Biology subjects

Unge, J.

Publications and source records attributed to Unge, J..

3 recordsLinked to original sources

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↗

bioRxiv2022

MicroED structure of a protoglobin reactive carbene intermediate

Microcrystal electron diffraction (MicroED) is an emerging technique which has shown great potential for describing new chemical and biological molecular structures. [1] Several important structures of small molecules, natural products and peptides have been determined using ab initio methods. [2] However, only a couple of novel protein structures have thus far been derived by MicroED. [3, 4] Taking advantage of recent technological advances including higher acceleration voltage and using a low-noise detector in counting mode, we have determined the first structure of an Aeropyrum pernix protoglobin (ApePgb) variant by MicroED using an AlphaFold2 model for phasing. The structure revealed that mutations introduced during directed evolution enhance carbene transfer activity by reorienting an alphahelix of ApePgb into a dynamic loop making the catalytic active site more readily accessible. After exposing the tiny crystals to substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineered ApePgbs new-to-nature activity, a challenging carbene transfer from a diazirine via a putative metallo-carbene. The bound structure discloses how an enlarged active site pocket stabilizes the carbene bound to the heme iron and, presumably, the transition state for formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enables investigation of structures that were previously beyond reach.

biochemistry↗

bioRxiv2021

Benchmarking ideal sample thickness in cryo-EM using MicroED

The relationship between sample thickness and quality of data obtained by microcrystal electron diffraction (MicroED) is investigated. Several EM grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion-beam scanning electron microscope (FIB/SEM) where the crystals were then systematically thinned into lamellae between 95 nm and 1650 nm thick. MicroED data were collected at either 120, 200, or 300 kV accelerating voltages. Lamellae thicknesses were converted to multiples of the calculated inelastic mean free path (MFP) of electrons at each accelerating voltage to allow the results to be compared on a common scale. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined from crystalline lamellae only up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages but the quality was insufficient to yield structures. No diffraction data were observed from lamellae thicker than four times the calculated inelastic mean free path. The quality of the determined structures and crystallographic statistics were similar for all lamellae up to 2x the inelastic mean free path in thickness, but quickly deteriorated at greater thicknesses. This study provides a benchmark with respect to the ideal limit for biological specimen thickness with implications for all cryo-EM methods. SignificanceA systematic investigation of the effects of thickness on electron scattering from protein crystals was previously not feasible, because there was no accurate method to control sample thickness. Here, the recently developed methods for preparing protein crystals into lamellae of precise thickness by ion-beam milling are used to investigate the effects of increasing sample thickness on MicroED data quality. These experiments were conducted using the three most common accelerating voltages in cryo-EM. Data across these accelerating voltages and thicknesses were compared on a common scale using their calculated inelastic mean free path lengths. It is found that structures may accurately be determined from crystals up to twice the inelastic mean free path length in thickness, regardless of the acceleration voltage.

biochemistry↗