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Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein

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Science  06 May 2016:
Vol. 352, Issue 6286, pp. 725-729
DOI: 10.1126/science.aad5081

Visualizing a response to light

Many biological processes depend on detecting and responding to light. The response is often mediated by a structural change in a protein that begins when absorption of a photon causes isomerization of a chromophore bound to the protein. Pande et al. used x-ray pulses emitted by a free electron laser source to conduct time-resolved serial femtosecond crystallography in the time range of 100 fs to 3 ms. This allowed for the real-time tracking of the trans-cis isomerization of the chromophore in photoactive yellow protein and the associated structural changes in the protein.

Science, this issue p. 725

Abstract

A variety of organisms have evolved mechanisms to detect and respond to light, in which the response is mediated by protein structural changes after photon absorption. The initial step is often the photoisomerization of a conjugated chromophore. Isomerization occurs on ultrafast time scales and is substantially influenced by the chromophore environment. Here we identify structural changes associated with the earliest steps in the trans-to-cis isomerization of the chromophore in photoactive yellow protein. Femtosecond hard x-ray pulses emitted by the Linac Coherent Light Source were used to conduct time-resolved serial femtosecond crystallography on photoactive yellow protein microcrystals over a time range from 100 femtoseconds to 3 picoseconds to determine the structural dynamics of the photoisomerization reaction.

Trans-to-cis isomerization constitutes a major class of chemical reactions of critical importance to biology, an example of which is the light-dependent isomerization of a retinal chromophore that underlies vision (1). Because isomerization occurs on the femtosecond to picosecond time scale, ultrafast time-resolved methods are necessary to follow the reaction in real time. The spectral response after photon absorption reveals the dynamics of the molecules involved (25) but does not directly observe the associated structural changes, which have to be inferred by computational approaches (6). Until recently, it has been impossible to directly determine the structure of molecules on ultrafast time scales. With the recent availability of hard x-ray pulses on the femtosecond time scale emitted by free electron laser (FEL) sources such as the Linac Coherent Light Source (LCLS), the ultrafast femtosecond-to-picosecond time scale has become experimentally accessible (711). Photochemical reactions (12) are initiated by photon absorption, which promotes electrons into the excited state. Thereafter, the nuclei experience and the structure evolves on the excited state potential energy surface (PES) (13, 14). The shape of the surface controls the subsequent nuclear dynamics. After returning to the ground state PES, the reaction continues and is driven thermally. Although structures of longer-lived excited state intermediates have been characterized with ~100-ps time resolution at synchrotrons (1519), the femtosecond structural dynamics of ultrafast photochemical reactions can only be investigated with an x-ray FEL (11). The photoactive yellow protein (PYP) is an ideal macromolecular system with which to investigate ultrafast trans-to-cis isomerization. Its chromophore, p-coumaric acid (pCA), can be photoexcited by absorbing a photon in the blue region of the spectrum. Upon photon absorption, PYP enters a reversible photocycle involving numerous intermediates (Fig. 1A). The primary photochemical event that controls entry into the photocycle is the isomerization of pCA about its C2=C3 double bond (see Fig. 1B for the pCA geometry). The pCA chromophore remains electronically excited for a few hundred femtoseconds (3, 5, 20). Excited state dynamics is thought to drive the configurational change from trans to cis (3, 21). The chromophore pocket within the PYP protein is sufficiently flexible to allow certain relatively large atomic displacements, but also imposes structural constraints that may affect the pathway and dynamics of isomerization (22, 23). In particular, the pCA chromophore is constrained by a covalent bond to the Cys69 side chain of PYP (Fig. 1B), by unusually short hydrogen bonds between its phenolate oxygen and nearby glutamate and tyrosine side chains (24), and by a hydrogen bond between the carbonyl oxygen of its tail and the main-chain amide of Cys69.

Fig. 1 Structural dynamics of PYP.

(A) The PYP photocycle from the perspective of a time-resolved crystallographer. Approximate time scales are given. The femtosecond/picosecond time scale (in red) is structurally charted in this paper. (B) The chemical structure of the pCA chromophore. The red line marks the four atoms that define the torsional angle ϕtail about the C2=C3 bond. (C) Results of the positive control experiment at a 200-ns time delay. Reaction was initiated by femtosecond laser pulses. Negative (red) and positive (blue) DED features on the –3σ/3σ level. A mixture of the pR1 (magenta) and pR2 (red) structures is present. Main signature of pR1: features β1 and β2. Main signature of pR2: features γ1 and γ2. The structure of PYPref (dark) is in yellow.

