Microcrystal Electron Diffraction (MicroED) – Small Molecule & Macromolecules

Conference: 2020: 70th ACA Annual Meeting
08/02/2020: 12:00 PM  - 3:00 PM 
Oral Session 


Make MicroED an efficient tool for ultrahigh-resolution structural determination∠

12:00 PM - 12:20 PM 
Microcrystal electron diffraction (MicroED) is becoming a powerful tool in determining the crystal structures of biological macromolecules and small organic compounds. An advantage of electron diffraction is that its resolution is just weakly influenced by the performance of the objective lens. Therefore, even on a low-end electron microscope the electron diffraction can reach half angstrom resolution. By developing a stage-camera synchronization scheme to minimize the hardware requirements and enable the use of the conventional electron cryo-microscope with single-frame CCD camera, the acquisition of ultrahigh-resolution diffraction data is achieved on conventional microscopes, including the low-end 120kV microscopes. This method was demonstrated by the structure determination of both peptide and small organic compounds at ultrahigh resolution up to ~0.60 angstrom with unambiguous assignment of nearly all hydrogen atoms. Importantly, this work also demonstrates the capability of the low-end 120kV microscope with a CCD camera in solving ultra-high resolution structures of both organic compound and biological macromolecules. To develop a routinely-available method for high-quality MicroED sample, we used the focused ion beam (FIB) equipped on the scanning electron microscope (SEM) to mill a large crystal to thin lamella. The influences of the milling on the crystal lamella were observed and investigated, and used to optimize the FIB milling. FIB also makes MicroED available for crystals with large size. 

View Proposal 425


Xueming Li, Tsinghua University Beijing

Polymorph Evolution of Organic Molecules during Crystal Growth Studied by MicroED

12:20 PM - 12:40 PM 
Rapid, atomic resolution structure determination of nanometre-sized crystals of small organic molecules can be obtained using MicroED. In this work, we combined the capabilities of MicroED for the study of very small crystallites with [i]in situ[/i] crystal growth to follow the sequential polymorph evolution of glycine from an aqueous solution ([i]1[/i]). The three known polymorphs of glycine which exist under ambient conditions follow the stability order β < α < γ. Crystal growth in droplets of the glycine solution (3 μL) was stopped by solvent removal and plunge freezing after 3, 4 and 5 minutes. The ability to observe the crystallization process in time-slices of only one minute enabled the fast, dynamic process to be observed at shorter timescales than has previously been possible. The thermodynamically least stable β polymorph forms exclusively after 3 minutes, but this begins to yield to the α form after just one minute more. One crystallite of the γ form was observed after the drop was allowed to evaporate to dryness over the course of 1 hour. Using this gentle and non-invasive technique, we could rapidly identify and selectively determine the crystal structures of all three polymorphic forms from a single sample, despite their presence in vanishingly small quantities. In Zou's lab, we have developed serial electron diffraction (SerialED) to automate data collection by taking electron diffraction patterns of individual particles ([i]2[/i]). Diffraction data from up to 3500 crystals per hour can be collected on a standard TEM. We have also combined data collection by SerialED with rotation electron diffraction (RED) and developed SerialRED to perform fully automated data collection and data analysis for 3D electron diffraction ([i]3[/i]). This provides new possibilities for studying beam-sensitive crystals, quantitative phase analysis and detection of minor phases in the sample. These methods, along with [i]in situ[/i] crystal growth, can greatly accelerate polymorphic discovery and provide new possibilities to study reaction mixtures, dynamical processes and other applications where specific solid forms of materials are required. 1. E. Broadhurst, H. Xu, M. Clabbers, M. Lightowler, F. Nudelman, X. Zou, S.Parsons, [i]IUCrJ[/i] [b]7[/b], 5 (2020). 2. S. Smeets, X. Zou, W. Wan, [i]J. Appl. Cryst.[/i] [b]51[/b], 1262 (2018). 3. B. Wang, X. Zou, S. Smeets, [i]IUCrJ[/i] [b]6[/b], 854 (2019). 

