Name: microcrystal electron diffraction of small molecules
Uploaded: 2021-03-15T15:00:00
Description: Watch this Scientific Journal Video about Microcrystal Electron Diffraction of Small Molecules at JoVE.com

The Gonen lab is supported by funds from the Howard Hughes Medical Institute. This study was supported by the National Institutes of Health P41GM136508.

1. Preparing small molecule samples
<ol>
	<li>Transfer a small amount (0.01 - 1 mg) of powder, liquid, or solids into a small vial or tube.</li>
	<li>For samples already in powder form, seal the tube using the cap until the sample is needed. Dry the liquid samples into powders prior to attempts at method 1 (step 3) or 2 (step 4). 
	&#8203;NOTE: Samples dissolved in liquid may use method 3 (5.X) below</li>
</ol>
2. Preparing TEM grids
NOTE: Some TEMs with autoloader...

<ol>
	<li>Shi, D., Nannenga, B. L., Iadanza, M. G., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+electron+crystallography+of+protein+microcrystals.">Three-dimensional electron crystallography of protein microcrystals.</a> eLife. 2, 01345 (2013).</li><li>Nannenga, B. L., Shi, D., Leslie, A. G. W., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+structure+determination+by+continuous-rotation+data+collection+in+MicroED.">High-resolution structure determination by continuous-rotation data collection in MicroED.</a> Nature Methods. 11 (9), 927-930 (2014).</li><li>Hattne, J., Martynowycz, M. W., Penczek, P. A., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroED+with+the+Falcon+III+direct+electron+detector.">MicroED with the Falcon III direct electron detector.</a> IUCrJ. 6 (5), 921-926 (2019).</li><li>Hattne, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroED+data+collection+and+processing.">MicroED data collection and processing.</a> Acta Crystallographica Section A Foundations and Advances. 71 (4), 353-360 (2015).</li><li>Henderson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+and+limitations+of+neutrons,+electrons+and+X-rays+for+atomic+resolution+microscopy+of+unstained+biological+molecules.">The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules.</a> Quarterly Reviews of Biophysics. 28 (2), 171-193 (1995).</li><li>Martynowycz, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroED+structure+of+the+human+adenosine+receptor+determined+from+a+single+nanocrystal+in+LCP.">MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP.</a> BioRxiv. , 316109 (2020).</li><li>Martynowycz, M. W., Zhao, W., Hattne, J., Jensen, G. J., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection+of+continuous+rotation+MicroED+data+from+ion+beam-milled+crystals+of+any+size.">Collection of continuous rotation MicroED data from ion beam-milled crystals of any size.</a> Structure. 27 (3), 545-548 (2019).</li><li>Martynowycz, M. W., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+incorporation+into+protein+microcrystals+for+MicroED+by+on-grid+soaking.">Ligand incorporation into protein microcrystals for MicroED by on-grid soaking.</a> Structure. , (2020).</li><li>Martynowycz, M. W., Khan, F., Hattne, J., Abramson, J., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroED+structure+of+lipid-embedded+mammalian+mitochondrial+voltage-dependent+anion+channel.">MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel.</a> Proceedings of the National Academy of Sciences. 117 (51), 32380-32385 (2020).