Supplementary Material
Rational design of crystal contact-free space in protein crystals for analyzing
spatial distribution of motions within protein molecules.
Rei Matsuoka, Atsushi Shimada, Yasuaki Komuro, Yuji Sugita, and Daisuke Kohda
Table of Contents
Supplementary Figures
Figure S1. Concept of CCFS in protein crystals.
Figure S2. Model building to estimate the appropriate spacer length in the connector helix of
the MBP-Tom20 fusion protein.
Figure S3. Native PAGE of the soluble fractions of E. coli cell lysates, to monitor the
expression of MBP-Tom20 fusion proteins with different spacer lengths.
Figure S4. Stereo view of the structure of MBP<+4>Tom20-SS-pALDH.
Figure S5. Reaction scheme of N-succinimidyl-4-iodobenzoate with the amino groups in
proteins.
Figure S6. Effects of the truncation of high-resolution reflections prior to Fourier
transformation on the quality of the electron density in the difference map of the
5MBP<+4>Tom20-SS-pALDH crystal.
Figure S7. Effects of the choice of a free test set on the electron density in the difference maps
truncated at 7 Å in the 5MBP<+4>Tom20-SS-pALDH crystal.
Figure S8. Effects of the truncation of high-resolution reflections prior to Fourier
transformation on the quality of the electron density in the difference maps of the
5MBP<+4>Tom20-SS-pALDH(L15S),
1
5MBP<+4>Tom20-SS-pALDH(L18S),
and 5MBP<+4>Tom20-SS-pALDH(L19S) crystals.
Figure S9. Model building to estimate the appropriate spacer length in the connector helix of
the MBP-sAglB fusion protein.
Figure S10. Comparison of the experimental and model structures of the CCFS scaffolds.
Figure S11.
Effects of the truncation of high-resolution reflections prior to Fourier
transformation on the quality of the electron density in the difference map of the
MBP<+20>sAglB crystal.
Figure S12.
Effects of the free test set choice on the electron density in the difference maps
truncated at 7 Å for the MBP<+20>sAglB crystal.
Figure S13.
Effects of the bulk solvent corrections implemented in the different programs on
the shape and volume of the electron density in the truncated difference map of the
5MBP<+4>Tom20-SS-pALDH crystal.
Figure S14. Data-processing workflow for the generation of the Fo-Fc difference electron
density map with truncation and FreeR averaging.
Supplementary Tables
Table S1. Design, expression, and crystallization of the MBP-Tom20 fusion proteins
Table S2. Protein design, X-ray data collection, and refinement statistics (1)
Table S3. Protein design, X-ray data collection, and refinement statistics (2)
Table S4. Protein design, X-ray data collection, and refinement statistics (3)
2
Figure S1. Concept of CCFS in protein crystals. Two cases, a mobile segment in a protein
molecule and a mobile ligand in the binding site of a protein molecule, are shown. (A) In a
conventional protein crystal lattice, crystal contacts with neighboring molecules inhibit the
movements of the segment/ligand, and thus a snapshot is obtained. (B) If the mobile
segment/ligand is fortuitously located in the CCFS, then the motions of the mobile
segment/ligand are preserved in the protein crystals.
3
Figure S2. Model building to estimate the appropriate spacer length in the connector helix of the
MBP-Tom20 fusion protein. MBP, Tom20, and the pALDH peptide are colored green, cyan, and
magenta, respectively. The maltose molecule bound to MBP is shown as a stick model. The
connector helix, which consists of the C-terminal helix of MBP, the variable-length spacer, and
the N-terminal helix of Tom20, is highlighted in yellow. The number in the angle brackets
denotes the spacer length. A negative number means a deletion of amino acid residues. The
orange transparent ovals indicate the predicted CCFS in the models of the fusion proteins with
spacer lengths of -4, 0, +4, and +8.
4
Figure S3. Production of recombinant MBP-Tom20 fusion proteins. Native PAGE of the soluble
fractions of E. coli cell lysates, to monitor the expression of MBP-Tom20 fusion proteins with
different spacer lengths.
