News
Chemistry World highlights MicroED
November 13, 2024
British news magazine Chemistry World traces the history of MicroED from its beginnings with aquaporin to its promising future, where dedicated instruments streamline the method even further.
Johan Unge to chair ACA MicroED special interest group
November 11, 2024
Johan Unge was elected chair of the MicroED special interest group. He will transition to chair The Structural Science Society’s interest group in 2025.
Johan Hattne chair elect of ACA Best Practices special interest group
November 11, 2024
Johan Hattne is the new chair elect for the Best Practices special interest group of the ACA: The Structural Science Society. He is slated to chair the group next year.
Design and implementation of suspended drop crystallization
April 18, 2023
Cody Gillman, William J Nicolas, Michael W Martynowycz, Tamir Gonen
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.
Read the publication: Design and implementation of suspended drop crystallization
The structure of the neurotoxin palytoxin determined by MicroED
April 18, 2023
Cody Gillman, Khushboo Patel, Johan Unge, Tamir Gonen
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.
Read the publication: The structure of the neurotoxin palytoxin determined by MicroED
Protoglobin from Directed Evolution: MicroED Structure of a Reactive Carbene Intermediate
April 5, 2023
Emma Danelius , Nicholas J Porter , Johan Unge , Frances H Arnold , Tamir Gonen
Microcrystal electron diffraction (MicroED) is an emerging technique that has shown great potential for describing new chemical and biological molecular structures. Several important structures of small molecules, natural products, and peptides have been determined using ab initio methods. However, only a couple of novel protein structures have thus far been derived by MicroED. 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 α helix of ApePgb into a dynamic loop, making the catalytic active site more readily accessible. After exposing the tiny crystals to the substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineered ApePgb’s 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 the formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enable investigation of structures that was previously beyond reach.
Read the publication: MicroED Structure of a Protoglobin Reactive Carbene Intermediate
Plasma ion-beam milling is a powerful tool for preparing biological lamellae for MicroED data collection
February 25, 2023
Michael W Martynowycz , Anna Shiriaeva , Max T B Clabbers, William J Nicolas , Sara J Weaver, Johan Hattne , Tamir Gonen
Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae.
Read the publication: A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals
MicroED in drug discovery
February 21, 2023
Emma Danelius Khushboo Patel , Brenda Gonzalez , Tamir Gonen
The cryo-electron microscopy (cryo-EM) method microcrystal electron diffraction (MicroED) was initially described in 2013 and has recently gained attention as an emerging technique for research in drug discovery. As compared to other methods in structural biology, MicroED provides many advantages deriving from the use of nanocrystalline material for the investigations. Here, we review the recent advancements in the field of MicroED and show important examples of small molecule, peptide and protein structures that has contributed to the current development of this method as an important tool for drug discovery.
Read the publication: MicroED in drug discovery
Unlocking the potential of MICROCRYSTAL ELECTRON DIFFRACTION
June 30, 2022
Mike Martynowycz , Tamir Gonen
Atoms stick together in different ways to make the molecules that compose everything we touch and see. Our bodies are made of cells. Cells, in turn, are made of lipids, proteins, nucleic acids, metabolites, and water. Every one of those molecules is made from the same handful of atoms. But although the components are the same, the molecules differ in how many atoms they have and how those atoms are arranged in space.
Read the publication: Unlocking the potential of MICROCRYSTAL ELECTRON DIFFRACTION
While crystals for MicroED data collection needs to be thin, the window of ideal thickness is larger than might have been anticipated
December 7, 2021
Michael W Martynowycz, Max T B Clabbers, Johan Unge, Johan Hattne, Tamir Gonen
The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (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 and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae 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 data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.
Read the publication: Benchmarking the ideal sample thickness in cryo-EM
Ab initio phasing macromolecular structures using electron-counted MicroED data
October 18, 2021
CryoEM MicroED with electron counting. Protein structure at 0.8A resolution. Ab initio phasing for two proteins facilitated by electron counting on a falcon 4 direct electron detector.
Read the publication: Ab initio phasing macromolecular structures using electron-counted MicroED data
MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP
September 8, 2021
Michael W Martynowycz, Anna Shiriaeva, Xuanrui Ge, Johan Hattne, Brent L Nannenga, Vadim Cherezov, Tamir Gonen
Microcrystal electron diffraction (MicroED) is a cryogenic electron microscopy (cryo-EM) method that determines protein structures from submicron crystals. G protein–coupled receptors (GPCRs) are membrane proteins that are critically important drug targets. These proteins require crystallization in lipidic cubic phase (LCP), making standard MicroED approaches intractable for investigating these samples. Here, we show that GPCR microcrystals grown in an LCP can be made amenable for MicroED by converting the LCP to the sponge phase and then ion-beam milling the crystals into thin lamellae. Our findings provide the basis for solving GPCR structures using MicroED, with future applications in structure-based drug discovery.
Read the publication: MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP
MicroED structure of voltage-dependent anion channel
December 19, 2020
Michael W. Martynowycz, Farha Khan, Johan Hattne, Jeff Abramson, and Tamir Gonen
Microcrystal electron diffraction (MicroED) is an electron cryo-microscopy (cryo-EM) method for determining structures using submicron crystals. Until now, determining structures of membrane proteins by MicroED required that the protein crystals be in a solution amenable to standard cryo-EM blotting and vitrification protocols. Here, we show that membrane protein microcrystals grown in a viscous bicelle mixture can become amenable to MicroED analyses by using modified blotting procedures combined with focused ion-beam milling. Our findings provide a basis for solving membrane protein structures using crystals embedded in a viscous media by MicroED.
MicroED goes into Lipidic Cubic Phase
October 7, 2020
Zhu L, Bu G, Jing L, Shi D, Lee MY, Gonen T, Liu W, Nannenga BL.
One of expected areas where MicroED may have an outstanding role is for proteins and complexes where crystal growth tend to yield smaller crystals only. Membrane proteins puts many challenges to the crystallographer and often a two phase systems as Lipid Cubic Phase (LCP) is needed. The authors shows the feasibility of using LCP in the cryoEM setting required by MicroED
Read the publication: Structure Determination from Lipidic Cubic Phase Embedded Microcrystals