Previously, we showed that time-resolved pump-probe serial femtosecond crystallography (TR-SFX) could be successfully carried out on PYP on the nanosecond-to-microsecond time scale. Difference electron density (DED) maps of very high quality, which compare the structures before (dark) and after (light) absorption of a photon (25), were obtained at near-atomic (1.6 Å) resolution. These experiments used a nanosecond laser pulse to initiate isomerization and subsequent structural changes. An overall reaction yield as high as 40% (25) could be reached. However, achieving femtosecond time resolution requires that a femtosecond pump laser pulse be used, which restricts the reaction yield to the much lower value of the primary quantum yield (around 10%) and correspondingly reduces the structural signal. The energy of femtosecond pulses (i.e., the number of photons per pulse) must also be limited to avoid damaging effects from their significantly higher peak power. Here, we present results of TR-SFX experiments covering the time range from 100 fs to 3 ps. We directly followed the trans-to-cis isomerization of the pCA chromophore and the concomitant structural changes in its protein environment in real time. Full details of the experiment and data analysis are provided in the supplementary materials (SM). Light-initiated structural changes in PYP were investigated at the Coherent X-ray Imaging (CXI) instrument of the LCLS (26). Electronic excitation was initiated in microcrystals of PYP by femtosecond pump laser pulses [wavelength (λ) = 450 nm]. Permanent bleaching of the chromophore was avoided by limiting the laser pulse energy to 0.8 mJ/mm2 (5.7 GW/mm2). Laser pulse duration, spectral distribution, and phase were characterized by second harmonic generation frequency-resolved optical gating (SHG-FROG) (27). The pulse duration was 140 ± 5 fs and had both positive group delay dispersion and third-order dispersion to maximize the conversion to the excited state (28). Offline spectroscopic experiments on thin crushed crystals of PYP had established that photoexcitation with femtosecond laser pulses under comparable conditions could be as high as 10% without inducing damage (SM). The structural changes induced by the laser pulse were probed with 40-fs x-ray FEL pulses at 9 keV (1.36 Å). Both the pump-probe and the reference x-ray diffraction data were collected at the full 120-Hz pulse repetition rate of the LCLS to a resolution of 1.6 and 1.5 Å, respectively. To address concerns that the detector response might be influenced by the stray light of the intense femtosecond laser pulse, the reference data were collected as a negative time delay, where the femtosecond laser pulse arrived 1 ps after the x-ray pulse.

To assess whether femtosecond laser pulses excited a sufficiently large number of molecules under these experimental conditions, we first performed a positive control experiment with a 200-ns pump-probe time delay, where large structural differences between the light and dark states have been well characterized (25, 29). From the pump-probe TR-SFX data and the reference data, DED maps were calculated (SM). Figure 1C shows that the femtosecond laser pulses are able to initiate sufficient entry into the photocycle to produce strong, chemically meaningful features. The 200-ns DED map is essentially identical to maps determined earlier at both the LCLS (25) and at BioCARS (29) at a time delay of 1 μs, and can be interpreted with the same mixture of intermediates, pR1 and pR2. The extent of reaction initiation is 12.6% as determined by fitting a calculated “pR1 + pR2 minus pG” difference map to the 200-ns DED map, a value which agrees with the maximum extent of excitation determined spectroscopically (7 to 10%). The femtosecond time scale was explored by using nominal settings for the time delay of 300 and 600 fs. The timing jitter between the 140-fs laser pump and 40-fs x-ray probe pulses is ~280 fs (8). The jitter was measured for every x-ray pulse by a timing tool (30, 31), which was combined with adjustments that take longer-term experimental drift into account (SM). Thus, each individual diffraction pattern was associated with a definite “time stamp.” However, due to the drift, the time stamps were non-uniformly distributed in time (fig. S1). Because the quality of structure amplitudes and of the DED maps derived from them depends on the number of diffraction patterns, indexed time-stamped diffraction patterns were binned into eight different pump-probe delays with about the same number of patterns (40,000) in each bin, spanning the time range from 100 to 1000 fs (table S1B). A set of diffraction patterns at a time delay of 3 ps was also collected. Because the jitter and drift are much smaller than the delay, time stamping was not necessary for the 3-ps or 200-ns delays. The values of R-split (table S1) for all data sets are 7.5 to 9.9%, which indicates the high quality of the diffraction data and results in DED maps of comparable good quality for all delays. Maps at seven time delays are shown in Fig. 2. Visual inspection of these maps reveals an important qualitative result. The features in all maps at delays less than 500 fs are similar (compare Fig. 2, A to C), and features in all maps at delays greater than 700 fs are also similar (compare Fig. 2, D to G) but differ from those in the first set. Consequently, there must be a structural transition between the 455- and 799-fs time delays that gives rise to the two distinct sets of features.