Figure S4. Stereo view of the structure of MBP<+4>Tom20-SS-pALDH. MBP, Tom20, and the
4-residue spacer are colored green, cyan, and orange, respectively. Δ5 in the crystal structure of
MBP<+4>Tom20-SS-pALDH
denotes
the
deleted
segment
(yellow), A51ATGD55,
in
Δ5MBP<+4>Tom20 to enlarge the CCFS. Due to the undesired intramolecular interaction
between pALDH and the Δ5 segment (the dotted line), the electron density of the pALDH
presequence was clearly seen outside of the binding site, and so its model was constructed
(magenta).
5
Figure S5.
Reaction scheme of N-succinimidyl-4-iodobenzoate with the amino groups in
proteins. N-succinimidyl-4-iodobenzoate was used to label the N-terminus of the pALDH
peptide with an iodine atom.
Figure S6.
Effects of the truncation of high-resolution reflections prior to Fourier
transformation on the quality of the electron density in the difference map, contoured at +3σ. (A)
The electron density of the pALDH presequence peptide in CCFS, and (B) that of an arbitrarily
chosen α-helical segment (A186GAKAGLTFLVDLIK200) in MBP, as a control for a fixed
segment, are shown as a function of the truncation threshold, rmin. In (B), the MBP-Tom20
structure lacking the α-helix was used as the search model in the PHASER molecular
replacement and the subsequent REFMAC5 refinement. MBP, Tom20, and the 4-residue spacer
are colored green, cyan, and orange, respectively. The structure of the deleted α-helix is depicted
as blue sticks with the side chains. The comparison between (A) and (B) indicates that the lowpass filter by truncation improves the quality of the electron densities of atoms with large Bfactors, but as expected, has rather deteriorative effects on the electron densities of atoms with
small B-factors. Note that the maps were generated by the REFMAC5 run with the free test set of
default Set 0 (See Fig. S7).
6
7
(figure on the previous page)
Figure S7. Effects of the choice of a free test set on the electron density in the difference maps
truncated at 7 Å, contoured at +3σ. Maps were generated using the MTZ files from REFMAC5
runs with different free test sets from Set0 to Set19, and subsequent truncation with rmin = 7 Å.
(A) The pALDH presequence peptide in CCFS, and (B) an arbitrarily chosen α-helical segment
(A186GAKAGLTFLVDLIK200) in MBP, as a control for a fixed segment. In (B), the MBP-Tom20
structure lacking the α-helix was used as the search model in the PHASER molecular
replacement and the subsequent REFMAC5 refinement. MBP, Tom20, and the 4-residue spacer
are colored green, cyan, and orange, respectively. The structure of the deleted α-helix is depicted
as blue sticks with the side chains. The comparison between (A) and (B) indicates that the choice
of a free test set had minimal effects on the electron density of the immobile α-helix in MBP, but
a high impact on the quality of the electron density of the mobile presequence. This indicates that
the electron densities of atoms with large B-factors are influenced much more by the exclusion of
a small fraction of reflection data than those of atoms with small B-factors.
8
Figure S8.
Effects of the truncation of high-resolution reflections prior to Fourier
transformation on the quality of the electron density in the difference maps, contoured at +3σ, of
the MBP-Tom20 fusion protein crystals with mutated pALDH presequences: (A) pALDHmut1,
5MBP<+4>Tom20-SS-pALDH(L15S),
(B)
pALDHmut2,
5MBP<+4>Tom20-SS-
pALDH(L18S), and (C) pALDHmut3, 5MBP<+4>Tom20-SS-pALDH(L19S). Tom20 and the 4residue spacer are colored cyan and orange, respectively. The maps were generated by the
REFMAC5 run with the free test set of default Set 0.
9
Figure S9.
Model building to estimate the appropriate spacer length in the connector helix of
the MBP-sAglB fusion protein. MBP and Tom20 are colored green and cyan, respectively. The
maltose molecule bound to MBP is shown as a stick model. The connector helix, which consists
of the C-terminal helix of MBP, the variable-length spacer, and the N-terminal helix of sAglB, is
highlighted in yellow. The number in the angle brackets denotes the spacer length. The orange
transparent ovals indicate the predicted CCFS in the models of the fusion proteins with the
spacer lengths of +1, +16, and +20.
