Design and implementation of suspended drop crystallization

Abstract

We have developed a novel crystal growth method known as suspended drop crystallization. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber which we designed, allowing for vapor diffusion to occur from both sides of the drop. A UV transparent window above and below the grid enables the monitoring of crystal growth via light, UV, or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for x-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, we grew crystals of the enzyme proteinase K and determined its structure by MicroED following FIB/SEM milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress, and/or suffering from preferred orientation on EM grids.

Figure 1.. Suspended drop crystallization

(a) A support-free EM grid is clipped into an autogrid cartridge and mounted between the arms of the suspended drop screening tool. The sample and crystallization solution are dispensed onto the grid. (b) The chamber is immediately sealed to allow vapor diffusion. (b) The incubation chambers are inserted into a screening tray for efficient storing and monitoring of crystallization progress by light, fluorescence and UV microscopy. (d) EM grids containing crystals are retrieved from the screening tool and frozen. (e) The specimen is then interrogated by MicroED or other methods such as tomography, x-ray crystallography, or general microscopy. FIB milling is optional depending on the application.

Figure 2.. MicroED structure of suspended drop Proteinase K

(a) The suspended drop viewed from the top and imaged by (b) Light microscopy or (c) UV. A frozen suspended drop specimen was loaded into the FIB/SEM and imaged normal to the grid surface by (d) SEM and (e) iFLM with the 385 nm LED to locate submerged crystals. (f) The targeted crystal site was milled into a 300 nm thick lamella. (g) Example of MicroED data acquired from the crystal lamella. The highest resolution reflections visible to 2.1 Å (red arrow). Resolution ring is shown at 2.0 Å (blue). (H) Cartoon representation of the Proteinase K colored by rainbow with blue N terminus and red C terminus. The 2mFo–DFc map of a selected alpha-helix is highlighted, which was contoured at 1.5 σ with a 2-Å carve.