Fig. 2 Trans-to-cis isomerization in PYP.

Weighted DED maps in red (–3σ) and blue (3σ); front (upper) and side view (lower). Each map is prepared from about the same number of diffraction patterns, except the 3-ps map (table S1, B and C). The reference dark structure is shown in yellow throughout; structures before the transition and still in the electronic excited state PES are shown in pink; structures after the transition and in the electronic ground state PES are shown in light green. Important negative difference density features are denoted as α and positive features as β in (B) and (G). Pronounced structural changes are marked by arrows. (A to C) Time delays before the transition. (A) Twisted trans at 142 fs, ϕtail 154°. (B) Twisted trans at 269 fs, ϕtail 140°, some important residues are marked; dotted lines: hydrogen bond of the ring hydroxyl to Glu46 and Tyr42. (C) Twisted trans at 455 fs, ϕtail 144°; dotted line: direction of C2=C3 double bond. (D to G) Time delays and chromophore configuration after the transition. (D) Early cis at 799 fs, ϕtail 50°. (E) Early cis at 915 fs; dotted line: direction of C2 = C3 double bond. (F) Early cis at 1023 fs; for (E) and (F), ϕtail ~ 65°. (G) 3-ps delay; dashed line: direction of C2=C3 double bond, feature β1; ϕtail is 35°.

To identify with more precision the time delay at which this transition occurs, the time-stamped diffraction patterns were re-binned into 16 narrower time bins with about 20,000 patterns in each bin (table S1A). The resultant time series of 16 DED maps in the femtosecond time range (together with the map for the 3-ps time delay) were subjected to singular value decomposition (SVD; fig. S2B) (32). The volume occupied by the pCA chromophore, the Cys69 sulfur, and the Glu46 carboxyl was included in the analysis. When a time series exhibits a change, a corresponding change should be even more readily recognizable in the right singular vectors (rSVs). This change is evident in the magnitude of both the first and second rSVs around 550 fs (red arrow in fig. S2B). The substantial increase in the magnitude of the first rSV after 155 fs (fig. S2B) shows the earliest (fastest) evolution of the structure after excitation. We tentatively associate the structural transition at around 550 fs, which is qualitatively evident from inspection of the DED maps and more quantitatively in their SVD analysis, with the trans–to-cis isomerization of the pCA chromophore. The transition occurs within ~180 fs (fig. S2B), but its exact duration needs to be further established. Rate kinetics would require that after a ~500-fs dwell time, the transition time would be stretched beyond the bandwidth-limited rate. Yet the observed transition time matches the experimental bandwidth of 3.15 THz. Therefore, the ensemble phase relation imparted by the optical pulse appears to be maintained for the duration of the dwell time, which may be supported by coherent motion. Although no oscillatory motion was detected in the TR-SFX data (they may be masked by the non-uniform data sampling), the time delay is, however, within the vibrational dephasing time of the PYP S1 state (3) and ground state modes in proteins (33). We further propose that at ~550 fs, the system lies at or very close to a conical intersection (20) (fig. S8), a branch point from which molecules either continue toward the cis configuration and enter the photocycle, or revert to the trans configuration and return to the resting (dark) state.