10
Figure S10. Comparison of the experimental and model structures of the CCFS scaffolds. The
model structures were constructed by assuming an ideal -helical conformation of the spacer in
the connector helix. The MBP structures (green) of the fusion proteins were superimposed on
each other. (A) The large deviation of the relative orientation of sAglB (cyan) from the model
structure (blue) was seen in MBP<+16>sAglB. (B) The deviation of the relative orientation was
smaller and acceptable in MBP<+20>sAglB, in spite of the longer spacer length. (C) The
deviation of the relative orientation of Tom20 in 5MBP<+4>Tom20-SS-pALDH was also
small. Taken together, the model building is effective, but must be tested experimentally,
particularly for long spacers (> 10 residues).
11
Figure S11.
Effects of the truncation of high-resolution reflections prior to Fourier
transformation on the quality of the electron density in the difference map, contoured at +3σ. The
electron density of the THL segment, in the CCFS created in the MBP<+20>sAglB crystal, is
shown as a function of the truncation threshold, rmin. The maps were generated by the REFMAC5
run with the free test set of default Set 0 (See Fig. S12). sAglB and the 20-residue spacer are
colored cyan and orange, respectively.
One-half (3-residue turn + 9-residue helix) of the mobile THL segment was clearly visualized in
the map at rmin = 7 Å. The other half (12-residue loop) except for 1 or 2 C-terminal residues
remained invisible, indicating that the movement was larger than the detection limit (>1.5 Å
rmsds from the mean positions). Close inspection of the map at rmin = 2 Å revealed that the
electron density corresponding to the turn part and the N-terminal half of the helical part of the
THL segment marginally appeared. This indicated that the visualized half of the THL segment at
rmin = 7 Å is further divided into two parts with respect to the mobility: The spatial distributions
of the turn part and the N-terminal half of the helical part are narrower than that of the Cterminal half of the helical part, which is invisible in the map truncated at rmin = 2 Å. Note that
the C-terminal tail segment is also invisible in the same map at rmin = 2 Å.
12
Figure S12. Effects of the free test set choice on the electron density in the difference maps
truncated at 7 Å, contoured at +3σ. The THL segment was visualized in the CCFS created in the
MBP<+20>sAglB crystal. Maps were generated using the MTZ files from REFMAC5 runs with
different free test sets from Set0 to Set19, and subsequent truncation with rmin = 7 Å. sAglB and
the 20-residue spacer are colored cyan and orange, respectively.
13
Figure S13. Effects of the bulk solvent corrections implemented in the different programs on the
shape and volume of the electron density in the FreeR-averaged difference maps truncated at 7
Å, contoured at +3σ. (A) The electron density of the pALDH presequence peptide in CCFS, and
(B) that of an arbitrarily chosen α-helical segment (A186GAKAGLTFLVDLIK200) in MBP, as a
control for a fixed segment, are shown. In (B), the MBP-Tom20 structure lacking the α-helix was
used as the search model in the PHASER molecular replacement and the subsequent REFMAC5
refinement. MBP, Tom20, and the 4-residue spacer are colored green, cyan, and orange,
respectively. The structure of the deleted α-helix is depicted as blue sticks with the side chains. A
REFMAC5 map with the flat mask-based bulk solvent correction was generated, by using the
keywords “SOLVENT YES” and “SCALE TYPE SIMPLE”. The default setting of REFMAC5
(“SOLVENT YES” and “SCALE TYPE BULK”) provides a map with the two types of
corrections (the exponential type correction + the flat mask-based bulk solvent correction), but
the generated map (Fig. 5A) was almost identical to the map obtained with only the flat maskbased bulk solvent correction. The CCFS scaffold (MBP-Tom20) structure refined by the
program REFMAC5 was used as input for the programs CNS ver. 1.3 and PHENIX ver. 1.9, with
the default settings of the flat mask-based bulk solvent corrections. In the CNS calculation,
model_map.inp was used to generate a sigma-A weighted Fo-Fc difference map with truncation,
by using the keyword “high_res=7.0”. In the PHENIX calculation, phenix.maps was used to
generate the MTZ file that contained the sigma-A weighted Fourier coefficients. SFTOOLS and
FFT in CCP4 were subsequently used for truncation and map generation.
