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

Christopher G. Jones,

†

#

Michael W. Martynowycz,

‡

#

Johan Hattne,

‡

Tyler J. Fulton,

§

Brian M. Stoltz,

*

§

Jose A. Rodriguez,

*

†

∥

Hosea M. Nelson,

*

†

and Tamir Gonen

*

‡

†

Department of Chemistry and Biochemistry,

‡

Howard Hughes Medical Institute, David Ge

en School of Medicine, Departments of

Biological Chemistry and Physiology, and

∥

UCLA-DOE Institute, University of California, Los Angeles, California 90095, United

States

§

The Warren and Katharine Schlinger Laboratory of Chemistry and Chemical Engineering, California Institute of Technology,

Pasadena, California 91125, United States

*

Supporting Information

ABSTRACT:

In the many scienti

c endeavors that are driven

by organic chemistry, unambiguous identi

cation of small

molecules is of paramount importance. Over the past 50 years,

NMR and other powerful spectroscopic techniques have been

developed to address this challenge. While almost all of these

techniques rely on inference of connectivity, the unambiguous

determination of a small molecule

’

s structure requires X-ray

and/or neutron di

raction studies. In practice, however, X-ray

crystallography is rarely applied in routine organic chemistry

due to intrinsic limitations of both the analytes and the

technique. Here we report the use of the electron cryo-microscopy (cryoEM) method microcrystal electron di

raction

(MicroED) to provide routine and unambiguous structural determination of small organic molecules. From simple powders,

with minimal sample preparation, we could collect high-quality MicroED data from nanocrystals (

∼

100 nm,

∼

−

g) resulting

in atomic resolution (<1 Å) crystal structures in minutes.

he history of organic chemistry closely parallels the

development of new methods for structural character-

ization. The earliest studies were driven by melting point

determination, and over the past 175 years more complex

methods for interrogation of structure have been developed.

Techniques such as polarimetry,

−

vis,

and infrared

spectroscopy,

coupled with electron paramagnetic resonance,

vibrational circular dichroism,

circular dichroism,

and mass

spectrometry

have been commonly employed over the years,

dramatically expanding our ability to assign structures. In the

past 50 years, however, the explosion of NMR spectroscopy

and the accompanying abun

dance of individual NMR

experiments have produced a wealth of detailed structural

information for organic chemists. Indeed, NMR is a mainstay

in chemistry and the most predominant method employed in

both routine synthetic chemistry experiments and in advanced

structural elucidation of complex small molecules. In the

current state of the art, only single crystal X-ray di

raction

holds a higher place in terms of precision, producing

unequivocal structural infor

mation about the position,

orientation, connectivity, and placement of individual atoms

and bonds within a given molecule.

For decades, small molecule X-ray analysis has been the

nitive tool for structural analysis.

This technique, however,

is not without limitations. The process is considered by many

art

, where the production of high-quality crystals suitable

for X-ray di

raction requires uncodi

“

tricks of the trade

”

well as a certain amount of luck! Additionally, even after a

substance has been successfully crystallized, there is no

guarantee that the particular crystal form will be amenable to

X-ray di

raction. Since crystal growth is both a slow and

arduous process, X-ray di

raction has not been an e

ective

tool for rapid, on-the-

y structural determination of small

molecules. For this reason, X-ray di

raction is generally not

implemented as a routine analytical tool for the practicing

organic chemist, despite the fact that the structural data

provided are far superior to any other characterization method

to date.

■

RESULTS AND DISCUSSION

Herein, we employ the recently developed electron cryo-

microscopy (CryoEM) method microcrystal electron di

rac-

tion (MicroED)

to address the long-standing need for fast

and reliable structure determination in organic chemistry.

Recently, electron di

raction was used to solve the structure of

a methylene blue derivative, although no scope studies were

undertaken to allow the reader to assess the applicability of the

methodology.

Moreover, a specialized detector was used for

their experiments, limiting the broad adaptability of their

Received:

October 17, 2018

Published:

November 2, 2018

Research Article

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approach to the wider synthetic community.

We demonstrate

that with minimal sample preparation and experiment time,

simple powders and seemingly amorphous materials (in some

cases, solids isolated via silica gel chromatography and rotary

evaporation) could be directly used in MicroED studies,

leading to rapid, high-quality structural elucidation of several

classes of complex molecules with atomic resolution, in many

cases better than 1 Å. Moreover, we utilize a commercially

available microscope that is already in use at universities

around the world. MicroED has the potential to dramatically

accelerate and impact the

elds of synthetic chemistry, natural

product chemistry, drug discovery, and many others by

delivering rapid, high-resolution atomic structures of complex,

small molecules with minimal sample preparation or formal

crystallization procedures.