To identify the isomerization, refined structures before and after the transition are required. Initially, data from bins with 40,000 indexed diffraction patterns each were used, and preliminary PYP structures were refined against these data. Refinement details are in the SM. The three bins with the shortest delays can be interpreted as having chromophores in a twisted trans configuration (Fig. 2, A to C). After 700 fs, the configuration is near cis (Fig. 2, D and E). The time course of the refined ϕtail torsional angles can be fit with a transition time identical to that observed in the second rSV (Fig. 3). We took advantage of the similarity of the DED maps for extended time ranges before and after the transition to further increase the accuracy of the refined structures. We combined the diffraction patterns into two bins: the fast time scale (100 to 400 fs, with 81,237 patterns) and a slower time scale (800 to 1200 fs, with 157,082 patterns) (table S1C). We refined the structure denoted PYPfast against the 100- to 400-fs data, and that denoted PYPslow against the 800- to 1200-fs data. The refinement statistics are presented in table S2. The DED maps are shown in insets in Fig. 3 (see also fig. S9, B and D), with the corresponding refined structures of PYPfast and PYPslow in pink and light green, respectively. The 3-ps DED map and the refined PYP3ps structure are shown in Fig. 2G. We used as many diffraction patterns as possible to refine PYPslow (fig. S12, B and D) and PYP3ps, because at the transition, roughly 30% of the excited molecules return directly to the dark state, no longer contribute to the DED maps, and reduce the signal. We emphasize that the refinement of transient structures populated on an ultrafast time scale is challenging, because these structures are very far from equilibrium and likely to be highly strained. Restraints in standard libraries are derived from structures at equilibrium and are therefore not applicable. In order to provide restraints more appropriate for this refinement, we used excited state quantum mechanics/molecular mechanics (QM/MM) calculations on PYP (20, 34) (SM). In addition, we used an iterative procedure, in which improved difference phases ϕΔF,calc were obtained and used with observed difference structure factor amplitudes during refinement (SM). The structural results of the refinement are summarized in Table 1. For the shortest time delays (up to about 450 fs), the PYP chromophore tail adopts a highly strained, twisted trans configuration, in which the C1–C2=C3–C1′ torsional angle ϕtail (shown by the red line spanning these four atoms in Fig. 1B) is ~140°. The position of the C2 = C3 double bond in PYPfast is displaced by ~1 Å behind the chromophore plane (loosely defined by the Cys69 sulfur, the tail carbonyl oxygen, and the atoms of the phenyl ring; Fig. 2, A to C). Hydrogen bonds to Glu46 and Tyr42, which are unusually short in the reference (dark) structure (24), are substantially elongated from 2.5 to 3.4 Å (Table 1). This structure is primed for the transition to cis. During the structural transition, substantial rotation about the double bond takes place. The head of the chromophore pivots about tail atom C2 and thereby aligns the C2C3 bond along the tail axis. Simultaneously, the head rotates about the C3 - C1′ single bond. (The complex motions can be effectively illustrated by using an educator’s stick model set, see fig. S3). The phenolate oxygen (Fig. 1B, O4′) moves even further away (3.6 Å, Table 1) from Glu46 (Fig. 2, D to F, and fig. S9, C and D), thereby breaking the hydrogen bond. At time delays longer than about 700 fs, ϕtail has decreased to ~50° (PYPslow, Fig. 3), which is characteristic of a cis configuration. PYPslow relaxes further toward the 3-ps structure (PYP3ps), in which the hydroxyl oxygen of the head reestablishes its hydrogen bond with Glu46 (Fig. 2G). ϕtail changes slightly to ~35°. The PYP3ps structure is already very similar to the early structures derived with 100-ps time resolution by independent synchrotron-based approaches (Table 1; Protein Data Bank entries 4I38 and 4B90) (22, 23) and has evolved only slightly from PYPslow by establishing shorter hydrogen bonds to Tyr42 and Glu46.

Fig. 3 Chromophore tail torsional angle dynamics.

Pink: twisted trans on excited state PES; light green: cis on ground state PES. Torsional angle ϕtail (solid spheres) is from structural refinement at various delays (table S3). Gray region: not time-resolved. Dashed line: fit with eq. S2, with a transition time of about 590 fs (fig. S2). Insets: structures of PYPfast (pink), PYPslow, and PYP3ps (light green), and dark-state structure PYPref in yellow. Difference electron density is shown in red (–3σ) and blue (3σ).

Table 1

Geometry of PYP structures. The PYPfast structure was refined using a data bin spanning 100 to 400 fs with 81,327 snapshots and the PYPslow structure from a bin spanning 800 to 1200 fs with 157,082 snapshots (table S1B). Structures of IT, pR0, and pB1 are from the Protein Data Bank, with the accession code in parentheses (22, 23, 47). Uncertainties of the torsional angles can be estimated to be ±20° by displacing the four atoms that define the angle with the coordinate error (0.2 Å). na, not applicable; nd, not determined.

