The collection of MicroED data for macromolecular crystallography

Abstract

The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.

Figure 1

Structures determined by MicroED. The proteins solved by MicroED are shown along with their Protein Data Bank (PDB) IDs and the final resolutions of the resulting structures.

Figure 2

Flow diagram for MicroED data collection protocol. The protocol begins with identifying suitable microcrystals using light microscopy and then preparing frozen samples by either automated vitrification systems or manual methods. After the microscope has been set up for low-dose diffraction and microcrystals identified on the grid, initial diffraction patterns are collected to assess crystal quality. When a high-quality crystal is found, diffraction data sets are collected.

Figure 3

Representative protein microcrystal images. Crystallization conditions that produce microcrystals can be identified by light microscopy in cases. Shown are examples in which small crystals are visible in light microscopy (a–c) and their corresponding images in negative-stain electron microscopy (EM; e–g), as well as an example in which only granular aggregates are observed by light microscopy (d), but the sample contains nanocrystals when investigated by EM (h). Scale bar, 500 nm (in a–d) and 400 nm (in e–h).

Figure 4

Assembled Vitrobot coolant container. Before liquid nitrogen is added to the coolant container outer reservoir (1), as described in Step 10A(iii), the cryo-gridbox (2), inner brass ethane container (3) and aluminum bridge (4) should be properly assembled, as shown above.

Figure 5

Setting the eucentric height correctly. When the eucentric height has been set properly, features at the center of the screen do not move when the microscope is tilted. (a,b) The crystal seen at the center of the untilted sample (a) remains centered after the stage is tilted when the eucentric height is set correctly (b). (c) If the eucentric height is not set correctly (+16 μm in this example), the crystal swings away from the image center during tilting. The dashed yellow line indicates the tilt axis.

Figure 6

Low-dose settings menu on the FEI Tecnai F20 microscope. Screenshot showing representative low-dose settings used during MicroED data collection. The microscope is currently in exposure mode, which is indicated by ‘Exposure’ being highlighted in yellow.

Figure 7

Example images from search, focus and exposure modes from a model MicroED sample. Search mode shows many well-separated microcrystals across a large area of the grid. White arrows are pointing to a few example microcrystals. When Focus mode is used to search at higher magnification, individual microcrystals, indicated by white arrows, can be located and centered for data collection. The high-quality initial diffraction pattern taken in exposure mode shows a single lattice with tight well-separated spots, indicating that a complete rotation data set should be collected from this crystal.