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Time-resolved x-ray crystallography (TR-X) at synchrotrons and free electron lasers is a promising technique for recording dynamics of molecules at atomic resolution. While experimental methods for TR-X have proliferated and matured, data analysis is often difficult. Extracting small, time-dependent changes in signal is frequently a bottleneck for practitioners. Recent work demonstrated this challenge can be addressed when merging redundant observations by a statistical technique known as variational inference (VI). However, the variational approach to time-resolved data analysis requires identification of successful hyperparameters in order to optimally extract signal. In this case study, we present a successful application of VI to time-resolved changes in an enzyme, DJ-1, upon mixing with a substrate molecule, methylglyoxal. We present a strategy to extract high signal-to-noise changes in electron density from these data. Furthermore, we conduct an ablation study, in which we systematically remove one hyperparameter at a time to demonstrate the impact of each hyperparameter choice on the success of our model. We expect this case study will serve as a practical example for how others may deploy VI in order to analyze their time-resolved diffraction data., Competing Interests: The authors have no conflicts to disclose., (© 2024 Author(s).)

Time-resolved X-ray crystallography (TR-X) at synchrotrons and free electron lasers is a promising technique for recording dynamics of molecules at atomic resolution. While experimental methods for TR-X have proliferated and matured, data analysis is often difficult. Extracting small, time-dependent changes in signal is frequently a bottleneck for practitioners. Recent work demonstrated this challenge can be addressed when merging redundant observations by a statistical technique known as variational inference (VI). However, the variational approach to time-resolved data analysis requires identification of successful hyperparameters in order to optimally extract signal. In this case study, we present a successful application of VI to time-resolved changes in an enzyme, DJ-1, upon mixing with a substrate molecule, methylglyoxal. We present a strategy to extract high signal-to-noise changes in electron density from these data. Furthermore, we conduct an ablation study, in which we systematically remove one hyperparameter at a time to demonstrate the impact of each hyperparameter choice on the success of our model. We expect this case study will serve as a practical example for how others may deploy VI in order to analyze their time-resolved diffraction data.

Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to poor experimental access. This shortcoming is evident with Escherichia coli dihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we describe ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments to map the conformational dynamics of the Michaelis complex of DHFR. We resolve coupled global and local motions and find that these motions are engaged by the protonated substrate to promote efficient catalysis. This result suggests a fundamental design principle for multistep enzymes in which pre-existing dynamics enable intermediates to drive rapid electrostatic reorganization to facilitate subsequent chemical steps., Competing Interests: Competing interests statement:The authors declare no competing interest.

A major goal in biomedical science is to move beyond static images of proteins and other biological macromolecules to the internal dynamics underlying their function. This level of study is necessary to understand how these molecules work and to engineer new functions and modulators of function. Stemming from a visionary commitment to this problem by Keith Moffat decades ago, a community of structural biologists has now enabled a set of x-ray scattering technologies for observing intramolecular dynamics in biological macromolecules at atomic resolution and over the broad range of timescales over which motions are functionally relevant. Many of these techniques are provided by BioCARS, a cutting-edge synchrotron radiation facility built under Moffat leadership and located at the Advanced Photon Source at Argonne National Laboratory. BioCARS enables experimental studies of molecular dynamics with time resolutions spanning from 100 ps to seconds and provides both time-resolved x-ray crystallography and small- and wide-angle x-ray scattering. Structural changes can be initiated by several methods-UV/Vis pumping with tunable picosecond and nanosecond laser pulses, substrate diffusion, and global perturbations, such as electric field and temperature jumps. Studies of dynamics typically involve subtle perturbations to molecular structures, requiring specialized computational techniques for data processing and interpretation. In this review, we present the challenges in experimental macromolecular dynamics and describe the current state of experimental capabilities at this facility. As Moffat imagined years ago, BioCARS is now positioned to catalyze the scientific community to make fundamental advances in understanding proteins and other complex biological macromolecules., Competing Interests: The authors have no conflicts to disclose., (© 2024 Author(s).)

Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to lack of experimental access. This shortcoming is evident with E. coli dihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we present ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments that enable identification of coupled conformational changes in DHFR. We identify a global hinge motion and local networks of structural rearrangements that are engaged by substrate protonation to regulate solvent access and promote efficient catalysis. The resulting mechanism shows that DHFR's two-step catalytic mechanism is guided by a dynamic free energy landscape responsive to the state of the substrate., Competing Interests: Declaration of Interests The authors declare no competing interests.

Serial x-ray crystallography can uncover binding events, and subsequent chemical conversions occurring during enzymatic reaction. Here, we reveal the structure, binding and cleavage of moxalactam antibiotic bound to L1 metallo-β-lactamase (MBL) from Stenotrophomonas maltophilia. Using time-resolved serial synchrotron crystallography, we show the time course of β-lactam hydrolysis and determine ten snapshots (20, 40, 60, 80, 100, 150, 300, 500, 2000 and 4000 ms) at 2.20 Å resolution. The reaction is initiated by laser pulse releasing Zn 2+ ions from a UV-labile photocage. Two metal ions bind to the active site, followed by binding of moxalactam and the intact β-lactam ring is observed for 100 ms after photolysis. Cleavage of β-lactam is detected at 150 ms and the ligand is significantly displaced. The reaction product adjusts its conformation reaching steady state at 2000 ms corresponding to the relaxed state of the enzyme. Only small changes are observed in the positions of Zn 2+ ions and the active site residues. Mechanistic details captured here can be generalized to other MBLs., (© 2022. The Author(s).)

Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B., (© Tadeo Moreno-Chicano et al. 2022.)

Photosynthetic water oxidation is a fundamental process that sustains the biosphere. A Mn 4 Ca cluster embedded in the photosystem II protein environment is responsible for the production of atmospheric oxygen. Here, time-resolved x-ray emission spectroscopy (XES) was used to observe the process of oxygen formation in real time. These experiments reveal that the oxygen evolution step, initiated by three sequential laser flashes, is accompanied by rapid (within 50 μs) changes to the Mn Kβ XES spectrum. However, no oxidation of the Mn 4 Ca core above the all MnIV state was detected to precede O-O bond formation, and the observed changes were therefore assigned to O-O bond formation dynamics. We propose that O-O bond formation occurs prior to the transfer of the final (4th) electron from the Mn 4 Ca cluster to the oxidized tyrosine Y Z residue. This model resolves the kinetic limitations associated with O-O bond formation, and suggests an evolutionary adaptation to avoid releasing of harmful peroxide species.

Silkworm silk has attracted considerable attention in recent years due to its excellent mechanical properties, biocompatibility, and promising applications in biomedical sector. However, a clear understanding of the molecular structure and the relationship between the excellent mechanical properties and the silk protein sequences are still lacking. This study carries out a thorough comparative structural analysis of silk fibers of four silkworm species ( Bombyx mori, Antheraea pernyi, Samia cynthia ricini, and Antheraea assamensis). A combination of characterization techniques including scanning electron microscopy, mechanical test, synchrotron X-ray diffraction, Fourier transform infrared spectroscopy (FTIR), and NMR spectroscopy was applied to investigate the morphologies, mechanical properties, amino acid compositions, nanoscale organizations, and molecular structures of various silkworm silks. Furthermore, the structure-property relationship is discussed by correlating the molecular structural features of silks with their mechanical properties. The results show that a high content of β-sheet structures and a high crystallinity would result in a high Young's modulus for silkworm silk fibers. Additionally, a low content of β-sheet structures would result in a high extensibility.