The applicability of MicroED was initially tested on the

naturally occurring steroid progesterone (

) as a model system

(

Figure 1

). The sample was obtained as a powder from

chemical supplier Preparations Laboratories Inc. (we estimate

the bottle to be more than 20 years old). Small quantities of

the seemingly amorphous solid were transferred directly from

the bottle onto glass cover slides and ground between another

slide to produce a

ne powder. The powder was then

deposited on a holey carbon

−

copper grid, cooled to liquid

nitrogen temperatures, and transferred to a cryoelectron

microscope operating at an acceleration voltage of 200 kV

(Thermo Fisher Talos Arctica). An overview of the preparation

is shown in

Figure 1

. Upon imaging, thousands of nanocrystals

were easily discernible on the grid surface providing ample

nanocrystals to investigate for di

raction. Typically, for

samples such as these, the vast majority of nanocrystals

racted to

∼

1 Å resolution or better (

Figure 1

). Through

continuous rotation of the sample stage, 140 degrees of

raction data could be collected from a single nanocrystal,

while the improved autoloader and piezo stage of the Talos

Arctica allowed us to travel through the zero degree point

without introducing errors in crystal position. Typically, the

stage was rotated at approximately 0.6 deg/s, and an entire

data set was collected in less than 3 min as a movie (

Video 1

)

using a bottom mount CetaD CMOS detector (Thermo

Fisher)

tted with a thick scintillator for di

raction studies.

Software adapted from previous studies

was used to convert

the di

raction movie frames into SMV format for expeditious

processing in the readily available XDS software package

commonly used for X-ray crystallography.

By collecting data

from just a single nanocrystal, the structure of steroid

was

resolved to an impressive 1 Å resolution. The entire process,

from bottle to structure, was easily accomplished in less than

30 min.

Encouraged by these results, we wanted to investigate a wide

range of natural products to fully explore the scope and

applicability of this powerful structural determination method

for small molecules (

Figure 2

A). The Talos Arctica was

particularly amenable to our studies as it is capable of storing

up to 12 di

erent grids at once, providing e

ortless swapping

of sample grids for rapid investigation of multiple compounds.

Once a reliable sample prep routine had been established,

over-the-counter medications were purchased from local

pharmacies for investigation. Tablets of CVS-branded acet-

aminophen and Kroger-branded ibuprofen were crushed using

a mortar and pestle, and the ground powder was placed on

electron microscopy grids as described above. Despite the

heterogeneity of such pharmaceuticals, which typically include

a multitude of coatings, binders, and other formulation agents,

we were astonished to obtain such clearly resolved atomic

resolution structures of both acetaminophen (

) and ibuprofen

(

) in rapid succession. Just as impressively, structures of the

sodium channel blocker carbamazepine (

) and the macro-

cyclic polypeptide antibiotic thiostrepton (

) were also

determined from seemingly amorphous powders used as

received from Sigma-Aldrich. We went on to further study

several commercially available natural products and derivatives.

Once again, compounds were used as received, without any

crystallization, to yield atomic resolution structures of biotin

(

), ethisterone (

), cinchonine (

), and brucine (

). Of the

11 di

erent commercial bioactives examined, all 11 yielded

processable MicroED data. Of those 11 compounds, 10 were

amenable to rapid structure determination by direct

methods,

while one was determined by molecular replace-

ment.

As mentioned previously, all structures were obtained

without any crystallization attempts or chemical modi

cations

to compounds examined. While these pharmaceutical and

commercial natural products were likely recrystallized for

puri

cation purposes by the manufacturer, the powders

examined by MicroED possessed nanocrystals a billionth the

size (

∼

100 nm) of crystals typically needed for X-ray

crystallography. This was powder to structure.

Figure 1.

Process of applying MicroED to small molecule structural analysis. Here commercial progesterone (

) was analyzed, and an atomic

resolution structure was determined at 1 Å resolution. Grid holes are 1

μ

m in diameter.

