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A Simple Pressure-Assisted Method for Microed Specimen Preparation

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A Simple Pressure-Assisted Method for Microed Specimen Preparation ARTICLE https://doi.org/10.1038/s41467-021-25335-7 OPEN A simple pressure-assisted method for MicroED specimen preparation ✉ Jingjing Zhao 1, Hongyi Xu 1 , Hugo Lebrette2, Marta Carroni 2,3, Helena Taberman4,5, ✉ Martin Högbom2 & Xiaodong Zou 1 Micro-crystal electron diffraction (MicroED) has shown great potential for structure deter- mination of macromolecular crystals too small for X-ray diffraction. However, specimen 1234567890():,; preparation remains a major bottleneck. Here, we report a simple method for preparing MicroED specimens, named Preassis, in which excess liquid is removed through an EM grid with the assistance of pressure. We show the ice thicknesses can be controlled by tuning the pressure in combination with EM grids with appropriate carbon hole sizes. Importantly, Preassis can handle a wide range of protein crystals grown in various buffer conditions including those with high viscosity, as well as samples with low crystal concentrations. Preassis is a simple and universal method for MicroED specimen preparation, and will sig- nificantly broaden the applications of MicroED. 1 Department of Materials and Environmental Chemistry, Stockholm University, Stockholm, Sweden. 2 Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden. 3 Science for Life Laboratory, Stockholm University, Solna, Sweden. 4 Max Delbrück Centrum for Molecular Medicine, ✉ Berlin, Germany. 5 Macromolecular Crystallography, Helmholtz-Zentrum Berlin, Berlin, Germany. email: [email protected]; [email protected] NATURE COMMUNICATIONS | (2021) 12:5036 | https://doi.org/10.1038/s41467-021-25335-7 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25335-7 hree-dimensional electron diffraction, known as micro- removed through the EM grid with the assistance of pressure. Tcrystal electron diffraction (MicroED)1,2, has shown great Preassis is applicable for a wide range of protein crystal suspen- potential for structure determination of macromolecules sions with both low and high viscosities. It can preserve up to two from nano- and micron-sized crystals, which are too small for orders of magnitude more crystals on the TEM grid compared X-ray diffraction3–5. This method has attracted large interest and with Vitrobot, which has a unique advantage to study samples extensive attention in structural biology, structural chemistry, and with low crystal concentrations. The ice thicknesses can be con- the pharmaceutical industry during the past years. However, until trolled by tuning the pressure in combination with appropriate now, only a few macromolecular structures solved by MicroED carbon hole sizes of EM grids. We provide detailed experimental are reported in the PDB database and all except R2lox3 had been guidance in finding appropriate parameters for preparing previously determined by X-ray diffraction. The R2lox structure3 MicroED specimens of new protein crystal samples. More was solved by molecular replacement using a search model of importantly, the Preassis method is simple and easy to imple- 35% sequence identity (PDB code 6QRZ). MicroED data collec- ment, making it widely accessible to cryo-EM labs at a very tion is usually very fast (1–3 min)2,6,7 by applying continuous low cost. rotation of crystals and a hybrid pixel detector or complementary metal-oxide semiconductor detector in movie mode. Data pro- cessing and structure determination can be done using standard Results and discussions X-ray crystallographic software suites. Despite those advances in The basic concept of Preassis is to pull a portion of sample sus- MicroED experiments, the major bottleneck has been specimen pension through an EM grid and simultaneously remove excess preparation, which is most delicate and time-consuming. liquid from the backside of the grid with the assistance of suction/ In MicroED experiments, sub-micrometre thick crystals are pressure. In its simplest setup (Fig. 1 and Supplementary Fig. 1), needed in order to allow the electron beam to penetrate through an EM grid is placed on a filter paper which is rested on the the specimen and minimize multiple scattering. The surrounding mouth of a Buchner flask and pumped with a certain pumping ice also needs to be as thin as possible to improve the signal-to- speed. A droplet of the sample suspension is then deposited onto noise ratio while still protecting the protein crystals from dehy- the grid. Due to the suction underneath the filter paper, the excess dration. Suitable crystal size can be obtained by adjusting the liquid is “pulled” through the grid. Then the grid is manually crystallization conditions8,9, by segmenting large crystals using picked up using a tweezer and plunged into liquid ethane. Details mechanical forces (e.g., vigorous pipetting, sonication, vortexing