1
Supplementary Materials for
The CryoEM method MicroED as a powerful tool for small molecule
structure determination
Christopher G. Jones
1†
, Michael W. Martynowycz
2†
, Johan Hattne
2
, Tyler
J.
Fulton
3
, Brian M.
Stoltz
3$
, Jose A. Rodriguez
1$
, Hosea M. Nelson
1$
, Tamir Gonen
2$
1
Department of Chemistry and Biochemistry, University of California, Los Angeles, 90095,
USA
2
Howard Hughes Medical Institute, Departments of Biological Chemistry an
d Physiology,
University of California, Los Angeles, 90095, USA
3
The Warren and Katharine Schlinger Laboratory of Chemistry and Chemical Engineering,
California Institute of Technology, Pasadena, 91125, USA
$
Correspondence to:
stoltz@caltech.edu
,
jrodriguez@chem.ucla.edu
,
hosea@chem.ucla.edu
,
tgonen@ucla.edu
This PDF file includes:
Materials and Methods
Supplementary Text
Figs.
S1
to
S11
Captions for Movies
S1
References
Other Supporting Online Material for this manuscript includes the following:
Movies
S1
Table of Contents
2
1.
Materials and Methods
................................
................................
................................
................
1
1.1
Sample Preparation
................................
................................
................................
........
2
1.2 Instrument P
arameters
................................
................................
................................
...
3
1.3 Data Collection
Procedure
................................
................................
.............................
4
2. Compound Data and Statistics
................................
................................
................................
.....
5
3. Caption for
Movie S1
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, Inc. Proges
terone was
purchased from Preparations Laboratories Inc. Thiostrepton was purchased from EMD
Millipore.
CVS
®
-
brand ac
etaminophen and Kroger
®
brand ibuprofen were used as over
-
the
-
counter medications.
(
+
)
-
Limaspermadine and H
K
L
-
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
pre
-
clipped
Quantifoil R2
/
2 Cu300
or Quantifoil
R1/4 Cu300 mesh grid.
The TEM grid was then
removed from the Eppendorf tube
and
gently
tapped
against a
filter paper to remove excess powder. Non
-
commercial samples of
HKL
-
I
-
029
and
(+)
-
limaspermadine were concentrated under vacuum to yield
a
dry film
and
solid
powder
respectively
. Sample grids of
HKL
-
I
-
029 were prepared by adding a TEM grid directly to a 20
mL scintillation vial with gentl
e
shaking.
(+)
-
Limaspermadine
grids
were prepared by scraping
the residue off the side of a 20 mL scintil
lation 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,
carbamazepine
, 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
All data were collected on a Thermo
-
Fischer Talos Artica electron
cryo
microscope operating at
an acceleration voltage of 200keV, corresponding to a wavelength of ~0.0251
Å
. Screening of
3
the
TEM
grid
s
for micro
crystals
was done by operating the microscope in
over focused
diffraction
mode to minimize diffraction and hysteresis between screening and diffraction
operational modes.
1.
3
Data Collection Procedure
MicroED
data collection was collected in rolling shutter using a Thermo
-
Fischer
CetaD
CMOS
4k x 4k camera. Images were collected as a movie as the crystal was continuously rotated in the
electron beam
(
3
)
. Typical data collection was performed using
a constant tilt rate of ~0.6
°
s
-
1
d
over an angular wedge of ~60
°
between the minimum and maximum tilt ranges of
-
72
°
to +7
2
°
degrees, respectively. During
continuous
rotation the camera integrated frames continuously at a
rate of 1
-
3s per frame. The dose rate was calibrated to
<
0.0
3
e
-
Å
-
2
s
-
1
.
Crystals selected for data
collection were isolated by a
selected
area aperture to reduce the background noise
contributions, and calibrated to eucentric height to stay in the aperture over the
entire
tilt range.