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Next we decided to investigate compounds that were never

crystallized, but instead were puri

ed by

ash column

chromatography. Since silica gel chromatography is the most

common method of puri

cation in early stage research for

complex molecules in drug discovery, natural product isolation

orts, and synthesis e

orts in general, we were interested to

see whether solid samples prepared in such a way would be

amenable to analysis by MicroED. Four compounds, puri

by chromatographic methods, were collected from our

laboratories, and samples of these seemingly amorphous solids

were analyzed. Here, two of four compounds di

racted,

yielding atomic resolution structures at or below 1 Å (

and

Figure 2

A,B). While the success rate for these compounds

was 50%, it is worthy to note that no crystallization procedures

were employed in the isolation of these materials. Notably,

(+)-limaspermidine (

), an alkaloid natural product synthe-

sized by our laboratories,

was resolved from a residue of only

milligram quantities of material following

ash chromatog-

Figure 2.

erent types of small molecules solved by MicroED. (A) Several pharmaceuticals, vitamins, commercial natural products, and synthetic

samples resolved through MicroED. (B) Example of an amorphous

lm utilized in this study leading to 1 Å resolution data. (C) Protons could be

observed for several compounds through MicroED. Green density are

−

maps showing positive density belonging to hydrogen atoms of the

molecule.

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raphy and rotary evaporation from a scintillation vial (

Figure

B). Furthermore, while it is extremely challenging to observe

protons in X-ray structures, electrons interact with matter more

strongly than X-rays and are a

ected by charge, making them

relatively common to observe in MicroED data.

−

For all

structures resolved from our samples, at least some, if not

most, protons could be observed on the molecule. In

particular, the density maps obtained for limaspermidine

(

) and carbamazepine (

) after re

nement showed protons

associated with almost all atoms in these molecules (

Figure

C).

Astounded by the ease with which such high-quality data

were obtained and the apparent generality of MicroED to small

molecules, we undertook studies to examine heterogeneous

samples (mixtures of compounds). In the case of heteroge-

neous samples, single crystal X-ray di

raction precludes the

study of mixtures, and NMR is poorly suited for this task. For

this experiment, mixtures of four compounds (

and

, cf.

Figure 3

) were crushed together and deposited on a holey

copper

−

carbon. Several crystal forms belonging to the

erent compounds in the mixture were visually identi

on the grids (

Figure 3

). MicroED data were collected from

several nanocrystals, and the identity of each species was

resolved within minutes by con

rmation of unit cell

parameters. After compound identi

cation, atomic resolution

structures could be rapidly determined for all small molecules

present in the mixture (

Figure 3

). (Note: no unexpected or

unusually high safety hazards were encountered.)

The results described here introduce a powerful new

characterization tool into the organic chemist

’

s toolbox.

While MicroED was initially developed for structure

determination of biological materials such as proteins in a

frozen, hydrated state,

we demonstrate that cross

pollination of macromolecular structural methods of CryoEM

are powerful tools for chemical synthesis, drug character-

ization, and drug discovery. Prior to this work, MicroED has

allowed for the structural characterization of several proteins

from crystals which had generally been unsuitable for X-ray

crystallography due to their small size or morphologies.

−

Despite this success, the MicroED method has largely gone

unnoticed in the small molecule communities. On the basis of

our

ndings, we anticipate that MicroED will be enthusiasti-

cally received by many types of small molecule chemists in

both academia and industry. Here we have shown that a variety

of seemingly amorphous solid materials can lead to rapid

atomic resolution structure determination by MicroED with

little or no additional sample preparation or crystallization.

The fact that a solid

lm in a

ask, following solvent removal

from a

ash chromatography puri

cation, can lead to an

atomic resolution molecular structure, is evidence that

MicroED will likely have a profound e

ect on the structural

characterization work-

ow of organic chemists. Although the

past 50 years have seen huge advances in the state of the art,

no completely new techniques have been introduced that alter

the routine structural interrogation of organic substances.

NMR,

IR,

−

vis,

and X-ray di

raction

have been

routinely in place since the 1960s and are still utilized today

as the most common methods for structure determination in

chemistry. We believe that electron di

raction is potentially

the next big advance in the

eld and are enthusiastic about the

prospects of expanding its utility as a routine analytical

technique for chemists.

■

POST PREPRINT ADDENDUM AND BACKGROUND

MicroED was developed for the determination of atomic

resolution protein structures from submicron thick, frozen-

hydrated crystals that are typically too small for X-ray

raction.

In MicroED, crystals are illuminated by an

extremely low dose (typically <0.01 e

−

/Å

/s) electron beam,

while the crystals are continuously rotated and di

raction is

collected on a fast camera as a movie.