</li><li>Jones, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CryoEM+method+MicroED+as+a+powerful+tool+for+small+molecule+structure+determination.">The CryoEM method MicroED as a powerful tool for small molecule structure determination.</a> ACS Central Science. 4 (11), 1587-1592 (2018).</li><li>Dick, M., Sarai, N. S., Martynowycz, M. W., Gonen, T., Arnold, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+tryptophan+synthase+TrpB+for+selective+quaternary+carbon+bond+formation.">Tailoring tryptophan synthase TrpB for selective quaternary carbon bond formation.</a> Journal of the American Chemical Society. 141 (50), 19817-19822 (2019).</li><li>Gallagher-Jones, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-&#229;ngstr&#246;m+cryo-EM+structure+of+a+prion+protofibril+reveals+a+polar+clasp.">Sub-&#229;ngstr&#246;m cryo-EM structure of a prion protofibril reveals a polar clasp.</a> Nature Structural &amp; Molecular Biology. 25 (2), 131-134 (2018).</li><li>Ting, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+scaffold+peptide+in+the+biosynthesis+of+amino+acid-derived+natural+products.">Use of a scaffold peptide in the biosynthesis of amino acid-derived natural products.</a> Science. 365 (6450), 280-284 (2019).</li><li>de la Cruz, M. J., Martynowycz, M. W., Hattne, J., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroED+data+collection+with+SerialEM.">MicroED data collection with SerialEM.</a> Ultramicroscopy. 201, 77-80 (2019).</li><li>Mastronarde, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+electron+microscope+tomography+using+robust+prediction+of+specimen+movements.">Automated electron microscope tomography using robust prediction of specimen movements.</a> Journal of Structural Biology. 152 (1), 36-51 (2005).</li><li>Schorb, M., Haberbosch, I., Hagen, W. J. H., Schwab, Y., Mastronarde, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Software+tools+for+automated+transmission+electron+microscopy.">Software tools for automated transmission electron microscopy.</a> Nature Methods. 16 (6), 471-477 (2019).</li><li>Kabsch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XDS.">XDS.</a> Acta Crystallographica Section D Biological Crystallography. 66 (2), 125-132 (2010).</li><li>Winter, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DIALS:+Implementation+and+evaluation+of+a+new+integration+package.">DIALS: Implementation and evaluation of a new integration package.</a> Acta Crystallographica Section D. 74 (2), 85-97 (2018).</li><li>de la Cruz, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic-resolution+structures+from+fragmented+protein+crystals+with+the+cryoEM+method+MicroED.">Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.</a> Nature Methods. 14 (4), 399-402 (2017).</li><li>Shi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+collection+of+MicroED+data+for+macromolecular+crystallography.">The collection of MicroED data for macromolecular crystallography.</a> Nature Protocols. 11 (5), 895-904 (2016).</li><li>Nannenga, B. L., Shi, D., Hattne, J., Reyes, F. E., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+catalase+determined+by+MicroED.">Structure of catalase determined by MicroED.</a> eLife. 3, 03600 (2014).</li><li>Martynowycz, M. W., Zhao, W., Hattne, J., Jensen, G. J., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+Analyses+of+Polishing+and+Precoating+FIB+Milled+Crystals+for+MicroED.">Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED.</a> Structure. 27 (10), 1594-1600 (2019).</li></ol>

