Terahertz radiation induces non-thermal structural changes associated with
Fröhlich condensation in a protein crystal
Ida V. Lundholm1†, Helena Rodilla2†, Weixiao Y. Wahlgren1, Annette Duelli1, Gleb Bourenkov3,
Josip Vukusic2, Ran Friedman4, Jan Stake2, Thomas Schneider3 and Gergely Katona1*
1
Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg,
Sweden.
2
Department of Microtechnology and Nanoscience, Chalmers University of Technology,
Gothenburg, Sweden.
3
European Molecular Biology Laboratory Hamburg Outstation, EMBL c/o DESY, Notkestrasse
85, 22603 Hamburg, Germany.
4
Department of Chemistry and Biomedicinal Sciences and Centre for Biomaterials Chemistry,
Linnaeus University, Kalmar, Sweden.
*Correspondence to: Correspondence and requests for materials should be addressed to G.K.
(gergely.katona@cmb.gu.se)
† These authors contributed equally to this work.
Quality of reticularly sampled data
We compared the data quality of continuous data integration strategy with the data derived from
integrating only every second image (reticularly integrated data). To make the comparison
meaningful we compared data collected using approximately the same X-ray dose: for the
continuous data integration case we integrated the first and second half (180° each) of a 360°
dataset whereas for the reticular data integration strategy only every second frame was used of
the same dataset. The statistical analysis of the two approaches is presented in Supplementary
Table 1. In general, the quality indicators of the reticularly integrated data lies in between the
first and second half of the continuously integrated data indicating that the radiation damage has
stronger influence on the data quality than how the data is integrated as long as the oscillation is
small. The data quality was still acceptable when only every third image was used (not shown).
The reticular sampling of the reflection rocking curves appears to affect Rmerge more than other
quality indicators, but one has to keep in mind that the reticularly integrated data has higher
multiplicity, which automatically leads to a higher Rmerge even if the data quality is the same (1).
Our experience with serial femtosecond crystallographic data is similar: even completely random
sampling of the ”rocking curve” (with uninterpretable high Rmerge) leads to accurate estimations
of reflection intensities (2-4).
Validation of the difference Fourier map
The <F0.4THz>-<Foff> difference Fourier map is based on 10 different datasets and to ensure that
the electron density difference peaks around helix 3 is not only caused by one of the datasets, 10
new <F0.4THz>-<Foff> maps were calculated excluding one dataset at a time. The new maps still
indicate difference Fourier peaks at the same positions, although their contrast to the noise, as
expected, is less pronounced (Supplementary Fig. 1A). An improved method for the estimation
of structure-factor amplitude differences was also applied in the electron density map calculation
which retains and sharpens the electron density features (Supplementary Fig. 1B)(5).
Figure S1. Validation of THz radiation induced difference electron density peaks. (a)
F0.4THz-Foff electron density maps calculated when data from one crystal is excluded from the
dataset. The map calculation is repeated 9 times with every crystal involved in the study. The
positive electron density peaks are shown in different colors. Negative differences are excluded
for clarity. The map is contoured at 3.4 σ. (b) F0.4THz-Foff difference electron density map
calculated from Bayesian estimation of difference structure factor amplitude (12). Positive
difference shown in cyan and negative difference shown in yellow. The F0.4THz-Foff difference
electron density map from Fig. 2 is shown in green (positive difference) and red (negative
difference) for comparison.
Figure S2. Difference electron density map, Fodd-Feven, calculated from reference data.
Figure S3. Illustration of the experimental setup. (a) Picture taken at beamline P13, PetraIII
(b) Idealized close up view showing the illuminated face of lysozyme crystal, its position in the
sample holding loop and the position and dimensions of the X-ray beam relative to the crystal
spindle. The inset shows a photograph of the crystal in the loop with the spindle and X-ray beam
position indicated.
Figure S4. Simulation of terahertz radiation absorption. (a) Simulated electric field for 0.4
THz radiation coming from the top where the capillary and crystal have been included. (b)
Normalized absorbed power in the crystal when the terahertz radiation comes from above. The
experimental incident angle and polarization were considered in the simulations. Only the
Gaussian part of the beam (84% of the total power) is considered in the simulations and the
crystal is approximated with bulk water.
Table S1. Data statistics from integration of continuous and reticular data
frames from a one, single crystal dataset.
Every 2nd
0-180°
180-360°
image
Resolution (Å)
15.0–1.7
15.0–1.7
15.0–1.7
<I/σ(I)> a
Rmerge(%)
CC1/2(%)
a, b
a
Completeness(%)
a
32.6(5.5)
26.6(3.3)
30.8(3.9)
4.7(29.9)
5.7(47.6)
7.1(52.7)
100.0(98.2)
99.9(96.5)
99.9(97.1)
97.5(92.9)
97.1(92.0)
96.7(88.9)
Redundancy
12.4
11.9
24.3
a
Values for the highest resolution shell (1.70-1.74Å) are shown in parentheses.
b
Rmerge= ƩhƩi|I(h)i-<I(h)>|/ƩhƩi|I(h)i| where I(h)i is the ith intensity measurement of a
reflection and I(h) the average intensity from multiple reflections.
Table S2. Crystal and structure refinement data for
reference data (12 crystals merged)
Even
Odd
Data Collection
Space group
P43212
P43212
Cell dimensions
a,b (Å)
79.2
79.2
c(Å)
37.5
37.5
α,β,γ(º)
90,90,90
90,90,90
Resolution(Å)
15.0-1.7
15.0-1.7
Rmerge(%)a,b
8.0(57.2)
8.0(56.9)
CC1/2a
100.0(99.3)
100.0(99.2)
<I/σ(I)> a
84.5(13.0)
84.0(13.3)
Completeness(%)a 93.4(74.8)
93.6(76.3)
Redundancy
182.0
184.2
Refinement
No. reflections
12140
12140
Rwork/Rfree(%)c
16.2/18.4
16.1/18.4
No. Atoms
(protein)
1042
1042
R.m.s. Deviations
Bond lengths(Å)
0.0231
0.0231
Bond angles(º)
2.0222
2.0224
a
Values for the highest resolution shell (1.70-1.74Å) are
shown in parentheses.
b
Rmerge= ƩhƩi|I(h)i-<I(h)>|/ƩhƩi|I(h)i| where I(h)i is the ith
intensity measurement of a reflection and I(h) the average
intensity from multiple reflections.
c
R-factor= Ʃ|Fo-Fc|/ƩFo
Table S3. The 10 strongest peaks in the
<F0.4THz>-<Foff> difference electron
density map. Peaks in helix 3 are
highlighted in red.
Rank
1
2
3
4
5
6
7
8
9
10
Peak σ
4.79
4.70
4.49
4.36
4.19
4.05
3.97
3.84
3.82
3.80
Closest residue
S86
W123
S91
A90
F34
N92
I55
K33
V120
L56
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