14
Figure S14. Data-processing workflow for the generation of the Fo-Fc difference electron
density map with truncation and FreeR averaging.
15
Table S1. Design, expression, and crystallization of the MBP-Tom20 fusion proteins
MBP<n>Tom20
Expression of
Crystallizationd
<n>a
spacer sequence at the junctionb
-4
AQTRI-....F
+
-3
AQTRI-...KF
+
-2
AQTRI-..QKF
+
-1
AQTRI-.VQKF
±
0
AQTRI-AVQKF
±
+1
AQTRI-K-AVQKF
±
+2
AQTRI-KE-AVQKF
++
+3
AQTRI-KEA-AVQKF
++
+4
AQTRI-KEAL-AVQKF
+
+5
AQTRI-KEALQ-AVQKF
±
+6
AQTRI-KEALQE-AVQKF
+
+7
AQTRI-KEALQEL-AVQKF
+
√
+8
AQTRI-KEALQELA-AVQKF
+
√
Crystal structuree
soluble proteinc
√
√
√
√
√
a
<n> denotes the length of the spacer.
b
The MBP sequence is shown in plain text, the spacer sequence is italicized, and the Tom20
sequence is in bold text. The basic spacer sequence is KEALQELA.
c
±, +, and ++ indicate rough estimates of the protein amounts in the soluble fractions of E. coli
cell lysates, based on the Coomassie Brilliant Blue stained native PAGE gel in Fig. S3.
d
√, Crystallized. Crystallization conditions: for <0>, 8% PEG400, 0.1 M sodium acetate (pH
4.6); for <+2> and <+4>, in Table S2; <+7>, 20% PEG4000, 5% isopropanol, 0.1 M sodium
citrate; <+8>, 16% PEG6000, 0.2 M calcium chloride, 0.1 M HEPES (pH 7.0).
e
√, Structures of the fusion proteins were determined. See Table S2.
16
Table S2. Protein design, X-ray data collection, and refinement statistics (1)
MBP<+2>Tom20
MBP<+4>Tom20
MBP<+4>Tom20-SSpALDH
Fusion protein
MBP-KE-Tom20
MBP-KEAL-Tom20
MBP-KEAL-Tom20
pALDH peptide
none
none
Crystallization
conditions
16% PEG3350
0.2 M disodium malonate
0.1M HEPES (pH 7.3)
50% PEG400
0.1 M phosphate citrate (pH
4.4)
Ac-GPRLSRLLSYAGCNH2
24% PEG8000
20% glycerol
0.04 M potassium
thiocyanate
Beamline
SPring-8 BL32XU
PF BL5A
PF BL17A
Cryo conditions
20% glycerol
20% glycerol
15% glycerol
Wavelength, Å
1.0000
1.0000
1.0000
Oscillation range, °
180
180
180
Space group
C2
I222
P43212
Cell dimensions
a = 129.9 Å, b = 81.2 Å, c =
109.9 Å, = 106.4°
a = 63.1 Å, b = 113.9 Å, c =
125.8 Å
a = b = 69.0 Å, c = 212.6 Å
No. of molecules in
AU
2
1
1
Resolution range, Å
50.0 – 2.56 (2.60 – 2.56)
50.0 – 1.97 (2.00 – 1.97)
50.0 – 1.70 (1.73 – 1.70)
Observed reflections
135990
188262
784858
Unique reflections
35889
30663
56782
Redundancy
3.8 (3.7)
6.1 (3.4)
13.8 (13.4)
Completeness, %
99.9 (99.9)
93.1 (64.4)
98.4 (99.6)
Rmerge(I)a
0.076 (0.436)
0.106 (0.591)
0.128 (0.734)
I / (I)
11.0 (3.65)
14.6 (1.35)
22.1 (3.38)
24.7– 2.56
(2.61 – 2.56)
6752
42.2 – 1.97
(2.01 – 1.97)
3367
42.1 – 1.70
(1.74 – 1.70)
3367
20
101
407
Rwork/Rfreeb
0.220/0.279
0.207/0.254
0.177/0.211
rmsdc from ideality
bond length, Å
0.009
0.019
0.018
angles, °
Ramachandran
d
,%
1.175
1.939
1.800
Protein design
Data
collection
statistics
Refinement
statistics
Resolution range, Å
No. of protein atoms
No.
