Microcrystal electron diffraction, or MicroED, is a

CryoEM 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 sol ...

method that was developed by the Gonen laboratory in late 2013 at the

Janelia Research Campus Janelia Research Campus is a scientific research campus of the Howard Hughes Medical Institute that opened in October 2006. The campus is located in Loudoun County, Virginia, near the town of Ashburn. It is known for its scientific research and m ...

of the

Howard Hughes Medical Institute The Howard Hughes Medical Institute (HHMI) is an American non-profit medical research organization based in Chevy Chase, Maryland. It was founded in 1953 by Howard Hughes, an American business magnate, investor, record-setting pilot, engineer, fil ...

. MicroED is a form of

electron crystallography Electron crystallography is a method to determine the arrangement of atoms in solids using a transmission electron microscope (TEM). Comparison with X-ray crystallography It can complement X-ray crystallography for studies of very small crystals ...

where thin 3D crystals are used for structure determination by

electron diffraction Electron diffraction refers to the bending of electron beams around atomic structures. This behaviour, typical for waves, is applicable to electrons due to the wave–particle duality stating that electrons behave as both particles and waves. Si ...

. The method was developed for structure determination of proteins from

nanocrystal A ''nanocrystal'' is a material particle having at least one dimension smaller than 100 nanometres, based on quantum dots (a nanoparticle) and composed of atoms in either a single- or poly-crystalline arrangement. The size of nanocrystals dist ...

s 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. The samples are frozen hydrated as for all other CryoEM modalities but instead of using the

transmission electron microscope Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a gr ...

(

TEM Tem or TEM may refer to: Acronyms * Threat and error management, an aviation safety management model. * Telecom Expense Management * Telecom Equipment Manufacturer * TEM (currency), local to Volos, Greece * TEM (nuclear propulsion), a Russian ...

) in imaging mode one uses it in

diffraction Diffraction is defined as the interference or bending of waves around the corners of an obstacle or through an aperture into the region of geometrical shadow of the obstacle/aperture. The diffracting object or aperture effectively becomes a s ...

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 while diffraction is collected on a fast camera as a movie. MicroED data is then processed using traditional software for

X-ray crystallography X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles ...

without the need for specialized software for structure analysis and refinement. 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. The structure of

lysozyme Lysozyme (EC 3.2.1.17, muramidase, ''N''-acetylmuramide glycanhydrolase; systematic name peptidoglycan ''N''-acetylmuramoylhydrolase) is an antimicrobial enzyme produced by animals that forms part of the innate immune system. It is a glycoside ...

, a classic test protein in

. The Abrahams group independently reported rotation

data collection using a

Medipix Medipix is a family of photon counting and particle tracking pixel detectors developed by an international collaboration, hosted by CERN. Design These are hybrid detectors as a semiconductor sensor layer is bonded to a processing electronics lay ...

quantum area detector on

crystals but were unable to solve the structure.

Experimental setup

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

Instrumentation

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 Selected area (electron) diffraction (abbreviated as SAD or SAED), is a crystallographic experimental technique typically performed using a transmission electron microscope (TEM). It is a specific case of electron diffraction used primarily in ma ...

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.

Data collection

Still diffraction

The initial proof of concept publication on MicroED used lysozyme crystals. 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. 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. 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. 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) and Rotation Electron Diffraction (RED). 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. ADT uses specialized hardware for precession and scanning transmission electron microscopy for crystal tracking. RED is done in TEM but the goniometer is coarsely tilted in discrete steps and beam tilting is used to fill in the gaps. Specialized software is used to process ADT and RED data. 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, small proteins, peptides, membrane proteins, organic molecules, and inorganic compounds. In many of these examples hydrogens and charged ions were observed.

Novel structures of α-synuclein of Parkinson's disease

The first novel structures solved by MicroED were published in late 2015. These structures were of peptide fragments that form the toxic core of α-synculein, the protein responsible for

Parkinson's disease Parkinson's disease (PD), or simply Parkinson's, is a long-term degenerative disorder of the central nervous system that mainly affects the motor system. The symptoms usually emerge slowly, and as the disease worsens, non-motor symptoms becom ...

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. 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 annua
MicroED Imaging Center Course at UCLA
or th

. For more up to date information about upcoming meetings and workshops related to

Cryogenic electron microscopy 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 so ...

methods as a whole, please check th
3DEM Meetings and Workshops page
Several universities and companies offer MicroED services, including th
MEDIC – Microcrystal Electron Diffraction Imaging Center at UCLA
an
Nanoimaging Services
Several electron microscope systems are capable of recording MicroED data including those developed by JEOL; and Thermo Fisher/FEI.