A simple pressure-assisted method for MicroED specimen preparation

Abstract

Cryo-electron microscopy (cryo-EM) has made great impacts on structural biology. However, specimen preparation remains a major bottleneck. Here, we report a simple method for preparing cryo-EM specimens, named Preassis, in which the excess liquid is removed by introducing a pressure gradient through the EM grid. We show the unique advantages of Preassis in handling samples with low concentrations of protein single particles and microcrystals in a wide range of buffer conditions. 2 Main textSingle particle cryo-EM is a powerful tool for structure determination of biological macromolecules at near-atomic resolution, which has been revolutionising the field of structural biology. Cryo-EM has undergone enormous technological innovations in both hardware and software over the past decades. However, the most widely used specimen preparation method has still been the pipetting-blotting-plunging routine first reported in 1981 1 . A major drawback of the method is the large sample consumption because more than 99.9% of the sample is lost to filter papers 2,3 . Therefore, it is of great importance to develop new methods that handle proteins samples only available at low concentrations, which is often the case for membrane proteins and complexes extracted from biological sources.Recently, a number of blot-less methods have been reported, such as multiple glass capillaries 4 , contact pin-printing 5 , and ink-jet spotter/Spotiton 2,6 . However, their implementations require special instrumentations and often non-standard EM grids.Recently 3D electron diffraction, known as MicroED 7,8 , has shown great potential for the determination of biomolecule structures from crystals too small for X-ray diffraction 9,10 .Protein crystallisation is often conducted in viscous media by introducing e.g. polyethylene glycols (PEGs) 11,12 , which is difficult to remove by the paper-blotting method. Currently, there is no report of efficient methods that can handle highly viscous samples. There is an urgent need to develop new cryo-EM specimen preparation methods for handling protein micro-crystals grown in a wide range of buffer conditions.Here, we demonstrate a simple pressure-assisted method, named Preassis, for preparing cryo-EM specimens of both single particles and micro-crystals. By using Preassis, suitable cryo-EM specimens can be prepared from proteins with concentrations as low as 0.18 mg/ml. In addition, Preassis can handle protein crystal suspensions with both low and high viscosity.1 6 9 were collected on the same microscope using the same detector. The conditions used to collect SAED patterns were: spot size 3, camera length 60 cm, and exposure time 2 s.

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

“…This viscous medium is problematic for traditional cryoelectron microscopy (cryoEM) blotting methods. In an attempt to circumvent these limitations, investigators have utilized nanoliter deposition through pin printing (6), vacuuming away the excess media using a pressure differential (7), liquid wicking grids (8), and changing the phase of the media using other less viscous detergents (9). Although these approaches have been successful for soluble protein crystals, there has been little progress for membrane protein crystals, likely stemming from crystal dehydration or damaging the crystal lattice.…”

Polymorph evolution during crystal growth studied by 3D electron diffraction

“…3 ml aliquots of the solution were pipetted onto a TEM grid (Quantifoil R3.5/1) and allowed to stand at ambient conditions (298 K, 21% humidity). The water was removed by pressure-assisted blotting (Zhao et al, 2019) at 3, 4 and 5 min and the sample immediately vitrified in liquid ethane to arrest further crystallization and protect the sample from beam and vacuum damage when under the microscope. A figure summarizing the procedure is available in the supporting information ( Fig.…”

Macromolecular crystallography using microcrystal electron diffraction

“…4). Recently, a pressure-assisted back-side blotting method was introduced specifically for MicroED specimen preparation (Zhao et al, 2019;Fig. 4).…”