of
molecules
water
plot
17
Favored region
93.6
97.7
98.4
Allowed region
5.1
2.1
1.2
Outlier region
1.3
0.2
0.1
5AZ6
5AZ7
5AZ8
PDB code
Values in parentheses are for the highest resolution shell.
a
Rmerge(I) = (|Ii - <I>|)/Ii, where Ii is the intensity of the ith observation and <I> is the mean
intensity.
b
Rwork/Rfree = ∑|FO - FC|/∑|FO|. Rwork was calculated from the working set (95% of the total
reflections). Rfree was calculated from the test set, using 5% of the total reflections. The test set
was not used in the refinement.
c
rmsd, root mean square deviation.
d
Calculated using the program RAMPAGE in the CCP4 program suite.
18
Table S3. Protein design, X-ray data collection, and refinement statistics (2)
5MBP<+4>To
m20-SSpALDH
5MBP<+4>To
m20-SS-(Niodo)pALDH
Protein design
Fusion protein
pALDH peptide
Crystallization
conditions
5MBP<+4>To
m20-SSpALDH(L15S)
5MBP<+4>To
m20-SSpALDH(L18S)
5MBP<+4>To
m20-SSpALDH(L19S)
5MBP-KEAL-Tom20
AcGPRLSRLLSY
AGC-NH2
4-iodobenzoylGPRLSRLLSY
AGC-NH2
AcGPRSSRLLSY
AGC-NH2
AcGPRLSRSLSY
AGC-NH2
AcGPRLSRLSSY
AGC-NH2
20% PEG3350
0.2 M potassium
nitrate
20% PEG3350
0.2 M potassium
iodide
20% PEG3350
0.2 M
ammonium
nitrate
20% PEG6000
0.2 M lithium
chloride
24% PEG3350
0.2 M sodium
iodide
SPring-8
BL44XU
15% glycerol
0.9000
SPring-8
BL26B1
20% glycerol
1.6000
SPring-8
BL44XU
20% glycerol
0.9000
SPring-8
BL26B1
20% glycerol
1.0000
SPring-8
BL44XU
20% glycerol
0.9000
180
720
180
180
180
C2
a = 160.5 Å, b =
71.4 Å, c = 47.5
Å, = 104.9°
C2
a = 160.6 Å, b =
71.4 Å, c = 47.6
Å, = 105.2°
C2
a = 157.1 Å, b =
70.8 Å, c = 86.1
Å, = 102.5°
P21
a = 70.4 Å, b =
83.5 Å, c = 86.5
Å, = 113.9°
P21
a = 70.8 Å, b =
84.0 Å, c = 86.5
Å, = 113.9°
1
1
2
2
2
39.6 – 1.82
(1.86 – 1.82)
50.0 – 2.71
(2.76 – 2.71)
50.0 – 2.13
(2.17 – 2.13)
50.0 – 1.78
(1.81 – 1.78)
50.0 – 1.62
(1.65 – 1.62)
169218
188174
188545
314494
430860
46512
14001
51375
87479
116783
3.6 (3.8)
99.8 (99.8)
0.083 (0.765)
14.0 (1.8)
13.4 (12.8)
98.2 (98.7)
0.077 (0.146)
29.9 (16.8)
3.7 (3.5)
99.4 (99.0)
0.114 (0.386)
10.8 (2.86)
3.6 (3.5)
99.8 (100.0)
0.064 (0.716)
18.7 (2.23)
3.7 (3.6)
98.2 (92.0)
0.082 (0.502)
13.9 (2.45)
no
no
no
Pseudomerohed
ral, (h, -k, -h-l),
= 0.18
Pseudomerohed
ral, (h, -k, -h-l),
= 0.25
39.6 – 1.82
41.5 – 2.13
30.0 – 1.78
39.5 – 1.62
3323
6696
6696
6696
172
224
234
260
0.1950.0005/0.