Diffraction movies saved as SER files were converted to SMV format using in
-
house software
developed for th
e CetaD and
made freely available online
(
https://cryoem.ucla.edu/pages/MicroED
). Frames were indexed and integrated in XDS, and
multiple datasets were scaled and merged using XSCALE
(
4
,
5
)
. The intensities were converted
to SHELX format using XDSCONV
(
5
)
. All structures except thiostrepton
(see below)
were
solved by
ab initio
direct methods in SHELXT, and refined in SHELXL as previously described
(
6
,
7
)
.
Four
datasets from thiostre
p
ton were indexed and integrated in MOSFLM through its graphical
user interface, iMosflm
(
8
,
9
)
. Data were merged in AIMLESS, and phased by molecular
replacement in MOLREP using 1E9W as a search model
(
10
,
11
)
. The solut
i
on was refined
using REFMAC5 with electron scattering factors to a resolution of
1.9Å
with the
free
R
set
copied from the initial search model
(
12
)
.
2.
Compound Data and Statistics
Individual integration and refinement statistics can be found for each compound in SI Figures 1
-
1
1
along with corresponding densities.
3.
Movie S1.
Continuous rotation MicroED data from a
carbamazepine nanocrystal with
corresponding resolution rings.
4
acetaminophen
Stoichiometric formula
C
8
N
1
O
2
Temperature (K)
100
Space group
P
2
1
/n
Unit cell lengths 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
Figure S1.
Data processing statistics and final structure of acetaminophen.
biotin
Stoichiometric formula
C
10
N
2
O
3
S
Temperature (K)
100
Space group
P
2
1
2
1
2
1
Unit cell lengths 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
Figure S2.
Data processing statistics and final structure of biotin.
brucine
Stoichiometric formula
C
23
N
2
O
4
Temperature (K)
100
Space group
P
2
1
Unit cell lengths 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
H
3
CO
H
3
CO
N
O
N
O
H
H
H
7,
brucine
Figure S3.
Data processing statistics and final structure of brucine.
5
carbamazepine
Stoichiometric formula
C
15
N
2
O
Temperature (K)
100
Space group
P
2
1
/n
Unit cell lengths 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
)
1.0
Completeness (%)
88.3 (84.9)
Total exposure (e
-
Å
-
2
)
~3
R
0.1931
wR2
0.3902
GooF
2.398
Figure S4.
Data processing statistics and final structure of carbamazepine.
cinchonine
Stoichiometric formula
C
19
N
2
O
Temperature (K)
100
Space group
P
2
1
/n
Unit cell lengths 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
)
1.0
Completeness (%)
77.4 (78.9)
Total exposure (e
-
Å
-
2
)
~3
R
0.1793
wR2
0.3907
GooF
1.831
Figure S5.
Data processing statistics and final structure of cinchonine.
ethisterone
Stoichiometric formula
C
21
O
2
Temperature (K)
100
Space group
P
2
1
Unit cell lengths 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
Figure S6.
Data processing statistics and final structure of
ethisterone
.
6
HKL
-
I
-
029
Stoichiometric formula
C
21
N O
3
Temperature (K)
100
Space group
P
2
1
/n
Unit cell lengths 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 (#)
3369 (446)
Unique reflections (#)
1970 (262)
R
obs
14.1 (22.8)
R
meas
18.4 (29.5)
CC
1/2
94.5 (84.8)
Resolution (
Å
)
1.0
Completeness (%)
55.3 (55.7)*
Total exposure (e
-
Å
-
2
)
~3
R
0.2366
wR2
0.4762
GooF
2.656
Figure S7.
Data processing statistics and final structure of HKL
-
I
-
029
.
*The completeness of this compound was limited due to preferred orientation
.
ibuprofen
Stoichiometric formula
C
13
O
2
Temperature (K)
100
Space group
P
2
1
/c
Unit cell lengths a, b, c (
Å
)
14.65(3), 7.88(2), 10.73(2)
angles
⍺
,
β
,
ɣ
(
o
)
90.00(3), 99.7(3), 90.00(3)
Reflections (#)
1452 (402)
Unique reflections (#)
506 (138)
R
obs
14.7 (20.8)
R
meas
17.8 (25.2)
CC
1/2
97.8 (89.9)
Resolution (
Å
)
1.1
Completeness (%)
54.3 (53.1
)*
Total exposure (e
-
Å
-
2
)
~3
R
0.2559
wR2
0.5282
GooF
2.686
Figure S8.
