Biology subjects

Gillman, C.

Publications and source records attributed to Gillman, C..

3 recordsLinked to original sources

bioRxiv2024

Eliminating the missing cone challenge through innovative approaches

Microcrystal electron diffraction (MicroED) has emerged as a powerful technique for unraveling molecular structures from microcrystals too small for X-ray diffraction. However, a significant hurdle arises with plate-like crystals that consistently orient themselves flat on the electron microscopy grid. If, as is typically the case, the normal of the plate correlates with the axes of the crystal lattice, the crystal orientations accessible for measurement are restricted because the grid cannot be arbitrarily rotated. This limits the information that can be acquired, resulting in a missing cone of information. We recently introduced a novel crystallization strategy called suspended drop crystallization and proposed that this method could effectively address the challenge of preferred crystal orientation. Here we demonstrate the success of the suspended drop crystallization approach in eliminating the missing cone in two samples that crystallize as thin plates: bovine liver catalase and the COVID-19 main protease (Mpro). This innovative solution proves indispensable for crystals exhibiting preferred orientations, unlocking new possibilities for structure determination by MicroED.

biochemistry↗

bioRxiv2023

The structure of the neurotoxin palytoxin determined by MicroED

Palytoxin (PTX) is a potent neurotoxin found in marine animals that can cause serious symptoms such as muscle contractions, haemolysis of red blood cells and potassium leakage. Despite years of research, very little is known about the mechanism of PTX. However, recent advances in the field of cryoEM, specifically the use of microcrystal electron diffraction (MicroED), have allowed us to determine the structure of PTX. It was discovered that PTX folds into a hairpin motif and is able to bind to the extracellular gate of Na,K-ATPase, which is responsible for maintaining the electrochemical gradient across the plasma membrane. These findings, along with molecular docking simulations, have provided important insights into the mechanism of PTX and can potentially aid in the development of molecular agents for treating cases of PTX exposure.

biochemistry↗

bioRxiv2023

Design and implementation of suspended drop crystallization

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.

biochemistry↗