2320.006
0.1990.0007/
0.2510.005
0.2410.0006/
0.2880.005
0.2290.0006/
0.2650.004
Data collection
statistics
Beamline
Cryo conditions
Wavelength, Å
Oscillation
range, °
Space group
Cell dimensions
No. of molecules
in AU
Resolution range,
Å
Observed
reflections
Unique
reflections
Redundancy
Completeness, %
Rmerge(I)a
I / (I)
Refinement
statistics
twinning
Resolution range,
Å
No. of protein
atoms
No. of water
molecules
Rwork/Rfreeb,e
19
rmsdc,e
from
ideality
bond
length, Å
angles, °
Ramachandran
plotd,e, %
Favored region
Allowed region
Outlier region
PDB code
a, b, c, d
e
0.0190.0008
0.0160.0003
0.0180.0002
0.0190.0006
1.900.04
1.720.03
1.890.02
1.890.04
96.90.24
2.40.21
0.60.13
5AZ9
97.30.34
2.00.32
0.60.08
-
96.00.40
3.20.35
0.80.24
-
97.70.20
1.90.18
0.50.06
-
No structure
See the legends of Table S2.
Average and standard deviation of the 20 refinement calculations with different free test sets.
20
Table S4. Protein design, X-ray data collection, and refinement statistics (3)
Protein design
Spacer
Crystallization conditions
Data collection statistics
Beamline
Cryo conditions
Wavelength, Å
Oscillation range, °
Space group
MBP<+16>sAglB
MBP<+20>sAglB
AEAAAKAEAAAKAAAA
AAAKAEAAAKAEAAAKAAAA
17% PEG10000
0.1 M ammonium acetate
0.1 M bis-Tris, pH 5.5
1.0 M lithium chloride
14% PEG4000
0.1 M sodium acetate, pH 4.5
0.1M manganese(II) chloride
SPring-8 BL44XU
20% glycerol
0.9000
360
P1
a = 54.85 Å, b = 62.56 Å, c = 136.4
Å, α= 102.0°, = °,γ= 92.9°
2
50.0 – 2.60 (2.64 – 2.60)
208533
53073
3.9 (3.8)
99.3 (95.1)
0.102 (0.406)
12.8 (3.4)
SPring-8 BL32XU
20% glycerol
1.0000
300
P212121 f
40.57 – 2.60
13018
48
61
43.61 – 2.08
6580
23
106
0.1910.0006/0.2570.005
0.1960.0006/0.2450.005
rmsd from ideality bond
length, Å
0.0130.0004
0.0170.0004
angles, °
1.6960.027
1.9020.030
Favored region
95.70.23
97.00.20
Allowed region
Outlier region
PDB code
3.40.20
0.90.11
-
2.60.26
0.40.15
5AZA
Cell dimensions
No. of molecules in AU
Resolution range, Å
Observed reflections
Unique reflections
Redundancy
Completeness, %
Rmerge(I)a
I / (I)
Refinement statistics
Resolution range, Å
No. of protein atoms
No. of ligands
No. of water molecules
Rwork/Rfreeb,e
c,e
a = 66.5 Å, b = 100.5 Å, c = 140.9 Å
1
50.0 – 2.08 (2.12 – 2.08)
694036
57642
12.0 (10.9)
100.0 (100.0)
0.142 (>1.00)
18.7 (2.0)
Ramachandran plotd,e, %
a, b, c, d
See the legends of Table S2.
e
Average and standard deviation of the 20 refinement calculations with different free test sets.
f
After three micro-seeding steps, the space group changed from P1 to P212121.
21