1
Title:
The CryoEM method MicroED as a powerful tool for small molecule structure
determination
Authors:
Christopher G. Jones
1†
, Michael W. Martynowycz
2†
, Johan Hattne
2
, Tyler Fulton
3
,
Brian M. Stoltz
3$
, Jose A. Rodriguez
1$
, Hosea M. Nelson
1$
, Tamir Gonen
2$
Affiliations:
5
1
Department of Chemistry and Biochemistry, University of California, Los Angeles, 90095,
USA
2
Howard Hughes Medical Institute, Departments of Biological Chemistry and Physiology,
University of California, Los Angeles, 90095, USA
3
The Warren and Katharine Schlinger Laboratory of Chemistry and Chemical Engineering,
10
California Institute of Technology, Pasadena, 91125, USA
$
Correspondence to: stoltz@caltech.edu, jrodriguez@chem.ucla.edu, hosea@chem.ucla.edu,
tgonen@ucla.edu
15
†These authors contributed equally.
Abstract:
In the many scientific endeavors that are driven by organic chemistry, unambiguous
identification 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
20
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 diffraction 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 CryoEM method
MicroED to provide routine and unambiguous structural determination of small organic
25
molecules. From simple powders, with minimal sample preparation
, we
could collect high
quality MicroED data from nanocrystals (~100x100x100 nm, ~10
–15
g) resulting in atomic
resolution (<1 Å) crystal structures in minutes.
Main Text:
The history of organic chemistry clos
ely parallels the development of new methods
for structural characterization. The earliest studies were driven by melting point determination
30
and over the past 175 years more complex methods for interrogation of structure have been
developed. Techniques such as polarimetry
(
1
), UV-vis
(
2
), and infrared spectroscopy
(
3
),
coupled with EPR
(
4
), VCD
(
5
), CD
(
6
), and mass spectrometry(
7
) 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
(
8
) and the accompanying abundance of
35
individual NMR experiments have produced a wealth of structural information. Indeed, NMR is
a mainstay in chemistry and the most predominant method employed in both routine sy
nthetic
chemistry experiments and in advanced structural elucidation of complex small molecules. In
the current state of the art, only single crystal
X-ray diffraction holds a higher place in terms of
2
precision, producing unequivocal structural informati
on 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 definitive tool for structural
analysis
(
9
). This technique, however, is not wit
hout limitations. The process is considered by
many an
art,
where the production of high quality crystals suitable for x
-ray diffraction requires
5
uncodified “tricks of the trade” and
a certain amount of luck! Additionally, even once a
substance has been successfully crystallized, there is no guarantee that the particular crystal form
will be amenable to x-ray diffraction. Since crystal growth is slow and arduous, X
-ray diffraction
has not been useful for on-the-fly structure determination of chemical structure. For this reason,
X-ray diffraction is not routinely used by most practicing organic chemists, despite the fact the
10
structural data provided is superior to any other method thus far.
Herein, we employ the recently developed cryoEM method MicroE
D
(
10
) to address the
need for fast and reliable structure determination in organic ch
emistry. We demonstrate that with
minimal sample preparation and experiment time, simple powders and amorphous materials (in
some cases, solids isolated
via
silica gel chromatography and rotary evaporation) could be
15
directly used in MicroED studies, rapidly leading to high quality molecular structures often at
atomic resolutions (better than 1Å). MicroED has the potential to dramatically accelerate and
impact the fields of synthetic chemistry, natural product
chemistry, drug discovery, among many
others, by rapidly delivering high quality atomic resolution structures of complex, small
molecules.
20
We initially tested the applicability of MicroED to a model system,
progesterone (
1
)
(Fig.
1). The sample was obtained as a powder from chemical supplier Preparations Laboratories Inc.
Small quantities of
this amorphous solid were placed in between two glass cover slides and
crushed. The fine powder was deposited on a holey carbon
copper grid, cooled to liquid nitrogen
temperatures, and transferred to a cryo electron microscope operating at an acceleration voltage
25
of 200kV (Thermo Fisher Talos Arctica). An overview of the preparation is shown in Figure 1.