Sample preparation is typically an iterative process, where optimizations are made after sessions of screening and data collection. For small-molecule samples, it is often prudent to first attempt grid preparation without glow-discharging the grids, since many pharmaceuticals tend to be hydrophobic10,11. If the grids have too few nanocrystalline deposits, it is a good idea to try again after first glow-discharging the grids. It may be the case that the crystals f...

Microcrystal electron diffraction (MicroED) is an electron cryo-microscopy (cryo-EM) method for determining atomic resolution structures from sub-micrometer sized crystals1,2. Crystals are applied to standard transmission electron microscope (TEM) grids and frozen by either plunging into liquid ethane or liquid nitrogen. Grids are then loaded into a TEM operating at cryogenic temperatures. Crystals are located on the grid and screened for initial diffraction quality. Continuous rotation MicroED data are collected from a subset of the screened crystals, where the data are saved using a fast camera as a movie3. These movies are converted to a standard crystallographic format and processed almost identically as an X-ray crystallography experiment4.
MicroED was originally developed to investigate protein microcrystals1,2. A bottleneck in protein crystallography is growing large, well-ordered crystals for traditional synchrotron X-ray diffraction experiments. As electrons interact with matter orders of magnitude stronger than X-rays, the limitations of the crystal size needed to produce detectable diffraction is considerably smaller5. Additionally, the ratio of elastic to inelastic scattering events is more favorable for electrons, suggesting that more useful data can be collected with a smaller overall exposure5. Constant developments have allowed for MicroED data to be collected from the most challenging microcrystals6,7,8,9.
Recently, MicroED has been shown to be a powerful tool for determining the structures of small molecule pharmaceuticals from apparently amorphous materials10,11,12,13. These powders can come straight from a bottle of purchased reagent, a purification column, or even from crushing a pill into a fine powder10. These powders appear amorphous by eye, but may be either entirely composed of nanocrystals or merely contain trace amounts of nanocrystalline deposits in a greater non-crystalline, amorphous fraction. Application of the material to the grid is facile, and the subsequent steps of crystal identification, screening, and data collection might even be automated in the near future14. While others may use different methods for sample preparation and data collection, here the protocols developed and used in the Gonen laboratory for preparing samples of small molecules for MicroED and for data collection are detailed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1-1.5mL Eppendorf tubes</td>
			<td>Fisher Scientific</td>
			<td>14-282-300</td>
			<td>Any vial or tube will do.</td>
		</tr>
		<tr>
			<td>Autogrid clips</td>
			<td>Thermo-Fisher</td>
			<td>1036173</td>
			<td>Clipped grids are not required for MicroED. They are required for Thermo-Fisher TEMs equipped with an autoloader system.</td>
		</tr>
		<tr>
			<td>Autogrid C-rings</td>
			<td>Thermo-Fisher</td>
			<td>1036171</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbamazapine</td>
			<td>Sigma</td>
			<td>C4024-1G</td>
			<td>Any amount will suffice for these experiments</td>
		</tr>
		<tr>
			<td>CMOS based detector</td>
			<td>Thermo-Fisher</td>
			<td>CetaD 16M</td>
			<td>We used a CetaD 16M, but any detector with rolling shutter mode or sufficiently fast readout is acceptable.&#160;</td>
		</tr>
		<tr>
			<td>Delphi software</td>
			<td>Thermo-Fisher</td>
			<td>N/A</td>
			<td>Software on Thermo-Fisher TEM systems that allows for manual rotation of the sample stage</td>
		</tr>
		<tr>
			<td>EPU-D software</td>
			<td>Thermo-Fisher</td>
			<td>N/A</td>
			<td>Commercial software for the acquisition of MicroED data</td>
		</tr>
		<tr>
			<td>Glass cover slides</td>
			<td>Hampton</td>
			<td>HR3-231</td>
			<td></td>
		</tr>
		<tr>
			<td>Glow discharger</td>
			<td>Pelco</td>
			<td>easiGlow</td>
			<td></td>
		</tr>
		<tr>
			<td>High PrecisionTweezers</td>
			<td>EMS</td>
			<td>78325-AC</td>
			<td>Any high precision tweezer will do</td>
		</tr>
		<tr>
			<td>Liquid nitrogen vessel</td>
			<td>Spear Lab</td>
			<td>FD-800</td>
			<td>A standard foam vessel for handling specimens under liquid nitrogen - 800mL</td>
		</tr>
		<tr>
			<td>SerialEM software</td>
			<td>UC Boulder</td>
			<td>N/A</td>
			<td>Free software distributed by D. Mastronarde. Department of Molecular, Cellular, and Developmental Biology</td>
		</tr>
		<tr>
			<td>TEM grids</td>
			<td>Quantifoil/EMS</td>
			<td>Q310CMA</td>
			<td>Multi-A 300 mesh grids were used here, but any thin carbon grids will work. For these small molecules, we suggest starting with continuous carbon.&#160;</td>
		</tr>
		<tr>
			<td>transmission electron microscope (TEM)</td>
			<td>Thermo-Fisher</td>
			<td>Talos Arctica</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman circular filter paper</td>
			<td>Millipore-Sigma</td>
			<td>WHA1001090</td>
			<td>90mm or larger</td>
		</tr>
	</tbody>
</table>

microcrystal electron diffraction of small molecules

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.

MicroED is a cryoEM method that leverages the strong interactions between electrons and matter, which allows for the investigation of vanishingly small crystals12,13. After these steps, it is expected to have a diffraction movie in crystallographic format collected from microcrystals (Movie 1). Here, the technique is demonstrated using carbamazepine12. The results show a continuous rotation MicroED dataset from a carbamaze...

Watch this Scientific Journal Video about Microcrystal Electron Diffraction of Small Molecules at JoVE.com

Microcrystal Electron Diffraction of Small Molecules

Here, we describe the procedures developed in our laboratory for preparing powders of small molecule crystals for microcrystal electron diffraction (MicroED) experiments.

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been ...

