Microcrystal electron diffraction

Last updated May 18, 2024

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]

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 experimental science of determining the arrangement of atoms in crystalline solids. Crystallography is a fundamental subject in the fields of materials science and solid-state physics. 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 that 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.

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.

The Protein Data Bank (PDB) is a database for the three-dimensional structural data of large biological molecules such as proteins and nucleic acids, which is overseen by the Worldwide Protein Data Bank (wwPDB). These structural data are obtained and deposited by biologists and biochemists worldwide through the use of experimental methodologies such as X-ray crystallography, NMR spectroscopy, and, increasingly, cryo-electron microscopy. All submitted data are reviewed by expert biocurators and, once approved, are made freely available on the Internet under the CC0 Public Domain Dedication. Global access to the data is provided by the websites of the wwPDB member organisations.

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.

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.

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.

Resolution in the context of structural biology is the ability to distinguish the presence or absence of atoms or groups of atoms in a biomolecular structure. Usually, the structure originates from methods such as X-ray crystallography, electron crystallography, or cryo-electron microscopy. The resolution is measured of the "map" of the structure produced from experiment, where an atomic model would then be fit into. Due to their different natures and interactions with matter, in X-ray methods the map produced is of the electron density of the system, whereas in electron methods the map is of the electrostatic potential of the system. In both cases, atomic positions are assumed similarly.

Protein crystallization is the process of formation of a regular array of individual protein molecules stabilized by crystal contacts. If the crystal is sufficiently ordered, it will diffract. Some proteins naturally form crystalline arrays, like aquaporin in the lens of the eye.

Macromolecular structure validation is the process of evaluating reliability for 3-dimensional atomic models of large biological molecules such as proteins and nucleic acids. These models, which provide 3D coordinates for each atom in the molecule, come from structural biology experiments such as x-ray crystallography or nuclear magnetic resonance (NMR). The validation has three aspects: 1) checking on the validity of the thousands to millions of measurements in the experiment; 2) checking how consistent the atomic model is with those experimental data; and 3) checking consistency of the model with known physical and chemical properties.

Structural chemistry is a part of chemistry and deals with spatial structures of molecules and solids. For structure elucidation a range of different methods is used. One has to distinguish between methods that elucidate solely the connectivity between atoms (constitution) and such that provide precise three dimensional information such as atom coordinates, bond lengths and angles and torsional angles.

In crystallography, direct methods is a set of techniques used for structure determination using diffraction data and a priori information. It is a solution to the crystallographic phase problem, where phase information is lost during a diffraction measurement. Direct methods provides a method of estimating the phase information by establishing statistical relationships between the recorded amplitude information and phases of strong reflections.

Cryogenic electron microscopy (cryo-EM) is a cryomicroscopy technique applied on samples cooled to cryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment of vitreous ice. An aqueous sample solution is applied to a grid-mesh and plunge-frozen in liquid ethane or a mixture of liquid ethane and propane. While development of the technique began in the 1970s, recent advances in detector technology and software algorithms have allowed for the determination of biomolecular structures at near-atomic resolution. This has attracted wide attention to the approach as an alternative to X-ray crystallography or NMR spectroscopy for macromolecular structure determination without the need for crystallization.

<span class="mw-page-title-main">Tamir Gonen</span> American biochemist and biophysicist

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.

Serial femtosecond crystallography (SFX) is a form of X-ray crystallography developed for use at X-ray free-electron lasers (XFELs). Single pulses at free-electron lasers are bright enough to generate resolvable Bragg diffraction from sub-micron crystals. However, these pulses also destroy the crystals, meaning that a full data set involves collecting diffraction from many crystals. This method of data collection is referred to as serial, referencing a row of crystals streaming across the X-ray beam, one at a time.

This is a timeline of crystallography.

Xiaodong Zou is a Chinese-Swedish chemist who is a professor at Stockholm University. Her research considers the development of electron diffraction for the three dimensional characterisation of materials. She is a member of the Nobel Committee for Chemistry. She was elected to the Royal Swedish Academy of Sciences and the Royal Swedish Academy of Engineering Sciences.

Hosea Nelson is an American chemist who is a professor at California Institute of Technology. His research investigates the design and total synthesis of complex molecules. He was a finalist for the 2021 Blavatnik Awards for Young Scientists.

References

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↑ 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.
↑ VAINSHTEIN, B.K. (1964), "Experimental Electron Diffraction Structure Investigations", Structure Analysis by Electron Diffraction, Elsevier, pp. 295–390, retrieved 2024-05-01
↑ Dorset, D. L. (1995-08-31). Structural Electron Crystallography. Springer Science & Business Media. ISBN 978-0-306-45049-5.
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↑ 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.
↑ 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.
↑ See also https://www.gatan.com/ccd-vs-cmos and https://www.gatan.com/techniques/imaging.
↑ 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.
↑ 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.
↑ 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.
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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.
↑ 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.
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)
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)
↑ 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.
↑ 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.
↑ 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.
↑ 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.