Abstract
In 2013 we established a cryo-electron microscopy (cryo-EM) technique called microcrystal electron diffraction (MicroED). Since that time, data collection and analysis schemes have been fine-tuned, and structures for more than 40 different proteins, oligopeptides and organic molecules have been determined. Here we review the MicroED technique and place it in context with other structure-determination methods. We showcase example structures solved by MicroED and provide practical advice to prospective users.
🔬 Techniques
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🧪 Sample Preparation
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💻 Software Details
💾 Data Repositories
🏛️ Research Organizations (ROR)
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📊 Figures
Figure 1:
Methods within the field of Cryo-EM.
The diverse imaging-based or diffraction-based techniques within cryo-electron microscopy can provide structural information from a wide range of samples. Images shown for the different techniques are...
Figure 2:
MicroED overview.
MicroED data are collected as movies while the stage of the cryo-EM is continuously rotated. This produces a series of high-resolution diffraction patterns that can be processed to produce high-resolu...
Figure 3:
Improvements in MicroED data quality.
Continual development of MicroED has led to steady improvements in the quality of structures obtained. This can be seen by the increases in resolution that are possible from similar lysosome microcrys...
Figure 4:
Examples of novel structures determined by MicroED.
(A) The NACore fragment from u03b1-synuclein was determined to 1.4 u00c5 resolution. (B) Gag-bevirimat MicroED structures (side and top views shown with a surface and ribbon representation, respective...
Figure 5:
Crystal identification and sample preparation for MicroED.
(A) Frequently, identification of microcrystals in drops that appear to have cloudy precipitates is difficult by visible light (left panel); however, when the drops are imaged using UV fluorescence, t...
Figure 6.
Cryo-FIB milling of a thick crystal.
(A) Sample preparation for MicroED using a cryo-FIB to mill down thick crystals to a few hundred nanometers (left and right). (B) Following cryo-FIB milling, the grid would be loaded into the TEM and ...
Figure 7:
Comparison of Proteinase K data collected with and without an energy filter.
The zero-loss data collected on a cryo-TEM equipped with an in-column energy filter shows much less diffuse scattering at lower resolutions.
Figure 8:
Dynamics probed in response to radiation damage.
When less than 1 e u2212 /u00c5 2 (left) was used for structure determination of Proteinase K (1.7u00c5, PDB ID: 6CL7), local radiation damage was minimal. When higher doses (right) were used (2.8u00c...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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