of this setup and specimen preparation procedures are available with beads)10, or by focused ion beam milling under cryogenic in Supplementary Methods. The overall vitrified ice thickness on conditions (cryo-FIB)11–14. High molecular weight polymers, like the EM grid can be tuned by changing the pressure, carbon hole polyethylene glycols (PEG), are common and popular agents to size of the EM grid, and the time over which the pressure is produce volume-exclusion effects for successful protein applied. While the pressure can be changed continuously, the crystallization8,9,15. However, their addition makes the buffer change of the carbon hole size is done by choosing the type of viscous. Lipid cubic phase (LCP) is commonly used for crystal- holey carbon EM grids. In the current setup, the pressure is lization of membrane protein, and they are extremely viscous as adjusted by changing the pumping speed, where the pressure is toothpaste13,16,17. It has been very challenging to prepare linearly proportional to the pumping speed in the range of MicroED samples for crystals grown in viscous buffers4,13,18. The 20–80% (Supplementary Fig. 2). It is worth mentioning that the pipetting-blotting-plunging rountine19, originally designed for ice layer thickness can vary throughout an EM grid prepared by single-particle cryo-EM specimen preparation, has two major the current Preassis setup, as shown in Supplementary Fig. 3. This drawbacks for MicroED specimen preparation; (1) many micro- may be due to non-uniform contact of the EM grid to the filter crystals are removed by blotting and (2) it is insufficient in paper. Such a gradient of ice thickness may not be a disadvantage, removing viscous liquids18. Manual back-side blotting18, direct and instead, it increases the chance of finding suitable crystals for crystallization on EM grids20, and cryo-FIB13,14,17,21 have been MicroED data collection. proposed as possible solutions to deal with the above problems. We compared EM specimens prepared by the double-side However, a simple and universal method for MicroED specimen blotting in Vitrobot and Preassis, by studying the crystal density, preparation is still missing. There is an urgent need to develop MicroED data quality, and ice thickness. We also studied the such a method in order to apply MicroED on a wide range of influence of humidity on ice thickness. We chose a non-viscous macromolecular crystals. suspension of tetragonal lysozyme microcrystals with a high Here, we describe a pressure-assisted method, named Preassis, crystal concentration for the study. For each method Preassis or for the preparation of MicroED specimens. The excess liquid is Vitrobot, at least three copies of grids were prepared using the same condition. We found Preassis requires much lower crystal concentrations than Vitrobot, as shown in Fig. 2 and Supple- mentary Figs. 4 and 5. When a crystal suspension with low concentration (i.e., diluted by 500×) was used, the grids prepared by Preassis had thousands of microcrystals (Fig. 2a–c and Sup- Air flow Plunge freezing plementary Figs. 4a–c and 5a–c), while those prepared by Liquid ethane Liquid nitrogen Vitrobot were almost empty (Fig. 2d–f and Supplementary Fig. 4d–f). To reach a sufficient crystal coverage for MicroED experiments, a crystal suspension with at least 250 times higher Buchner concentration (i.e., diluted by only 2×) was needed for Vitrobot, flask as shown in Fig. 2g–i and Supplementary Fig. 5d–f. This com- parison shows that Preassis can keep around two orders of Fig. 1 Schematic drawing of the Preassis setup. A drop of the microcrystal magnitude more crystals on the EM grid than Vitrobot. This is suspension is transferred onto an EM grid resting on a support (e.g., filter mainly because, by Preassis, excess liquid is removed through the paper). Simultaneously, the extra liquid is removed through the support carbon holes of an EM grid where crystals are kept by the holey with the assistance of pressure, and followed by manually plunge freezing carbon film. In the case of Vitrobot, most excess liquid was taken in liquid ethane for vitrification. away from the front side of the grid by a filter paper where 2 NATURE COMMUNICATIONS | (2021) 12:5036 | https://doi.org/10.1038/s41467-021-25335-7 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25335-7 ARTICLE Preassis Vitrobot Vitrobot Vitrobot 1:500 diluon 1:500 diluon 1:2 diluon a b c a d g 9.0 Å 100 μm 100 μm 100 μm b e h Preassis d e f 3.0 Å 10 μm 10 μm 10 μm 20 μm c f i Fig. 3 Comparison of ice thickness and data resolution obtained from the specimens prepared by Vitrobot and Preassis. a–f TEM images (a–b, d–e) and ED patterns (c, f) taken from specimens prepared from the same viscous R2lox crystal suspension by Vitrobot and Preassis, respectively. 2 μm 2 μm 2 μm The ice thickness can be reduced and ED data resolution was improved from 9.0 Å (c) to 3.0 Å (f) by applying Preassis.
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