Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED

Abstract

Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction limits the thickness of crystals that can be investigated by MicroED, mainly due to absorption. Recent studies have demonstrated that focused ion beam (FIB) can thin even very large crystals into ideal sized lamellae however it is not clear how to best apply FIB-milling for MicroED. Here, The effects of polishing the lamellae, whereby the last few nanometers are milled away using a low-current gallium beam, are explored in both platinum pre-coated and uncoated samples. Our results suggest that pre-coating samples with a thin layer of platinum followed by polishing the crystal surfaces prior to data collection consistently led to superior results as indicated by higher signal/noise ratio, higher resolution and better refinement statistics. This study lays the foundation for routine and reproducible methodology for sample preparation in MicroED.

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

“…Although the milling time decreased from ~60 min to ~20 min, the use of the higher current led to lower quality lamellae, diffracting to 7 -4 Å only. Of course, this could have been caused by other factors such as crystals grown to lower quality or damaged during transfer from crystallization probe to a TEM grid, nevertheless, it is worth mentioning that similar observations were reported by (Martynowycz et al, 2019b) in experiments on FIB-milled lamellae of proteinase K crystals. The authors utilized a cryo-FIB instrument similar to the one used in our present study and showed that focused Ga + beams with 10-pA and 30-pA current produced lamellae of different qualities, diffracting to 1.8 Å and 2.1 Å, respectively.…”

“…The specimen was kept at −192°C on the cryo-stage during the whole procedure. The lamella preparation protocol followed the approach published earlier (see (Schaffer et al, 2017;Martynowycz et al, 2019b;Zhou et al, 2019)). The crystal was coated with a conductive Pt-sputter (10 mA, 60 s) layer and a protective organometallic Pt-GIS (30°C, 80 s) layer.…”

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

“…Grid clips were marked with a dot on the top with a sharpie to indicate the milling direction. A thin layer of platinum (~10nm) was deposited on the grids by sputter coating prior to inspecting the grids using the SEM (Michael W Martynowycz et al, 2019). An additional layer (~300nm) of carbon-rich platinum was added on top of this using the gas injection system.…”

“…Crystals were identified in the SEM and FIB, brought to eucentric height, and milled using cleaning cross sections in steps into lamellae until a thickness of ~200nm as described (Duyvesteyn et al, 2018; Michael W. Martynowycz et al, 2019). The final polishing step used a gallium beam current of 10pA to remove the last few nm of crystal from the lamellae (Michael W Martynowycz et al, 2019).…”

“…MicroED data collection was performed very similarly to previous experiments (Hattne et al, 2019, 2018, 2015; Jason de la Cruz et al, 2019; Martynowycz et al, 2020; Michael W Martynowycz et al, 2019; Michael W. Martynowycz et al, 2019). Grids with milled crystals were transferred to a Thermo-Fisher Talos Arctica transmission electron microscope after rotating the grids by 90°.…”