Microcrystal electron diffraction

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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. Prior to this demonstration, macromolecular (protein) electron crystallography was mainly used on 2D crystals, for example. [3] [4] The method is one of several modern versions of approaches to determine atomic structures using electron diffraction first demonstrated for the positions of hydrogen atoms in NH4Cl crystals by W. E. Laschkarew and I. D. Usykin in 1933, [5] which has since been used for surfaces, [6] via precession electron diffraction, [7] with much of the early work described in the work of Boris Vainshtein [8] and Douglas L. Dorset. [9]

Contents

The method was developed for structure determination of proteins from nanocrystals that are typically not suitable for X-ray diffraction because of their size. [10] Crystals that are one billionth the size needed for X-ray crystallography can yield high quality data. [11] 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 a 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 software for X-ray crystallography for structure analysis and refinement. [12] The hardware and software used in a MicroED experiment are standard and broadly available. [13] [14]

Development

Electron diffraction to solve crystal structures date back to the earliest days of electron diffraction. The first successful demonstration of MicroED was reported in 2013 by the Gonen laboratory [1] for the structure of lysozyme, a classic test protein in X-ray crystallography.

Experimental setup

Detailed protocols for setting up the electron microscope and for data collections have been published. [15]

Instrumentation

Microscope

MicroED data is collected using transmission electron (cryogenic) microscopy. The microscope can be equipped with a selected area aperture but MicroED can also be done without a selected area aperture. While some structures have been reported without freezing, radiation damage is sometimes minimized and higher resolution obtained by using cryo cooling even for small molecules. [16]

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. [17] More recently, direct electron detectors have been successfully used in both linear and counting modes. [18] [19] In these examples electron counting allowed ab initio phasing and visualization of hydrogens in proteins.

Data collection

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 [20] to yield a 2.9Å resolution structure with good refinement statistics, enabling determination of the structure of a dose-sensitive protein from 3D microcrystals in cryogenic conditions.

Continuous rotation

MicroED uses 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. This led to several improvements in data quality and allowed data processing using standard X-ray crystallographic software. [2] Continuous rotation MicroED improves sampling of reciprocal space. [21]

Data processing

Detailed protocols for MicroED data processing have been published. [12] When MicroED data is collected using continuous stage rotation, standard crystallography software [14] can be used.

Differences between MicroED and other electron diffraction methods

Other electron diffraction methods that have been developed and demonstrated to work include Automated Diffraction Tomography (ADT) [22] and Rotation Electron Diffraction (RED [23] ). These methods differ slightly from MicroED: In ADT discrete steps of goniometer tilt are used to cover reciprocal space in combination with beam precession to reduce dynamical diffraction effects. [22] ADT uses hardware and software for precession and scanning transmission electron microscopy for crystal tracking. [22] RED is done in TEM but the goniometer is tilted in discrete steps and beam tilting is used to fill in the gaps. [23] Software is used to process ADT and RED data. [23]

Milestones

Method scope

MicroED has been used to determine the structures of large globular proteins, [24] small proteins, [2] peptides, [25] membrane proteins, [26] organic molecules, [27] [28] and inorganic compounds. [29] In many of these examples hydrogens and charged ions were observed. [25] [26]

Novel structures of α-synuclein of Parkinson's disease

The first structures solved by MicroED were published in late 2015. [25] 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. [30] 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 known structure.

Related Research Articles

<span class="mw-page-title-main">Crystallography</span> Scientific study of crystal structures

Crystallography is the branch of science devoted to the study of molecular and crystalline structure and properties. The word crystallography is derived from the Ancient Greek word κρύσταλλος, and γράφειν. In July 2012, the United Nations recognised the importance of the science of crystallography by proclaiming 2014 the International Year of Crystallography.

<span class="mw-page-title-main">Structural biology</span> Study of molecular structures in biology

Structural biology, as defined by the Journal of Structural Biology, deals with structural analysis of living material at every level of organization.

<span class="mw-page-title-main">X-ray crystallography</span> Technique used for determining crystal structures and identifying mineral compounds

X-ray crystallography is the experimental science of determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract in specific directions. By measuring the angles and intensities of the X-ray diffraction, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal and the positions of the atoms, as well as their chemical bonds, crystallographic disorder, and other information.