Thousands of nano crystals were easily discernable and were investigated for
diffraction.
Typically, for samples such as these, the vast majority of nano crystals diffracted to ~1Å
Fig. 1.
The process of ap
plying MicroE
D to small molecule structu
ral analysis. Here com
mercial progesterone (
1
) was
analyzed and an atomic resolution structure determined at 1Å resolution.
Grid holes are 1
μ
m in diameter.
3
resolution or better (Fig. 1). 140 degrees of diffraction data could be collected from a single nano
crystal by continuous rotation (
11
)
as the improved autoloader and piezo stage of the Talos
Arctica allowed us to travel through the 0 degree point without introducing errors in crystal
position. Typically, the stage was rotated at 0.5 degrees per second and an entire data set
collected in les
s than 3 minutes as a movie (video 1) using a bottom mount CetaD CMOS
5
detector. This detector was fitted with a thick scintillator for diffraction studies. Software written
to convert the movie frames into SMV format allowed for processing in XDS(
12
). From the
collected data, the structure of steroid
1
was determined to 1Å resolution from a single nano
crystal. The entire proces
s from powder to structure took less than 30 minutes.
Encouraged by these results, we wanted to explore the scope of this structural
10
determination method by investigating a wide range of small molecules (Fig. 2a). Up to 12
different samples could be loaded onto the Talos Arctica autoloader allowing us to investigate
up
to 12 different samples rapidly. Notably, over-the-counter medications, CVS
®
branded
acetaminophen and Kroger
®
branded ibuprofen tablets were obtained as
tablets and crushed as
described above allowing us to obtain atomic resolution structures of acetaminophen (
2
) and
15
ibuprofen (
3
) in a rapid fashion. Importantly, we were able to determine structures of
these active
pharmaceutical ingrediaents despite the heterogeneity of the tablets
resulting from the presence
of binders and other formulation agents. The structures of the sodium channel blocker
carbamazepine (
4
) and the macrocyclic polypetide antiobiotic
thiostrepton (
5
) were also
determined from seemingly amorphous powders, which were used as recieved from Sigma-
20
Aldrich. We went on to study several natural products obtained from commercial sources. Used
as recieved, without any crystallization, we were able to obtain
atomic resolution structures of
biotin (
6
), ethisterone (
7
), cinchonine (
8
), and brucine (
9
). Of the eleven different commercial
bioactives examined, all eleven yielded MicroED data and ten were amenable to rapid structure
determination by direct methods
(
13
) while one was determined by molecular replacement
(
14
).
25
Importantly, all were obtained with
out any crystallization attempts. While these pharmaceutical
and commercial natural products were likely recrystal
lized for purification purposes
by the
manufacturer, the powders examined by MicroED were nano crystals a billionth the size
(~100x100x100 nm) of crystals typically needed for X-ray crystallography. This was powder to
structure.
30
Next we decided to investigate compounds that were
never crystallized but instead were
purified by flash column chromatography. Since, silica gel chromatography is t
he most common
method of purification in early stage research for complex molecules in drug discovery, natural
product isolation efforts, and in general synthesis efforts, we were interested to determine
whether solid samples prepared in this way would be amenable to analysis by MicroED. Four
35
compounds, purified by chromatographic methods, were collected from our laboratories and
samples of these seemingly amorphous solids were analyzed. Here, two of four compounds
diffracted and we were able to resolve both strucutres at atomic resolution (
10
and
11
, Figs. 2A
and 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,
(–)
-
40
limaspermidine (
10
), an alkaloid natural product synthesized by our labs (
15
), was resolved from
a residue of only milligram quantities of material following flash chromotography and rotary
evaporation from a scintillation vial (Fig. 2B). Electrons interact with matter more strongly than
X-rays and they are affected by the charge
(
16
). While it is extremely challen
ging to observe
protons in X-ray structures, it is relatively common in MicroED data
(
17
–
20
). Several protons
45
were observed in all structures. For example, the density maps obtained for limaspermidine (
10
)
4
and carbamazepine (
4
) after refinement allowed us to identify protons associated with almost all
atoms in these molecules (Fig. 2C).