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The structures derived from the refinements confirm that the transition at around 550 fs is indeed associated with a trans–to-cis isomerization. Theoretical considerations (20) (fig. S8) suggest that during isomerization, the PYP chromophore relaxes through a conical intersection between the electronically excited state PES and the ground state PES. Accordingly, structures between 100 and 400 fs can be identified as electronically excited, whereas the structures at time delays >700 fs can be identified with the electronic ground state. In both the excited and ground states, structural changes (i.e., translation of atoms) may also have occurred. Our experiments identified the ultrafast dynamics of both the excited state structures and the ground state structures (Figs. 2 and 3). Because we restricted our pump laser pulses to moderate power, we avoided damaging nonlinear effects (e.g., two-photon absorption), and most excited molecules populate the excited state surface S1 (5). Part of the stored energy is used to rapidly displace the chromophore by about 0.7 Å within the crowded molecular environment in the interior of PYP (Fig. 2A and Table 1). If this initial displacement is complete after 250 fs, the chromophore must have experienced an acceleration of ~2 × 1015 m/s2 and attains a final velocity of 500 m/s (SM). Figure 1B shows that nine carbon atoms, two oxygen atoms, and seven hydrogen atoms (molecular mass = 147 g/mol) are displaced. During the first few hundred femtoseconds, the force on the chromophore is ~500 pN, which is enormous compared to forces in single molecules at thermal equilibrium, which are usually only a few piconewtons (35). The origin of the force is due to the change of the potential energy surface when the chromophore is excited to the electronic excited state, which affects the intra- and intermolecular interactions of the chromophore as also inferred from ultrafast Raman spectroscopy (3). The energy required to displace the chromophore is ~0.2 eV, which is ~10% of the blue photon energy (2.76 eV) that starts the reaction. It appears that by rapidly evolving down the excited state PES, part of the photon energy is initially converted into kinetic energy, which is then released by collision of the chromophore atoms with the surrounding protein atoms that make up the chromophore pocket. The excited chromophore loses 0.12 eV of energy by intramolecular vibrational energy redistribution on the sub–100-fs time scale (36), which can be roughly estimated from the Stokes shift by comparing absorption and fluorescence spectra (3). Accordingly, ~85% of the photon energy remains stored as strain and electronic excitation in the chromophore before isomerization occurs. On passing through the conical intersection (20), the molecules either revert toward the initial dark state (30% of the excited molecules, Table 1 and table S3) or continue relaxing toward the cis isomer (70%), gradually releasing the excess energy as heat. Because the chromophore pocket tightly restricts the chromophore head displacements, further structural changes must be volume-conserving; i.e., they minimize the volume swept out by the atoms as they move. Accordingly, the chromophore performs the complex motions described above (fig. S3). Although the energy stored in the chromophore is sufficient to break the hydrogen bonds (~0.1 eV), the spatial constraints imposed by the chromophore pocket direct the reformation of the hydrogen bonding network at longer time delays (Table 1). This is a macromolecular cage effect reminiscent of the solvent cage effect in liquid chemical dynamics (37). The macromolecular cage in PYP, however, is soft enough to allow certain specific, relatively large (up to 1.3 Å, Table 1) structural changes. This contrasts with crystals of small molecules, where the stronger crystal lattice constraints usually do not allow such large displacements. Hence, biological macromolecular crystallography aimed at elucidating biological function may also provide insight into the reaction mechanisms of small molecules.

To assess global conformational changes of PYP on the femtosecond time scale, we calculated the radius of gyration Rg from each refined structure (SM). Rg fluctuates by only 0.2% in all structures from 200 fs to 200 ns (Table 1). An increase of Rg by up to 1 Å, determined by others using x-ray scattering in solution upon photodissociation of CO from CO-myoglobin (9), was not observed in our PYP crystals. Concomitant systematic large volume changes were also not apparent in PYP crystals over the first 3 ps that our data span. Our data show no evidence for a protein quake (9, 10, 38), characterized by an ultrafast and large change in Rg that occurs significantly before a large volume change. The reason for this is unclear and will require further experiments.

Ultrafast fluorescence and transient absorption spectroscopy of PYP have shown that excited state decay is multi-phasic (3, 5, 39). The fast (sub-picosecond) time constants are significantly more productive in creating the cis-like photoproduct than the slow (picosecond) time constants; the long-lived excited state population primarily decays back to the ground state (5, 36). With excitation at 450 nm, at least 50% of the total isomerization yield is generated with a dominant ~600-fs time constant (5), which agrees with our observation of a transition at ~550 fs. It should be noted that a ground state intermediate with a 3- to 6-ps life time has been proposed by ultrafast spectroscopy (36). However, under the conditions used here, the peak concentration of this intermediate is expected to be small (5). In contrast to spectroscopic techniques that reported vibrational coherence with 50 cm−1 and 150 cm−1 frequency (3, 40), we could not unambiguously detect oscillations in our data. Intense femtosecond optical pumping of PYP crystals generates both excited state and ground state vibrational coherences within the 3.15-THz experimental bandwidth (41). It will be an important goal of future experiments to structurally characterize these coherences using femtosecond TR-SFX. Nevertheless, our data show that before 400 fs, there are large distortions corresponding to a Franck-Condon (FC) excited state (42). The nuclear dynamics of the FC excited state at 100 to 200 fs agrees with the conclusions from ultrafast spectroscopy (3, 4245) that also suggest a distortion of the C2=C3 double bond on similar time scales, as in the PYPfast structure. The isomerization at 550 fs through the conical intersection between the excited state and ground state PES is in reasonable agreement with the time scales for isomerization reported by others (3, 5, 42, 46). After passing through the conical intersection, the chromophore is cis-like and still highly strained. The transiently broken hydrogen bond is reestablished quickly as the structure relaxes, exemplified by the PYP3ps structure (Fig. 3). Further relaxation on the ground state PES completes the initial phase of the isomerization.

