenzyme R2-like ligand-binding oxidase (R2lox) from Sulfolobus acidocaldarius. The structure was solved at 3.0 Å resolution by molecular replacement using a model of 35% sequence identity built from the closest homolog with a know structure. This work demonstrated that MicroED could be used to obtain unknown structure of protein.

Access to MicroED education and services

To learn more about MicroED, one can attend the annual MicroED Imaging Center Course at UCLA or the MicroED Course at the Diamond Light Source . For more up to date information about upcoming meetings and workshops related to Cryogenic electron microscopy methods as a whole, please check the 3DEM Meetings and Workshops page.

Several universities and companies offer MicroED services, including the MEDIC – Microcrystal Electron Diffraction Imaging Center at UCLA and Nanoimaging Services.

References
  1. Shi, Dan; Nannenga, Brent L; Iadanza, Matthew G; Gonen, Tamir (2013-11-19). "Three-dimensional electron crystallography of protein microcrystals". eLife. 2: e01345. doi:10.7554/elife.01345. ISSN 2050-084X. PMC 3831942. PMID 24252878.
  2. Nannenga, Brent L; Shi, Dan; Leslie, Andrew G W; Gonen, Tamir (2014-08-03). "High-resolution structure determination by continuous-rotation data collection in MicroED". Nature Methods. 11 (9): 927–930. doi:10.1038/nmeth.3043. ISSN 1548-7091. PMC 4149488. PMID 25086503.
  3. de la Cruz, M Jason; Hattne, Johan; Shi, Dan; Seidler, Paul; Rodriguez, Jose; Reyes, Francis E; Sawaya, Michael R; Cascio, Duilio; Weiss, Simon C (2017). "Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED". Nature Methods. 14 (4): 399–402. doi:10.1038/nmeth.4178. ISSN 1548-7091. PMC 5376236. PMID 28192420.
  4. Hattne, Johan; Reyes, Francis E.; Nannenga, Brent L.; Shi, Dan; de la Cruz, M. Jason; Leslie, Andrew G. W.; Gonen, Tamir (2015-07-01). "MicroED data collection and processing". Acta Crystallographica Section A. 71 (4): 353–360. doi:10.1107/s2053273315010669. ISSN 2053-2733. PMC 4487423. PMID 26131894.
  5. Nederlof, I.; van Genderen, E.; Li, Y.-W.; Abrahams, J. P. (2013-07-01). "A Medipix quantum area detector allows rotation electron diffraction data collection from submicrometre three-dimensional protein crystals". Acta Crystallographica Section D. 69 (7): 1223–1230. doi:10.1107/S0907444913009700. ISSN 0907-4449. PMC 3689525. PMID 23793148.
  6. Shi, Dan; Nannenga, Brent L; de la Cruz, M Jason; Liu, Jinyang; Sawtelle, Steven; Calero, Guillermo; Reyes, Francis E; Hattne, Johan; Gonen, Tamir (2016-04-14). "The collection of MicroED data for macromolecular crystallography". Nature Protocols. 11 (5): 895–904. doi:10.1038/nprot.2016.046. ISSN 1754-2189. PMC 5357465. PMID 27077331.
  7. See also https://www.gatan.com/ccd-vs-cmos and https://www.gatan.com/techniques/imaging.
  8. Mugnaioli, E.; Gorelik, T.; Kolb, U. (2009). ""Ab initio" structure solution from electron diffraction data obtained by a combination of automated diffraction tomography and precession technique". Ultramicroscopy. 109 (6): 758–765. doi:10.1016/j.ultramic.2009.01.011. ISSN 0304-3991. PMID 19269095.
  9. Wan, Wei; Sun, Junliang; Su, Jie; Hovmöller, Sven; Zou, Xiaodong (2013-11-15). "Three-dimensional rotation electron diffraction: softwareREDfor automated data collection and data processing". Journal of Applied Crystallography. 46 (6): 1863–1873. doi:10.1107/s0021889813027714. ISSN 0021-8898. PMC 3831301. PMID 24282334.
  10. Nannenga, Brent L; Shi, Dan; Hattne, Johan; Reyes, Francis E; Gonen, Tamir (2014-10-10). "Structure of catalase determined by MicroED". eLife. 3: e03600. doi:10.7554/elife.03600. ISSN 2050-084X. PMC 4359365. PMID 25303172.
  11. Rodriguez, J.A.; Ivanova, M.; Sawaya, M.R.; Cascio, D.; Reyes, F.; Shi, D.; Johnson, L.; Guenther, E.; Sangwan, S. (2015-09-09). "MicroED structure of the segment, GVVHGVTTVA, from the A53T familial mutant of Parkinson's disease protein, alpha-synuclein residues 47-56". doi:10.2210/pdb4znn/pdb. Cite journal requires |journal= (help)
  12. Liu, S.; Gonen, T. (2018-09-12). "MicroED structure of NaK ion channel reveals a process of Na+ partition into the selectivity filter". doi:10.2210/pdb6cpv/pdb. Cite journal requires |journal= (help)
  13. Gallagher-Jones, Marcus; Glynn, Calina; Boyer, David R.; Martynowycz, Michael W.; Hernandez, Evelyn; Miao, Jennifer; Zee, Chih-Te; Novikova, Irina V.; Goldschmidt, Lukasz (2018-01-15). "Sub-ångström cryo-EM structure of a prion protofibril reveals a polar clasp". Nature Structural & Molecular Biology. 25 (2): 131–134. doi:10.1038/s41594-017-0018-0. ISSN 1545-9993. PMC 6170007. PMID 29335561.
  14. Jones, Christopher; Martynowycz, M; Hattne, Johan; Fulton, Tyler J.; Stoltz, Brian M.; Rodriguez, Jose A.; Nelson, Hosea; Gonen, Tamir (2018). "The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination" (PDF). doi:10.26434/chemrxiv.7215332.v1. Cite journal requires |journal= (help)
  15. Vergara, Sandra; Lukes, Dylan A.; Martynowycz, Michael W.; Santiago, Ulises; Plascencia-Villa, Germán; Weiss, Simon C.; de la Cruz, M. Jason; Black, David M.; Alvarez, Marcos M. (2017-10-31). "MicroED Structure of Au146(p-MBA)57 at Subatomic Resolution Reveals a Twinned FCC Cluster". The Journal of Physical Chemistry Letters. 8 (22): 5523–5530. doi:10.1021/acs.jpclett.7b02621. ISSN 1948-7185. PMC 5769702. PMID 29072840.
  16. Xu, Hongyi; Lebrette, Hugo; Clabbers, Max T. B.; Zhao, Jingjing; Griese, Julia J.; Zou, Xiaodong; Högbom, Martin (7 August 2019). "Solving a new R2lox protein structure by microcrystal electron diffraction". Science Advances. 5 (8): eaax4621. doi:10.1126/sciadv.aax4621.

Further reading
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