Astounded by the ease with which such high quality data was obtained
and the apparent
generality of MicroED to small molecules, we undertook studies to examine heterogeneous
samples (mixtures of compounds). Here, single crystal x-ray diffraction precludes the study of
5
mixtures and NMR is poorly suited for this task. Mixtures of four compounds (
4
,
6
,
8
and
9
,
cf.
Fig. 2.
Different types of small molecules solved by MicroED. A) Several Pharmaceutical, vitamins, commercial
natural products an
d synthetic samples solveds by MicroED, and
B) example of amorphous film utilized in this
study leading
to 1Å resolution MicroED data. C) many protons observed in compounds. Green spheres are Fo
-Fc
maps showing positive density belonging to hydrogens in the molecules.
5
Fig. 3) were crushed together and deposited on a holey carbon grid. Several crystal forms
belonging to the different materials in the mixture, were
visually identified on the grids (Fig. 3).
MicroED data was collected from several nano crystals and the identity of each
species identified
within minutes based on the diffraction and unit cell parameters. Atomic resolution structures
were determined for all small molecules in the mixture rapidly
(Fig. 3).
5
The results described here introduce a new characterization tool into the organic chemists
toolbox. While MicroED was developed for structure determination of biological material such
as proteins in a frozen hydrated state
(
21
,
22
), we demonstrate that cross pollination of
macromolecular structural methods of cryoEM are powerful tools for chemical synthesis and
drug characterization and discovery. MicroED has allowed for the structural charaterizarion of
10
several proteins
(
21
–
23
) from small crystals and crystals that are unsuitable for
X-ray
crystallography because of their size or pathologies
(
21
), but the method has largely gone
unnoticed in the small molecule communities. Based on our findings, we anticipate that
MicroED will be enthusiastically received by many types of small molecule chemists. We have
shown that a variety of amorphous solid materials can lead to rapid atomic resolution structure
15
determination by MicroED with little or no additional sample preparation or crystallization. The
fact that a solid film in a flask, following solvent removal from a flash chromatography
purification, can lead to an atomic resolution molecular structure is evidence that MicroED will
likely have a profound effect on the structural characterization work
-flow of organic chemists.
Although the past 50 years have seen huge advances in the state
-of-the-art, no completely new
20
Fig. 3.
Identification of compounds from heterogeneous mixtures. EM grid prepared as above with biotin, brucine,
carbamazepine, an
d cinchonine powders mixed together. All four compounds identified by unit cell parameters
using MicroED data from within the same grid square. All structures were solved to ~1Å resolution. Grid holes
are 2
μm in diameter.
6
techniques have been introduced that alter the routine structural interrogation of organic
substances. NMR
(
8
), IR
(
3
), UV-Vis
(
2
), and X-ray diffraction (
9
) have been routinely in place
since the 1960’s and are still utilized today as the most common methods for structure
determination in chemistry. We believe that MicroED
(
24
,
25
) is potentially the next big
advance in the field and are enthusiastic about the prospects of expanding its utility as a routine
5
method for chemists.
References and Notes:
1.
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Analysis of chiral organic molecules:
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Analytical Chemistry: Applications, Theory and Instrumentation
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Chirality
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Chem. Soc. Rev.
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(2000).
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H. Günther,
NMR spectroscopy: basic principles, concepts and applications in chemistry
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B. L. Nannenga, D. Shi, A. G. W. Leslie, T. Gonen, High-resolution structure
determination by continuous
-rotation data collection in MicroED.
Nat. Methods
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, 927–
30
930 (2014).
12.
D. Shi
et al.
, The collection of MicroED data for macromolecular crystallography.
Nat.
Protoc.
11
, 895–904 (2016).
13.
G. M. Sheldrick, SHELXT - Integrated space-group and crystal-structure determination.
Acta Crystallogr. Sect. A Found. Crystallogr.
71
, 3–8 (2015).
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7
14.
A. J. McCoy
et al.
