Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

Abstract

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.

Demonstration of electron diffraction from membrane protein crystals grown in a lipidic mesophase after lamella preparation by focused ion beam milling at cryogenic temperatures

“…We believe that the cryo-FIB milling method can be applicable to crystals of membrane or non-membrane proteins grown or embedded in various lipidic mesophases, including bicells and vesicles, or in other viscous crystallization media such as concentrated polyethylene glycol (PEG) solutions and media used for highviscosity injectors (e.g. media used in (Wolff et al, 2020)). Moreover, we believe that, the cryo-FIB milling technique can also be suitable for crystals of few microns or even of smaller sizes.…”

“…Electron crystallography of three-dimensional (3D) macromolecular crystals is a promising technique for solving structures at high resolution . Profiting from strong interactions of electrons with matter (Henderson, 1995) and utilizing a standard cryo-electron microscope (cryo-EM) (Shi et al, 2013(Shi et al, , 2016, electron diffraction provides an effective means of solving structures of proteins and peptides from very thin crystals, which would either be difficult to study by synchrotron-based methods or can only be investigated with X-ray free-electron lasers (XFELs) (Neutze et al, 2000;Chapman et al, 2011;Wolff et al, 2020). Moreover, atomic scattering factors for electrons, unlike those for X-rays, are strongly influenced by the charge state of the scattering atoms.…”

Efficient, high-throughput ligand incorporation into protein microcrystals by on-grid soaking

“…Crystals on the TEM grid were identified in the SEM after platinum coating as described previously (Martynowycz et al, 2019; Martynowycz et al, 2019; Wolff et al, 2020). The crystal density was high with crystals being present over both the holey carbon film and the film above the grid bars (Figure 1).…”

MicroED structure of lipid-embedded mammalian mitochondrial voltage dependent anion channel

“…All FIB/SEM experiments were performed on a Thermo-Fisher aquilos dual-beam FIB/SEM instrument at liquid nitrogen temperatures as described (Duyvesteyn et al, 2018;Martynowycz et al, 2019aMartynowycz et al, , 2019bWolff et al, 2020). The instrument was operated at 2kV and 3.1pA while using the SEM for imaging, and 30kV and 1.5 or 10pA while operating the FIB beam for imaging.…”

“…Recently two reports, one published and one posted online, attempted to determine the MicroED structure of a membrane protein embedded in LCP but were not successful likely because the sample preparation methods were not optimized (Zhu et al, 2020b;Polovinkin et al, 2020). Importantly, to date FIB milling coupled with successful structure determination by MicroED has only been demonstrated for soluble standard proteins, such as lysozyme and proteinase K (Beale et al, 2020;Duyvesteyn et al, 2018;Li et al, 2018;Martynowycz et al, 2019aMartynowycz et al, , 2019bWolff et al, 2020;Zhou et al, 2019).…”