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, QM/MM and MD simulations ? Excited state QM/MM simulations: calculation of protonation states ????????

, Excited state QM/MM simulations: molecular dynamics simulations ???????? 8 ? Excited state QM/MM simulations: method ?????????????????

, ? Excited state QM/MM simulations : chromophore models in vacuum ???????... 10 ? Excited state QM/MM simulations of the chromophore in rsEGFP2 ????????

, 13 ? Excited state classical MD simulations : MD protocol ?????????????.. 15 ? Excited state classical MD simulations: protein dynamics with the S 0 chromophore ??? 15 ? Excited state classical MD simulations, ? DFT calculations of the cationic ground-state chromophore in rsEGFP2 ??????? 13 ? Excited state classical MD simulations: force field ??????????????

, ? S 0 -S 1 energy gap and oscillator strengths calculated from QM and QM/MM simulations ? 16

, ? Microcrystal growth, preparation and injection ????????????????, vol.18

, Pre-illumination of microcrystals to convert rsEGFP2 from the on to the off state ???? 19

, ? Femtosecond pump-laser excitation of rsEGFP2 crystals in TR-SFX experiments ???, p.19

, ? SFX data collection and on-line monitoring ?????????????????.. 20 ? Calculation of absorbed X-ray dose ????????????????????, vol.21

, ? Time-sorting diffraction images based on arrival time diagnostics ?????????.. 21 ? Hit-finding and sorting of laser-on and laser-off data ??????????????, p.22

, 23 ? 1-ps intermediate-state structure: data collection, modeling and refinement ??????, p.26

, ? Difference Fourier maps calculated with data collected after a 3 ps pump-probe delay ??, p.32

, Site-directed mutagenesis, purification and spectroscopic characterisation of rsEGFP2 and rsEGFP2-V151A ?????????????????????, p.33

+. , We hypothesize that the similarity of model P to the off-state chromophore indicates that no major conformational changes in the protein scaffold accompanies the slight change in chromophore conformation. Consequently, conformational changes in the difference-refined laser-on-?1ps structure with respect to the off-state structure are interpreted as accompanying chromophore twisting towards model T. The most notable conformational change is that occurring in the side chain of Thr204 (?-strand-10), which rotates by 120° in model T (Supplementary Fig. 23). Hence, the on-state, the off-state and model T are each associated to a specific rotamer of Thr204, The chromophore conformation in model T, however, displays an imidazolinone ring that is nearly perpendicular to that of the phenol group (Fig. 4c, d)

, Å from Wat356 and Wat17, respectively. Wat17 is H-bonded to Gln184(N?1) and

, As Ser164O? is itself a donor in the H-bond it forms with Tyr52(O), the chromophore OH is an obligate donor in the H-bond it establishes with Wat17 in the off-state. The same applies to Wat356, Ser164(O?) in both the off-state structure and in model P

, Upon transition of the chromophore to model T, steric hindrance forces Thr204 side chain to rotate by 120°, resulting in the breaking of its H-bond with Wat356, and in the latter becoming available for H-bonding to the chromophore phenol-OH. Upon transition to model T and formation of this H-bond, that with Wat17 is forced to break, since the chromophore OH can only be donor in a single H-bond. Thus, this water was kept in model P, but not in model T (Supplementary Fig. 23). Also due to steric hindrance, His149 side chain draws back from the chromophore phenol, but no rotation of its side chain is suggested by the data. Around the imidazolinone ring of the chromophore, side chains of several residues display conformational changes, His149(O), Val151(N) and Leu202(O) and Thr204(OH) in the off-state structure of rsEGFP2

, This DDM illustrates that in addition to the above-described local-changes, the ?-barrel expands upon the transition from the off-state to model T, and thus offers a peek into concerted conformational changes across the protein, A distance difference matrix (DDM) was calculated between the difference refined laser-on?1ps structure and the off-state conformer (Supplementary Fig. 27a)

. Ligase, Digestion of the template methylated DNA was achieved by incubating the PCR product with 10 U DpnI

, µL/50 µL cells) for DNA minipreps and sequencing. Fluorescent proteins (rsEGFP2 and rsEGFP2-V151A) fused to an N-terminal polyhistidine tag were

, After cell lysis, the fluorescent proteins were purified by Ni-NTA affinity chromatography. Absorption spectra were recorded using a Jasco V-630 UV/VIS photospectrometer

E. Avantes, The Netherlands) coupled with optic fibres to a cuvette holder

, Photoinduced fluorescence switching cycles were measured as described 34 at low laser power densities: 6.7 mW/cm² at 488 nm (to both excite and switch-off fluorescence) and 1 mW/cm² at 405 nm (to promote off-to-on photoswitching), Molar extinction coefficients of on and off

, mJ/cm 2 ) and (e, j) pre-exponential factors associated to the kinetics at 385, 455, 530 and 650 nm

, Ultrafast transient difference absorption spectra of rsEGFP2 (off (a, d, g), from 0.4 to 1.4 ps (b, e, h), and from 1.6 to 9.9 ps (c, f, i) for three different pump power densities (13 GW cm -2 (left), 68 GW cm -2 (middle), and 140 GW cm -2 (right). are averaged over several, vol.4

, Raw data (blue) were fitted (red) using a kinetic model involving one-to-one reversible conversion between the on-and the off-state, Cyan and violet lines represent the illumination times at 488-and 405-nm, vol.30

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