, Phaser crystallographic software.
J. Appl. Crystallogr.
40
, 658–674
(2007).
15.
B. P. Pritchett, E. J. Donckele, B. M. Stoltz, Enantioselective Catalysis Coupled with
Stereodivergent Cyclization Strategies Enables Rapid Syntheses of (+)
-Limaspermidine
and (+)-Kopsihainanine A.
Angew. Chem. Int. Ed.
56
, 12624–12627 (2017).
5
16.
R. Henderson, The Potential and Limitations of Neutrons, Electrons and X
-Rays for
Atomic Resolution Microscopy of Unstained Biological Molecules.
Q. Rev. Biophys.
28
,
171–193 (1995).
17.
S. Vergara
et al.
, MicroED Structure of Au146(p-MBA)57at Subatomic Resolution
Reveals a Twinned FCC Cluster.
J. Phys. Chem. Lett.
8
, 5523–5530 (2017).
10
18.
J. A. Rodriguez
et al.
, Structure of the toxic core of
α
-synuclein from invisible crystals.
Nature
.
525
, 486–490 (2015).
19.
M. R. Sawaya
et al.
, Ab initio structure determination from prion nanocrystals at atomic
resolution by MicroED.
Proc. Natl. Acad. Sci.
113
, 11232–11236 (2016).
20.
J. Hattne
et al.
, Analysis of Global and Site-Specific Radiation Damage in Cryo-EM.
15
Structure
(2018), doi:10.1016/j.str.2018.03.021.
21.
M. J. de la Cruz
et al.
, Atomic-resolution structures from fragmented protein crystals with
the cryoEM method MicroED.
Nat. Methods
.
14
, 399–402 (2017).
22.
B. L. Nannenga, D. Shi, J. Hattne, F. E. Reyes, T. Gonen, Structure of catalase determined
by MicroED.
Elife
.
3
, e03600 (2014).
20
23.
M. Gallagher-Jones
et al.
, Sub-ångström cryo-EM structure of a prion protofibril reveals a
polar clasp.
Nat. Struct. Mol. Biol.
(2018), doi:10.1038/s41594-017-0018-0.
24.
M. W. Martynowycz, T. Gonen, From electron crystallography of 2D crystals to MicroED
of 3D crystals.
Curr. Opin. Colloid Interface Sci.
(2018), doi:10.1016/j.cocis.2018.01.010.
25.
B. L. Nannenga, T. Gonen, Protein structure determination by MicroED.
Curr. Opin.
25
Struct. Biol.
27
, 24–31 (2014).
Acknowledgments:
We thank Professors Doug Rees and Bil Clemmons for useful discussions.
We thank Byungkuk Yoo and Michael Takase. for technical assistance with data analysis.
Funding:
C. G. J. would like to acknowledge the National Science Foundation for a predoctoral
30
fellowship. 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
35
Hughes Medical Institute.;
Author contributions:
C.G. J. performed experiments, developed the
8
sample preparation techniques, refined structural data,
prepared figures and assisted with
manuscript preparation. M. W. M performed experiments, developed the sample preparation
techniques, collected data, refined structural data, prepared figures and assisted wit
h manuscript
preparation. T. F. performed experiments. J. H. wrote the software for image conversion,
participated in data analysis, refinement and s
tructure determination. J. A. R. performed
5
experiments, developed the sample preparation techni
ques, 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 wit
h manuscript and
figure preparation. T. G. performed experiments, collected data, developed the sample prep
10
techniques, developed microscope data collection parameters, provided the microscope and
expertise, and assisted in manuscript and figure preparation.
Competing interests:
The authors
declare no competing interests.
Data and materials availability:
MicroED density maps have
been deposited to the EMDB and CCDC.
15
1
Supplementary Materials for
The CryoEM method MicroED as a powerful tool for small molecule
structure determination
Christopher Jones, Mike Martynowycz, Johan Hattne, Tyler Fulton, Brian M. Stoltz*, Jose A.