In this procedure, we show how to proceed from a small molecule powder to a three-dimensional structure in minutes. This is a transformative technique for the chemistry community. Many compounds do not easily grow into large crystals.With MicroED, we can determine atomic resolution structures from nanometer size crystals. Demonstrating the procedure will be Dr.Mike Martynowycz who is a professor at UCLA. To begin, transfer a small amount of powder, liquid or solids into a small vial or tube.For samples already in powder form, seal the tube using the cap until the sample is needed. Similarly, dry the liquid samples into powders. Wrap plastic film around one end of a glass cover slide.Place the TEM grids onto the film on top of the cover slide with the brown color carbon side facing up. Then, place the slide with grids into the glow discharge chamber and glow discharge the cover slide for approximately 30 seconds using the negative setting at 15 picoamperes. Store the grids on the cover slide inside a glass Petri dish lined with filter paper prior to adding the sample to the grids.Remove the glow discharge TEM grid from the covered Petri dish using tweezers and place it onto a circular filter paper with the carbon side facing up. Using a small spatula, remove a very small scoop of powder and place it onto a small square glass cover slip just next to the TEM grid on the filter paper. Place another small square glass slide or cover slip on top of the powder.With fingers, gently rubbed the two glass slides together to make a fine powder. Angle the cover slips and position them just above the TEM grid on the filter paper and continue to rub the cover slips together just a few centimeters above the glow discharged TEM grid. Observe if the powder is falling toward the grid.Uncover the finely ground powder by removing one of the two glass cover slips. Then gently brush the fine powder off from the cover slip onto the TEM grid using a piece of filter paper. Grab the edge of the TEM grid using a set set of tweezers, assuring that the tips do not puncture any of the grid squares.Lift the grid one to two centimeters above the filter paper and angle the grid at 90 degrees to the paper below. Gently tap the tweezers while keeping the grid firmly tweezed to remove any loose powder. Freeze the grid by moving the tip of the tweezers with the grid directly into a liquid nitrogen container by hand and wait until the grid and tweezers stop boiling.Under liquid nitrogen, place the grid in the sample holder with the carbon side oriented such that the sample will be hit by the beam prior to the carbon support film. Load the sample holder into the TEM assuring that the grid is always kept at liquid nitrogen temperatures. For auto loader systems, place the clipped grids into a cassette in a liquid nitrogen cooled container, which allows the auto loader robotics to accept the cassette while keeping the samples safe for shuttling between the auto loader and the column.Open the TEM column valves and adjust the magnification using the hand panels to the lowest magnification possible. Find the beam by adjusting the intensity knob on the hand panels such that a round bright area is visible on the fluorescent screen. Take an all grid atlas at low magnification using appropriate software ensuring that the microscope is well aligned for both low and high magnification imaging prior to collecting high resolution MicroED data.Identify grid squares without broken carbon and visible black or dark grains on the film. Navigate around the grid either physically using the joystick on the hand panels or virtually on the collected atlas to search for grid squares that are not broken and contain micro crystals. Add the center of each of these squares to a list of grid locations for investigation, which can be added to a notebook in the microscope user interface or in the microscope automation software.Set the magnification between 500 to 1300x. Adjust the centric height at each stored grid location and update the saved z-value to the positions noted. Search either on the fluorescent screen or on a flat camera for small black spots or grains on the grid.A good sample will often have sharp edges at high magnification suggesting crystal in order. Move a located potential crystal to the center of the screen and increase the magnification such that the TEM enters high magnification mode. Insert a selected area aperture and change the aperture to a larger or smaller size to ensure that the selected area is larger than the crystal.Switch to defraction mode by pressing the defraction button on the TEM hand panels assuring that the fluorescent screen is inserted. Adjust the camera length using the magnification knob such that the edge is at least one angstrom resolution. Adjust the diffraction focus such that the central spot is as sharp and small as possible.Using the diffraction shift knobs, move the central beam to the center of the fluorescent screen. Insert the beam stop making sure the beam is behind it. Lift the fluorescent screen and take a short exposure on the camera.Inspect the corresponding defraction pattern, making sure that the pattern will have sharp spots that are regularly arranged in columns and rows. Save the crystal coordinates in either the TEM user interface or by writing them down, then repeat diffraction screening for all the potential crystals of interest on the current grid square. Center the screened crystal at a magnification greater than 1000x and adjust the centric height of the crystal using either an automatic routine or by hand.Insert the selected area aperture that best fits the crystal size and shape. Tilt the stage in the negative and positive directions until the image is occluded by the grid bars. And note these angles for data collection purposes by reading the data out every one second on a modern camera with a rolling shutter readout mode.Set the rotation rate by specifying the percent of the maximum tilt speed and when to stop in the microscope user interface or dedicated software. Save the data in a variety of formats says either individual frames or as a stack of images. A defraction movie in crystallographic format collected from micro crystals shows a continuous rotation MicroED dataset from a carbamazepine micro crystal identified on a TEM grid.After data collection, integration, and structure solution, a high resolution structure is determined and its clarity depends on the quality and completeness of the data. Applying the sample to the grid is critical for acquiring good data. Following this method, the data can be further processed using standard crystallographic software.Demonstrating that MicroED is a powerful method for small molecules has opened its use worldwide for solving new structures of critically important molecules.

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