Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin
- Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Japan
- Graduate School of Life Science, University of Hyogo, Japan
- Graduate School of Engineering, Nagoya Institute of Technology, Japan
- Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Germany
- RIKEN SPring-8 Center, Japan
- Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Japan
- Department of Bioengineering Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Japan
- Department of Chemistry, Graduate School of Science, Kobe University, Japan
- Japan Synchrotron Radiation Research Institute, Japan
- Department of Chemistry, Graduate School of Science, Kyoto University, Japan
Figures
Figure 1 with 1 supplement
Photocycle model of channelrhodopsin.
Schematic model of the C1C2 photocycle. Superscript of each reaction intermediate (P1(520), P2(390), P3(520), P4(480)) indicates the wavelength of maximum absorption. Open/close states of the …
Figure 1—figure supplement 1
Sequence alignment of channelrhodopsin (ChR) variants.
Amino acid sequences of major variants of ChRs are shown. Conserved residues are highlighted in gray and red. The residues in red indicate important residues for the initial trigger revealed by the …
Figure 2 with 3 supplements
Flash photolysis and flash photo activation measurement of C1C2.
(a) Transient absorption spectra of C1C2 reconstituted in POPE/POPG (protein/lipid molar ratio = 1/50), 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5% glycerol, and 0.01% cholesteryl hemisuccinate (CHS). …
Figure 2—figure supplement 1
Flash photolysis measurements of C1C2 solution in n-dodecyl-β-D-maltoside (DDM).
(a) Absorption spectrum of purified C1C2 solubilized in DDM detergent. (b) Transient absorption spectra of C1C2 in DDM. (c) Time traces of absorption changes of C1C2 in DDM at 445 (green), 375 …
Figure 2—figure supplement 2
Voltage-clamp recordings in HEK293 cells of photocurrents from C1C2.
Normalized, log-binned and averaged photocurrents of the C1C2 protein (mean ± SEM, n = 3 - 5) with pHi/e 6.0 (a), 7.2 (b), and 8.0 (c). Red arrows indicate the inward directed current caused by …
Figure 2—figure supplement 3
Flash photolysis measurements of C1C2 crystals and C1C2 solution in n-dodecyl-β-D-maltoside (DDM).
(a and b) Transient difference absorption spectra recorded from the C1C2 crystals (a) and C1C2 solution in DDM (b). The time evolution is indicated from red to blue. Dashed lines indicate the …
Figure 3 with 3 supplements
Microcrystals for serial femtosecond crystallography (SFX) experiment and C1C2 SFX structure.
(a) Lipidic cubic phase (LCP) crystals of C1C2 optimized for the time-resolved SFX (TR-SFX) experiments. The orange scale bar on the lower right indicates 50 μm, with 5 μm sub-scaling lines. The …
Figure 3—figure supplement 1
Electron density of retinal.
A stereo view of the 2Fo–Fc electron density map for the retinal binding pocket, shown as a mesh representation contoured at 0.9σ. The all-trans retinal and the surrounding residues are indicated by …
Figure 3—figure supplement 2
Comparison of the crystal packing between C1C2 and CrChR2.
(a and b) Comparison of the crystal packing between C1C2 (PDB: 3UG9) (a) and CrChR2 (PDB: 6EID) (b). The crystal packing of C1C2 shows minimal packing interactions, as compared to the CrChR2 crystal …
Figure 3—figure supplement 3
Schematic model of the time-resolved serial femtosecond crystallography setup.
(a) The lipidic cubic phase (LCP) microjet continuously transports microcrystals across the focused X-ray free electron laser (XFEL) beam. X-ray diffraction is recorded on a detector for each XFEL …
Figure 4 with 5 supplements
Difference Fourier electron density map and structural changes around TM7 and TM3.
Views of the |Fobs|light− |Fobs|dark difference Fourier electron density maps and the structural changes around TM7 (a–e) and TM3 (f–j) for 1 μs (a and f), 50 μs (b and g), 250 μs (c and h), 1 ms (d …
Figure 4—figure supplement 1
Overview of the |Fobs|light− |Fobs|dark difference Fourier electron density maps for five time points.
Green represents positive difference electron density (contoured at +3.1 to 3.3σ, where sigma represents the root mean square electron density of the unit cell) and purple represents negative …
Figure 4—figure supplement 2
Stereo views of the difference Fourier electron density maps and structural changes.
Stereo views of the |Fobs|light− |Fobs|dark difference Fourier electron density maps and the structural changes of C1C2. Each time point is indicated in the respective maps. The structural model …
Figure 4—figure supplement 3
Electron density of extrapolated map.
A stereo view of the extrapolated electron density map for the retinal binding pocket, shown as a mesh representation contoured at 1.2σ. The 13-cis retinal and the surrounding residues are indicated …
Figure 4—figure supplement 4
Calculated difference |Fcalc|light− |Fcalc|dark map and structural changes around TM7 and TM3.
Views of the structural changes around TM7 (a–e) and TM3 (f–j) for 1 μs (a and f), 50 μs (b and g), 250 μs (c and h), 1 ms (d and i), and 4 ms (e and j). Light green and orange mesh indicate …
Figure 4—figure supplement 5
Difference Fourier maps along the putative ion pore.
Difference maps of different time delays are shown for the inner-gate (left), central-gate (middle), and extracellular water access channel (right). Each time point and contour level is indicated in …
Figure 5
Conformational change of retinal and comparison with QM/MM model.
(a) Intracellular view of the |Fobs|light− |Fobs|dark difference Fourier electron density map and the structural changes around the retinal. Green and purple meshes indicate positive and negative …
Figure 6
Schematic model of the C1C2 channel opening.
The inner gate formed by Glu121, Glu122, His173, and Arg307 prevents water influx in the initial dark-adapted state. After the retinal isomerization reaction, the retinal twists and shifts toward …
Videos
Video 1
Overview of the |Fobs|light− |Fobs|dark difference.
Fourier electron density maps for five time points and structural changes. Views of the |Fobs|light− |Fobs|dark difference Fourier electron density maps and the structural changes. Green and purple …
Additional files
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Supplementary file 1
Crystallographic data and refinement statistics.
Values in parenthesis are those of the highest resolution shell.
- https://cdn.elifesciences.org/articles/62389/elife-62389-supp1-v1.docx
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Supplementary file 2
Refinement statistics associated with extrapolated data.
Because of the large variance of the extrapolated structure factor amplitude, the R values tend to be worse.
- https://cdn.elifesciences.org/articles/62389/elife-62389-supp2-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/62389/elife-62389-transrepform-v1.pdf