Rodriguez*, Hosea M. Nelson*, Tamir Gonen*
*Corresponding author. Email: , stoltz@caltech.edu, jrodriguez@chem.ucla.edu,
hosea@chem.ucla.edu, tgonen@g.ucla.edu
Table of Contents
1. Materials and Methods .................................................................................................................1
1.1 Sample Preparation ........................................................................................................
1
1.2
Instrument Parameters, Data Collection, and Analysis
................................................
2
2.
Compound Data and Statistics .....................................................................................................
.3
1.
Materials and methods
All commercial samples were used as received with no additional crystallization or chemical
modification. Ethisterone, cinchonine, carbamazepine, and biotin were purchased from Sigma-
Aldrich. Brucine was purchased from the The Matheson Company, In
c. Progesterone was
purchased from Preparations Laboratories Inc. Thiostrepton was purchased from
EMD
Millipore. CVS
®
brand acetaminophen and Kroger
®
brand ibuprofen were used as over-the-
counter medications. (
-
)-Limaspermadine and HML-I-029 were synthesized according to
previously reported literature procedures (
1
,
2
).
1.1
Sample Preparation
To prepare commercial compounds for MicroED, approximately 1 mg of product as received
was placed between two microscope slides and ground to a fine powder. The ground powder was
placed into an Eppendorf tube along with a prepared TEM grid and shaken. The loaded TEM
grid was then removed from the Eppendorf tube and gently tapped against a filter paper to
2
remove excess powder. Non-commercial samples of HML-I-029 and (+)-limaspermadine were
concentrated under vacuum to yield
a dry film and solid powder respectively. Sample grids of
HML-I-029 were prepared by adding a TEM grid directly to a 20 mL scintillation vial with
gentle shaking. (+)-Limaspermadine grids were prepared by scraping the residue off the side of a
20 mL scintillation vial over a TEM grid. Once sample grids were prepared, they were
subsequently plunged into liquid nitrogen, placed into the sample cartridge, and loaded into the
microscope for analysis. Heterogenous sample mixtures were prepared by adding ~1 mg of
biotin, carabamazepine, cinchonine, and brucine
to a glass cover slide and grinding to a fine
powder. The heterogenous powder was then added to an Eppendorf tube and the grid was
prepared in the same manner as the homogeneous samples.
1.2
Instrument Parameters, Data Collection, and Analysis
The
holey carbon
copper
grid, cooled to liquid nitrogen temperatures, and transferred to a cryo
electron microscope operating at an acceleration voltage of 200kV (Thermo
Fisher Talos Arctica). An
overview of the preparation is shown in Figure 1. 140 degrees of diffraction data could be collected
from a single nano crystal by continuous rotation
as the improved autoloader and piezo stage of the
Talos Arctica allowed us to
travel through the 0 degree point without introducing errors in crystal
position. Typically, the stage was rotated at 0.5 degrees per second and an entire data set collected in
less than 3 minutes as a movie using a bottom mount CetaD CMOS detector. This
detector was fitted
with a thick scintillator for diffraction studies. Software written to convert the movie frames into SMV
format allowed for processing in XDS
.
