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![[Figure 4]](ic5114fig4mag.jpg)
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Figure 4
Schematic overview of the specimen preparation involved in MicroED. The aim of specimen preparation is to rapidly vitrify the hydrated protein crystals, while keeping a thin layer of vitrified ice around the crystals to protect them from the vacuum and electron beam radiation during MicroED data collection (Dubochet et al., 1988  ; Shi et al., 2016). (a) The protein crystal suspension is typically deposited on a 3 mm TEM grid (Quantifoil or lacey carbon). Prior to vitrification, any excess liquid can be removed by back-side blotting (Martynowycz & Gonen, 2020), pressure-assisted blotting (Zhao et al., 2019) or (b) double-sided blotting (Shi et al., 2016). (c) Alternatively, a small amount of crystal suspension can be sprayed onto a self-wicking grid, leaving a line of thin liquid trail covering approximately 50 grid squares, as highlighted by the red lines in the figure (Jain et al., 2012; Klebl et al., 2020). The concentration of the crystals in the suspension needs to be tuned to avoid clogging the nozzle of the inkjet. (d) After the excess liquid has been removed from the grid by any of the methods introduced above (a–c), the grid is then rapidly plunged into liquid ethane. (e) If the prepared specimen is of suitable thickness, MicroED data can be collected straight away using TEM. (f) Otherwise, cryo-FIB milling can be used to make thin crystalline lamella of the sample suitable for MicroED data collection (Duyvesteyn et al., 2018; Zhou et al., 2019; Martynowycz et al., 2019a,b). |
![[Figure 4]](ic5114fig4.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
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