View Proposal 356


Molly Lightowler, Stockholm University Stockholm

Additional Author(s)

Edward Broadhurst, The University of Edinburgh Edinburgh
Hongyi Xu, Department of Materials and Environmental Chemistry Stockholm
Max Clabbers, Stockholm University Djursholm
Fabio Nudelman, The University of Edinburgh Edinburgh, -- SELECT -- 
Xiaodong Zou, Department of Materials and Environmental Chemistry Stockholm, -- SELECT -- 
Simon Parsons, The University of Edinburgh Edinburgh, -- SELECT -- 

MicroED on lipidic cubic phase embedded crystals

12:40 PM - 1:00 PM 
The lipidic cubic phase (LCP) technique is a proven method to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. Because crystals grown in LCP can be limited in size, improved techniques for structure determination from these small crystals are important. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-TEM to collect electron diffraction data and determine high-resolution structures from very thin micro and nanocrystals. However, the viscosity of the LCP matrix makes MicroED sample preparation extremely difficult. In this presentation, we will discuss our results and recent progress on using modified LCP and MicroED protocols to analyze crystals embedded in LCP. 

View Proposal 373


Brent Nannenga, Arizona State University Tempe, AZ 

Coffee Break

1:00 PM - 1:20 PM 

MyD88 TIR domain signalosome interactions revealed by MicroED

1:20 PM - 1:40 PM 
Microcrystal electron diffraction (MicroED) has recently shown potential for structural biology, enabling structure determination from submicron-sized crystals of macromolecules and naturally self-assembled filaments of short peptide fragments, revealing the underlying mechanisms of interactions within assemblies present in cells. Here, we present the MicroED structure of the MyD88 TIR domain signalosome. Most Toll-like receptors (TLRs) and /interleukin-1 receptors (IL-1Rs) signal through the adaptors MAL and MyD88 to activate pro-inflammatory cytokines. It was previously observed that MAL induced the formation of thin crystalline arrays of MyD88TIR protofilaments with a diameter of typically 100 nm. These microcrystals were too small for conventional x-ray crystallography, yet are highly suitable for structure elucidation by electron diffraction. We used MicroED to solve the structure via molecular replacement, based on a distantly related TIR domain homologue with only 30% sequence identity. The electrostatic potential map at 3.0 Å resolution was of sufficient quality for accurate interpretation and rebuilding of the structural model. Several loop regions were remodelled and show conformations distinctly different from crystal structures of monomeric MyD88TIR. In parallel, the microcrystals were also characterized by serial X-ray free electron laser (XFEL) crystallography (2.3 Å resolution), confirming the interpretation of the model. Our results provide novel insights into TIR domain binding interactions that form the structural basis for signaling in the TLR and interleukin-1 receptor pathways. We anticipate MicroED to complement and extend existing methods in structural biology for a variety of crystalline samples that are too small for x-ray and neutron diffraction. 

View Proposal 276


Max Clabbers, Stockholm University Djursholm

MicroED for small molecule and natural product research

1:40 PM - 2:00 PM 
The electron cryo-microscopy (cryo-EM) method Microcrystal Electron Diffraction (MicroED) allows the collection of high-resolution structural data from vanishingly small crystals, typically with a thickness in the nanometer range. Since its debut in 2013, data collection and analysis schemes have been fine-tuned, and there are now over 100 structures determined by MicroED. Although originally developed to study proteins, MicroED is clearly also powerful for smaller systems, with some recent and very promising examples of important natural products and peptides as well as small molecule pharmaceuticals. As for any structural investigation by X-ray crystallography, deriving the structures of small molecules and natural products is often prohibited due to difficulties in obtaining the large and well-ordered crystals required. On the other hand, the crystals used for MicroED are about a billionth the size as compared to those used for X-ray diffraction which means that they are much more easily obtained, as demonstrated by the many novel and previously unattainable structures. In fact, in MicroED atomic resolution structures can rapidly be derived directly from powders, i.e. without any prior crystallization. In these cases, seemingly amorphous solids from silica gel chromatography or directly from a chemical supplier were used. In this seminar I will show recent examples of MicroED in small molecule research including determining the stereochemistry of natural products, as well as deriving the atomic structures of molecules directly from compounds mixtures, where the latter example is not possible with other structural methods. Given the unique features of MicroED, its potential impact is multifold, and the method is expected to continue to greatly influence the field of natural product and small molecule research. 