acetaminophen
stoichiometric formula
C
8
N
1
O
2
temperature (K)
100
Space group
P 21/n
Unit cell a, b, c (
Å
)
6.630(2), 8.620(2), 10.790(2)
angles
⍺
, β, ɣ
(
o
)
90.00(3), 97.56(3), 90.00(3)
Reflections (#)
2300 (380)
Unique reflections (#)
874 (141)
R obs
18.3 (34.7)
R meas
22.8 (43.2)
CC
1/2
95.2 (83.6)
Resolution (
Å
)
0.8
Completeness (%)
69.9 (70.1)
Total exposure (e
-
Å
-2
)
~3
R
0.22
wR2
0.4462
GooF
2.003
0.9
Å
2
Compound Data and Statistics
biotin
stoichiometric formula
C10 N2 O3 S1
temperature (K)
100
Space group
P 21 21 21
Unit cell a, b, c (
Å
)
5.200(2), 10.310(2), 20.910(4)
angles
⍺
, β, ɣ
(
o
)
90.00 (3),90.00 (3),90.00 (3)
Reflections (#)
5498 (1081)
Unique reflections (#)
1323 (246)
R obs
20.3 (37.1)
R meas
23.3 (42.1)
CC
1/2
95.5 (78.4)
Resolution (
Å
)
0.9
Completeness (%)
82.6 (84.8)
Total exposure (e
-
Å
-2
)
~3
R
0.186
wR2
0.3458
GooF
1.818
1.0
Å
brucine
stoichiometric formula
C23 N2 O4
temperature (K)
100
Space group
P 21
Unit cell a, b, c (
Å
)
15.340(3), 7.540(2), 20.010(4)
angles
⍺
, β, ɣ
(
o
)
90.00(3), 112.49(3), 90.00(3)
Reflections (#)
12427 (814)
Unique reflections (#)
5858 (416)
R obs
18.2 (56.1)
R meas
24.2 (74.9)
CC
1/2
95.1 (25.9)
Resolution (
Å
)
0.9
Completeness (%)
95.3 (96.1)
Total exposure (e
-
Å
-2
)
~3
R
0.2244
wR2
0.4468
GooF
1.711
1.0
Å
carbamazepine
stoichiometric formula
C15 N2 O
temperature (K)
100
Space group
P 21/n
Unit cell a, b, c (
Å
)
7.460(2), 11.040(2), 13.760(3)
angles
⍺
, β, ɣ
(
o
)
90.00(3), 92.61(3), 90.00(3)
Reflections (#)
4682 (678)
Unique reflections (#)
1044 (146)
R obs
17.3 (22.1)
R meas
19.5 (24.7)
CC
1/2
97.3 (93.8)
Resolution (
Å
)
1
Completeness (%)
88.3 (84.9)
Total exposure (e
-
Å
-2
)
~3
R
0.1931
wR2
0.3902
GooF
2.398
0.9
Å
cinchonine
stoichiometric formula
C19 N2 O1
temperature (K)
100
Space group
P 21/n
Unit cell a, b, c (
Å
)
10.710(2), 7.060(2), 11.150(2)
angles
⍺
, β, ɣ
(
o
)
90.00(3), 109.66(3), 90.00(3)
Reflections (#)
1933 (399)
Unique reflections (#)
1289 (262)
R obs
11.0 (14.8)
R meas
15.6 (21.0)
CC
1/2
95.0 (89.2)
Resolution (
Å
)
1
Completeness (%)
77.4 (78.9)
Total exposure (e
-
Å
-2
)
~3
R
0.1793
wR2
0.3907
GooF
1.831
0.9
Å
ethisterone
stoichiometric formula
C21 O2
temperature (K)
100
Space group
P 21
Unit cell a, b, c (
Å
)
6.43(2), 21.17(4), 6.48(2)
angles
⍺
, β, ɣ
(
o
)
90.00(3), 105.6(3), 90.00(3)
Reflections (#)
1811 (231)
Unique reflections (#)
1506 (197)
R obs
10.0 (25.4)
R meas
14.1 (35.9)
CC
1/2
97.3 (56.1)
Resolution (
Å
)
0.9
Completeness (%)
60.8 (54.6)
Total exposure (e
-
Å
-2
)
~3
R
0.2481
wR2
0.5109
GooF
2.087
1.0
Å
HML
-I-029n0
stoichiometric formula
C21 N1 O3
temperature (K)
100
Space group
P 21/n
Unit cell a, b, c (
Å
)
8.280(2), 24.370(5), 8.810(2)
angles
⍺
, β, ɣ
(
o
)
90.00(3), 108.80(3), 90.00(3)
Reflections (#)
5455 (536)
Unique reflections (#)
2518 (249)
R obs
18.2 (33.2)
R meas
23.6 (42.2)
CC
1/2
93.1 (79.8)
Resolution (
Å
)
1
Completeness (%)
70.8 (68.6)
Total exposure (e
-
Å
-2
)
~3
R
0.2336
wR2
0.4011
GooF
1.912
0.82
Å