In physics, the phase problem is the problem of loss of information concerning the phase that can occur when making a physical measurement. The name comes from the field of X-ray crystallography, where the phase problem has to be solved for the determination of a structure from diffraction data. The phase problem is also met in the fields of imaging and signal processing. Various approaches of phase retrieval have been developed over the years.

Electron crystallography is a subset of methods in electron diffraction focusing just upon detailed determination of the positions of atoms in solids using a transmission electron microscope (TEM). It can involve the use of high-resolution transmission electron microscopy images, electron diffraction patterns including convergent-beam electron diffraction or combinations of these. It has been successful in determining some bulk structures, and also surface structures. Two related methods are low-energy electron diffraction which has solved the structure of many surfaces, and reflection high-energy electron diffraction which is used to monitor surfaces often during growth.

<span class="mw-page-title-main">Transmission electron cryomicroscopy</span>

Transmission electron cryomicroscopy (CryoTEM), commonly known as cryo-EM, is a form of cryogenic electron microscopy, more specifically a type of transmission electron microscopy (TEM) where the sample is studied at cryogenic temperatures. Cryo-EM, specifically 3-dimensional electron microscopy (3DEM), is gaining popularity in structural biology.

<span class="mw-page-title-main">Cryogenic electron tomography</span>

Cryogenic electron tomography (cryoET) is an imaging technique used to reconstruct high-resolution (~1–4 nm) three-dimensional volumes of samples, often biological macromolecules and cells. cryoET is a specialized application of transmission electron cryomicroscopy (CryoTEM) in which samples are imaged as they are tilted, resulting in a series of 2D images that can be combined to produce a 3D reconstruction, similar to a CT scan of the human body. In contrast to other electron tomography techniques, samples are imaged under cryogenic conditions. For cellular material, the structure is immobilized in non-crystalline, vitreous ice, allowing them to be imaged without dehydration or chemical fixation, which would otherwise disrupt or distort biological structures.

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<span class="mw-page-title-main">Protein crystallization</span>

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<span class="mw-page-title-main">Structure validation</span> Process of evaluating 3-dimensional atomic models of biomacromolecules

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<span class="mw-page-title-main">Cryogenic electron microscopy</span> Form of transmission electron microscopy (TEM)

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Tamir Gonen is an American structural biochemist and membrane biophysicist best known for his contributions to structural biology of membrane proteins, membrane biochemistry and electron cryo-microscopy (cryoEM) particularly in electron crystallography of 2D crystals and for the development of 3D electron crystallography from microscopic crystals known as MicroED. Gonen is an Investigator of the Howard Hughes Medical Institute, a professor at the University of California, Los Angeles, the founding director of the MicroED Imaging Center at UCLA and a Member of the Royal Society of New Zealand.

<span class="mw-page-title-main">Serial femtosecond crystallography</span> Crystallography technique

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This is a timeline of crystallography.

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<span class="mw-page-title-main">Virus crystallisation</span>