View Proposal 403


Emma Danelius, UCLA Los Angeles, CA 

Additional Author(s)

Johan Hattne, University of California, Los Angeles
Michael Martynowycz, HHMI/UCLA Los Angeles, CA 
Steve Halaby, UCLA Biological Chemistry Los Angeles, CA 
Tamir Gonen, HHMI/UCLA

Improved MicroED structures by cryo-FIB milling

2:00 PM - 2:20 PM 
Microcrystal electron diffraction is an electron cryo-microscopy method that determines crystal structures from vanishingly small crystals. The strong interaction between high energy electrons and matter limits the size of crystals to those typically thinner than perhaps 500 nanometers. Though some crystals will naturally form crystals small enough for MicroED, many will end up too large for MicroED and too small for synchrotron X-ray diffraction experiments. We demonstrate the combination of scanning electron microscopy (SEM) and cryogenic focused ion-beam (FIB) milling of protein microcrystals. Our approach results in ideally thick specimens with better crystallographic statistics, and opens MicroED to entirely new experiments. Here, we present the methods, rationale, and recent results using this approach. 

View Proposal 372


Michael Martynowycz, HHMI/UCLA Los Angeles, CA 

Small Molecule Microcrystal Electron Diffraction (MicroED) for the Pharmaceutical Industry

2:20 PM - 2:40 PM 
The emerging field of microcrystal electron diffraction (microED) is of great interest to the pharmaceutical industry. The promise of high-resolution structures without the need to grow large, single crystals makes this technique very attractive as it lowers sample requirements and in many cases eliminates the need for crystallization trials. Here we will present our experience collecting microED data from over 30 small molecule samples for clients across the pharmaceutical industry. We have adapted the TEM data acquisition software package Leginon to collect high-quality diffraction data in an automated manner. We have used this data acquisition pipeline coupled to our DIALS-based data reduction pipeline to solve over 20 small molecule crystal structures in-house. We will present some of the unique challenges posed by small molecule microED data processing and we will share our experience adapting tools built for processing protein diffraction data to particularly challenging cases. We hope to inspire conversation amongst developers of data processing packages to improve the tools available to those venturing into the world of electron diffraction. 

View Proposal 302


Jessica Bruhn, NanoImaging Services Oceanside, CA 

Additional Author(s)

Anchi Cheng, NanoImaging Services San Diego, CA 
Travis Nieusma, NanoImaging Services San Diego, CA 
Sargis Dallakyan, NanoImaging Services San Diego, CA 
Jeff Speir, NanoImaging Services San Diego, CA 
Anette Schneemann
Clint Potter, NanoImaging Services San Diego, CA 
Bridget Carragher, NanoImaging Services San Diego, CA 
Giovanna Scapin, NanoImaging Services San Diego, CA 

Microcrystal electron crystallographic analysis of organic solid solution (mixed crystal)

2:40 PM - 3:00 PM 
Solid solution (mixed crystal) is a crystal containing a second constituent (with a variable ratio) which fits into and is distributed in the lattice of the host crystal (IUPAC). It is widely found among inorganic substances, but rarely among organic compounds because it generally forms an amorphous solid or, at best, tiny crystals with a low degree of crystalline order. We examined the potential of microcrystal electron crystallography for the study of a solid solution of a racemic mixture (pseudoracemate) of a new molecule that we synthesized recently. Diffraction data of rather poor quality were collected for numerous crystal-like particles, and optimized for various parameter including the R(F) value. The datasets collected from approximately 20 particles each containing R and S isomers in different ratios afforded the molecular structure as well as the crystal packing of the specimen of interest. In this lecture, we will also touch on our recent analysis of a supramolecular organic polymer that defied X-ray analysis. Reference: Bull. Chem. Soc. Jpn., 93, 776–782 (2020). 

View Proposal 438


Takayuki Nakamuro

Additional Author(s)

Keitaro Yamashita, The University of Tokyo
Haruaki Yanagisawa, The University of Tokyo
Osamu Nureki, The University of Tokyo
Masahide Kikkawa, The University of Tokyo
Hiroyoshi Hamada, The University of Tokyo
Rui Shang, The University of Tokyo
Koji Harano, The University of Tokyo
Eiichi Nakamura, The University of Tokyo