MicroED data are then

processed using broadly available software for X-ray crystallog-

raphy without the need for specialized software for structure

analysis and re

nement.

Figure 3.

Identi

cation of compounds from heterogeneous mixtures. An EM grid was prepared as above with biotin, brucine, carbamazepine, and

cinchonine powders mixed together. All four compounds identi

ed by unit cell parameters using MicroED data from within the same grid square.

All structures were solved to

∼

1 Å resolution. Grid holes are 2

μ

m in diameter.

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Other electron di

raction methods include automated

raction tomography

(ADT) and rotation electron

raction

(RED). These methods di

er from MicroED in

the sampling of di

raction space, through tilt series and/or

precession. The broad applicability of MicroED has been

demonstrated since its conception

as structures of large

globular proteins,

small proteins,

peptides,

membrane

proteins,

and inorganic compounds

have been successfully

determined. In many of these examples, hydrogens were

observed and reported with the

rst example in 2015

followed by an independent report from Palatinus and co

workers two years later.

Most recently, the structure of a

small organic molecule, carbamezapine, was determined by

MicroED to sub-Angstrom resolution.

■

ASSOCIATED CONTENT

*

Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website

at DOI:

10.1021/acscents-

ci.8b00760

Movie of continuous rotation MicroED data from a

carbamazepine nanocrystal with corresponding resolu-

tion rings (

AVI

)

Procedures, chemical suppliers, and crystallographic data

(

PDF

)

Accession Codes

Data and materials availability: MicroED density maps have

been deposited to the PDB (thiostrepton, 6MXF), EMDB

(EMD-9282, EMD-9284, EMD-9285, EMD-9286, EMD-9287,

EMD-9288, EMD-9289, EMD-9290, EMD-9291, EMD-9292),

and CCDC (1876036, 1876037, 1876038, 1876039, 1876040,

1876041, 1876042, 1876043, 1876044, 1876045).

■

AUTHOR INFORMATION

Corresponding Authors

*

(B.M.S.) E-mail:

stoltz@caltech.edu

*

(J.A.R.) E-mail:

jrodriguez@chem.ucla.edu

*

(H.M.N.) E-mail:

hosea@chem.ucla.edu

*

(T.G.) E-mail:

tgonen@ucla.edu

ORCID

Brian M. Stoltz:

0000-0001-9837-1528

Author Contributions

#

C.G.J. and M.W.M. contributed equally. C.G.J. performed

experiments, developed the sample preparation techniques,

ned structural data, prepared

gures, and assisted with

manuscript preparation. M.

W.M performed experiments,

developed the sample preparation techniques, collected data,

ned structural data, prepared

gures, and assisted with

manuscript preparation. T.F. performed experiments. J.H.

wrote the software for image conversion, participated in data

analysis, re

nement, and structure determination. J.A.R.

performed experiments, developed the sample preparation

techniques, and assisted with manuscript preparation. B.M.S.

conceived of the project, designed experiments, and assisted

with manuscript preparation. H.M.N. conceived of the project,

designed experiments, performed experiments, developed the

sample preparation techniques, and assisted with manuscript

and

gure preparation. T.G. performed experiments, collected

data, developed the sample p

rep techniques, developed

microscope data collection parameters, provided the micro-

scope and expertise, and assisted in manuscript and

gure

preparation.

Funding

C.G.J. would like to acknowledge the National Science

Foundation for a predoctoral fellowship (DGE-1650604).

B.M.S. acknowledges the NIH-NIGMS for generous funding

(R01GM080269). J.A.R. is supported by DOE Grant DE-

FC02-02ER63421, NIH-NIGMS Grant R35GM128867, and

as a Beckman Young Investigator, a Searle Scholar, and a Pew

Scholar. H.M.N. thanks The Packard Foundation, The Sloan

Foundation, Pew Charitable Trusts, and the NIH-NIGMS

(R35 GM128936) for generous funding. The Gonen

laboratory is supported by funds from the Howard Hughes

Medical Institute.

Notes

The authors declare no competing

nancial interest.

■

ACKNOWLEDGMENTS

We thank Profesor Doug Rees (Caltech), Professor Bil

Clemons (Caltech), and Dr. Michael Sawaya (UCLA) for

useful discussions. We thank Byungkuk Yoo and Michael

Takase (Caltech) for technical assistance with data analysis.

We thank Beau Pritchett and Hendrik Klare for providing

synthetic samples.

■

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