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References

  1. 1 2 3 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. 1 2 3 4 5 6 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. ↑ Gonen, Tamir; Sliz, Piotr; Kistler, Joerg; Cheng, Yifan; Walz, Thomas (May 2004). "Aquaporin-0 membrane junctions reveal the structure of a closed water pore". Nature. 429 (6988): 193–197. doi:10.1038/nature02503. ISSN   1476-4687.
  4. ↑ Walz, Thomas; Hirai, Teruhisa; Murata, Kazuyoshi; Heymann, J. Bernard; Mitsuoka, Kaoru; Fujiyoshi, Yoshinori; Smith, Barbara L.; Agre, Peter; Engel, Andreas (June 1997). "The three-dimensional structure of aquaporin-1". Nature. 387 (6633): 624–627. doi:10.1038/42512. ISSN   0028-0836.
  5. ↑ Laschkarew, W. E.; Usyskin, I. D. (1933). "Die Bestimmung der Lage der Wasserstoffionen im NH4Cl-Kristallgitter durch Elektronenbeugung". Zeitschrift fur Physik (in German). 85 (9–10): 618–630. doi:10.1007/BF01331003. ISSN   1434-6001.
  6. ↑ Takayanagi, K.; Tanishiro, Y.; Takahashi, M.; Takahashi, S. (1985-05-01). "Structural analysis of Si(111)-7×7 by UHV-transmission electron diffraction and microscopy". Journal of Vacuum Science & Technology A: Vacuum, Surfaces, and Films. 3 (3): 1502–1506. doi:10.1116/1.573160. ISSN   0734-2101.
  7. ↑ Vincent, R.; Midgley, P.A. (1994). "Double conical beam-rocking system for measurement of integrated electron diffraction intensities". Ultramicroscopy. 53 (3): 271–282. doi:10.1016/0304-3991(94)90039-6.
  8. ↑ VAINSHTEIN, B.K. (1964), "Experimental Electron Diffraction Structure Investigations", Structure Analysis by Electron Diffraction, Elsevier, pp. 295–390, retrieved 2024-05-01
  9. ↑ Dorset, D. L. (1995-08-31). Structural Electron Crystallography. Springer Science & Business Media. ISBN   978-0-306-45049-5.
  10. ↑ Shi, Dan; Nannenga, Brent L; de la Cruz, M Jason; Liu, Jinyang; Sawtelle, Steven; Calero, Guillermo; Reyes, Francis E; Hattne, Johan; Gonen, Tamir (May 2016). "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.
  11. ↑ 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.
  12. 1 2 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.
  13. ↑ Zatsepin, Nadia A; Li, Chufeng; Colasurd, Paige; Nannenga, Brent L (October 2019). "The complementarity of serial femtosecond crystallography and MicroED for structure determination from microcrystals". Current Opinion in Structural Biology. 58: 286–293. doi:10.1016/j.sbi.2019.06.004. PMC   6778504 . PMID   31345629.
  14. 1 2 Nannenga, Brent L.; Gonen, Tamir (May 2019). "The cryo-EM method microcrystal electron diffraction (MicroED)". Nature Methods. 16 (5): 369–379. doi:10.1038/s41592-019-0395-x. ISSN   1548-7091. PMC   6568260 . PMID   31040436.
  15. ↑ 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.
  16. ↑ Christensen, Jeppe; Horton, Peter N.; Bury, Charles S.; Dickerson, Joshua L.; Taberman, Helena; Garman, Elspeth F.; Coles, Simon J. (2019-07-01). "Radiation damage in small-molecule crystallography: fact not fiction". IUCrJ. 6 (4): 703–713. doi:10.1107/S2052252519006948. ISSN   2052-2525. PMC   6608633 . PMID   31316814.
  17. ↑ See also https://www.gatan.com/ccd-vs-cmos and https://www.gatan.com/techniques/imaging.
  18. ↑ Martynowycz, Michael W.; Clabbers, Max T. B.; Hattne, Johan; Gonen, Tamir (June 2022). "Ab initio phasing macromolecular structures using electron-counted MicroED data". Nature Methods. 19 (6): 724–729. doi:10.1038/s41592-022-01485-4. ISSN   1548-7091. PMC   9184278 . PMID   35637302.
  19. ↑ Clabbers, Max T.B.; Martynowycz, Michael W.; Hattne, Johan; Gonen, Tamir (2022). "Hydrogens and hydrogen-bond networks in macromolecular MicroED data". Journal of Structural Biology: X. 6: 100078. doi:10.1016/j.yjsbx.2022.100078. PMC   9731847 . PMID   36507068.
  20. ↑ 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.
  21. ↑ Nannenga, Brent L; Shi, Dan; Leslie, Andrew G W; Gonen, Tamir (September 2014). "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.
  22. 1 2 3 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.
  23. 1 2 3 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.
  24. ↑ 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.
  25. 1 2 3 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}}: Cite journal requires |journal= (help)
  26. 1 2 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. S2CID   240183721.{{cite journal}}: Cite journal requires |journal= (help)
  27. ↑ 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.
  28. ↑ 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). ACS Central Science. 4 (11): 1587–1592. doi:10.26434/chemrxiv.7215332.v1. PMC   6276044 . PMID   30555912.
  29. ↑ 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.
  30. ↑ 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 . PMC   6685719 . PMID   31457106.

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