Data processing statistics and final structure of ibuprofen
.
*The completeness of this compound was limited due to preferred orientation
.
(+)
-
limaspermidine
Stoichiometric formula
C
19
N
2
O
Temperature (K)
100
Space group
P
2
1
2
1
2
1
Unit cell lengths a, b, c (
Å
)
7.620(2), 13.880(3), 15.200(3)
angles
⍺
,
β
,
ɣ
(
o
)
90.00(3), 90.00(3), 90.00(3)
Reflections (#)
8252 (387)
Unique reflections (#)
3430 (185)
R
obs
16.7 (68.2)
R
meas
21.6 (88.6)
CC
1/2
97.0 (34.2)
Resolution (
Å
)
0.77
Completeness (%)
93.0 (69.3)
Total exposure (e
-
Å
-
2
)
~3
R
0.2422
wR2
0.4309
GooF
1.541
Figure S9.
Data processing statistics and final structure of (+)
-
limaspermidine
.
7
References
1.
Y. Liu, S.
-
J. Han, W.
-
B. Liu, B. M. Stoltz, Catalytic Enantioselective Construction of
Quaternary Stereocenters: Assembly of Key Building Blocks for the Synthesis of
Biologically Active Molecules.
Acc. Chem. Res.
48
, 740
–
751 (2015).
2.
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. Chemie Int. Ed.
56
, 12624
–
12627 (2017).
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B. L. Nannenga, D. Shi, A.
G. W. Leslie, T. Gonen, High
-
resolution structure
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W. Kabsch, Integration, scaling, space
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G. M. Sheldrick, SHELXT
-
Integrated space
-
group and crystal
-
structure determination.
progesterone
Stoichiometric formula
C
21
O
2
Temperature (K)
100
Space group
P
2
1
2
1
2
1
Unit cell lengths a, b, c (
Å
)
10.380(2), 12.810(3), 13.890(3)
angles
⍺
,
β
,
ɣ
(
o
)
90.00(3), 90.00(3), 90.00(3)
Reflections (#)
4487(577)
Unique reflections (#)
1871 (238)
R
obs
14.7 (44.6)
R
meas
18.0 (53.9)
CC
1/2
98.0 (66.1)
Resolution (
Å
)
0.9
Completeness (%)
72.1 (68.6)
Total exposure (e
-
Å
-
2
)
~3
R
0.2045
wR2
0.4155
GooF
1.888
Figure S10.
Data processing statistics and final structure of progesterone.
thiostrepton
Resolution range (
Å
)
18.99
–
1.91 (2.13
–
1.91)
Space group
P
4
3
2
1
2
Unit cell lengths a, b, c (
Å
)
26.219, 26.219, 27.534
angles
⍺
,
β
,
ɣ
(
o
)
90, 90, 90
Total reflections
5578 (458)
Unique reflections
686 (93)
Multiplicity
8.1 (4.9)
Completeness (%)
78.6 (40.3)
Mean
I
/
σ
(
I
)
5.1 (3.4)
Wilson
B
-
factor
2.6
R
merge
0.236 (0.320)
R
meas
0.251 (0.353)
CC
1/2
0.985 (0.813)
Reflections used in refinement
620 (
92)
R
work
0.1818 (
0.2191)
R
free
0.2396 (
0.1766)
N
N
S
N
H
O
H
N
O
NH
2
O
HN
S
N
O
O
N
OH
NH
OH
H
N
O
N
H
O
H
N
O
O
NH
S
N
NH
O
HN
O
HO
S
N
HN
O
O
N
S
OH
OH
Figure S11.
Data processing statistics and final structure of
thiostrepton
.
8
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–
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G. M. Sheldrick, Crystal structure refinement with SHELXL.
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A new graphical interface for diffraction
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image processing with MOSFLM.
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