Correction (5 May 2016): Two new references were added, 84 and 85, and previous ref. 84 was renumbered as ref. 86. The new refs. are “84. A. A. Granovsky, Communication: An efficient approach to compute state-specific nuclear gradients for a generic state-averaged multi-configuration self consistent field wavefunction. J. Chem. Phys. 143, 231101 (2015). 85. A. A. Granovsky, Firefly version 8.1.0.”

Supplementary Materials

www.sciencemag.org/content/352/6286/725/suppl/DC1

Materials and Methods

Supplementary Text

Figs. S1 to S12

Tables S1 to S5

References (4884)

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Acknowledgments: This work is supported by the NSF Science and Technology Center BioXFEL (grant NSF-1231306); by NIH grants R01GM095583 (P.F.), R01EY024363 (K.M.), and R24GM111072 (V.S., R.H., and K.M.); Helmholtz Association Virtual Institute Dynamic Pathways (H.C.); and grant NSF-0952643 (M.S.). K.P. is partly supported by grant NSF-1158138 (to D. Saldin and M.S.) and Federal Ministry of Education and Research, Germany (BMBF) grant 05K14CHA (to H.C.). J.J.v.T. acknowledges support from the Engineering and Physical Sciences Research Council via grant agreement EP/M000192/1. G.G. and D.M. are supported by the Academy of Finland, D.O. by BMBF project 05K13GUK, and M.M. by the European Union through grant FP7-PEOPLE-2011-ITN NanoMem. Use of the LCLS, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract DE-AC02-76SF00515. Part of this work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory (LLNL) under contract DE-AC52-07NA27344, and M.F. was supported by LLNL Lab-Directed Research and Development Project 012-ERD-031. C.G. thanks the PIER Helmholtz Graduate School for financial support. Parts of the sample injector used at LCLS for this research were funded by NIH grant P41GM103393, formerly P41RR001209. We thank the Moscow State University supercomputing center and the Finnish IT Center for Science (CSC-IT) Center for Science, Finland, for computing resources. We thank M. Hunter for valuable discussions, T. Graen for help with the computer simulations, and C. Li for assistance with injectors. The PYPref, PYPfast, PYPslow, PYP3ps, and PYP200ns structures are deposited in the Protein Data Bank together with their respective weighted difference structure factor amplitudes under accession codes 5HD3, 5HDC, 5HDD, 5HDS, and 5HD5, respectively. M.S. prepared the proposal with input from J.J.vT., K.M., V.S., J.C.H.S., H.N.C., A.O., and P.F.; A.A., S.B., M.L., J.S.R., and J.E.K. operated the CXI instrument, including the time tool and the femtosecond laser; and K.P., A.B., J.T., S.B., T.A.W., N.Z., O.Y., and T.D.G. analyzed the SFX data. C.D.M.H and J.J.vT. set up the FROG at the CXI instrument; G.G. and D.M. performed QM/MM calculations; J.T., J.B., D.O., P.L.X., C.G., C.K., and M.S. prepared protein and grew nano- and microcrystals; D.DeP., C.K., C.C., S.R.-C., J.D.C., M.M., G. K., and U.W. provided and operated the injector system; M.F., R.F., M.S., J.T., P.F., D.O., and C.G. wrote the electronic log; M.F., M.S, J.T., J.S.R., J.J.vT., and K.M. discussed femtosecond laser excitation; J.T., M.S, V.S, R.H, C.D.M.H., and J.J.vT. performed preliminary ultrafast experiments on crystals; M.S. calculated and analyzed the difference maps; and M.S., K.P., K.M., G.G., P.F., and J.J.vT. wrote the manuscript, with improvements from all authors.
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