The internal mechanics of proteins-the coordinated motions of amino acids and the pattern of forces constraining these motions-connects protein structure to function. Here we describe a new method combining the application of strong electric field pulses to protein crystals with time-resolved X-ray crystallography to observe conformational changes in spatial and temporal detail. Using a human PDZ domain (LNX2 PDZ2 ) as a model system, we show that protein crystals tolerate electric field pulses strong enough to drive concerted motions on the sub-microsecond timescale. The induced motions are subtle, involve diverse physical mechanisms, and occur throughout the protein structure. The global pattern of electric-field-induced motions is consistent with both local and allosteric conformational changes naturally induced by ligand binding, including at conserved functional sites in the PDZ domain family. This work lays the foundation for comprehensive experimental study of the mechanical basis of protein function.

This study provides a detailed secondary structural characterization of major ampullate dragline silk from Latrodectus hesperus (black widow) spiders. X-ray diffraction results show that the structure of black widow major ampullate silk fibers is comprised of stacked β-sheet nanocrystallites oriented parallel to the fiber axis and an amorphous region with oriented (anisotropic) and isotropic components. The combination of two-dimensional (2D) (13)C-(13)C through-space and through-bond solid-state NMR experiments provide chemical shifts that are used to determine detailed information about the amino acid motif secondary structure in black widow spider dragline silk. Individual amino acids are incorporated into different repetitive motifs that make up the majority of this protein-based biopolymer. From the solid-state NMR measurements, we assign distinct secondary conformations to each repetitive amino acid motif and, hence, to the amino acids that make up the motifs. Specifically, alanine is incorporated in β-sheet (poly(Alan) and poly(Gly-Ala)), 3(1)-helix (poly(Gly-Gly-Xaa), and α-helix (poly(Gln-Gln-Ala-Tyr)) components. Glycine is determined to be in β-sheet (poly(Gly-Ala)) and 3(1)-helical (poly(Gly-Gly-X(aa))) regions, while serine is present in β-sheet (poly(Gly-Ala-Ser)), 3(1)-helix (poly(Gly-Gly-Ser)), and β-turn (poly(Gly-Pro-Ser)) structures. These various motif-specific secondary structural elements are quantitatively correlated to the primary amino acid sequence of major ampullate spidroin 1 and 2 (MaSp1 and MaSp2) and are shown to form a self-consistent model for black widow dragline silk.

It is well-known that biological samples undergo X-ray-induced degradation. One of the fastest occurring X-ray-induced processes involves redox modifications (reduction or oxidation) of redox-active cofactors in proteins. Here we analyze room-temperature data on the photoreduction of Mn ions in the oxygen-evolving complex (OEC) of photosystem II, one of the most radiation damage-sensitive proteins and a key constituent of natural photosynthesis in plants, green algae, and cyanobacteria. Time-resolved X-ray emission spectroscopy with wavelength-dispersive detection was used to collect data on the progression of X-ray-induced damage. A kinetic model was developed to fit experimental results, and the rate constant for the reduction of OEC Mn(III) and Mn(IV) ions by solvated electrons was determined. From this model, the possible kinetics of X-ray-induced damage at a variety of experimental conditions, such as different rates of dose deposition as well as different excitation wavelengths, can be inferred. We observed a trend of increasing dosage threshold prior to the onset of X-ray-induced damage with increasing rates of dose deposition. This trend suggests that experimentation with higher rates of dose deposition is beneficial for measurements of biological samples sensitive to radiation damage, particularly at pink beam and X-ray free electron laser sources.

The paradigm of "detection-before-destruction" was tested for a metalloprotein complex exposed at room temperature to the high x-ray flux typical of third generation synchrotron sources. Following the progression of the x-ray induced damage by Mn Kβ x-ray emission spectroscopy, we demonstrated the feasibility of collecting room temperature data on the electronic structure of native Photosystem II, a trans-membrane metalloprotein complex containing a Mn(4)Ca cluster. The determined non-damaging observation timeframe (about 100 milliseconds using continuous monochromatic beam, deposited dose 1*10(7) photons/µm(2) or 1.3*10(4) Gy, and 66 microseconds in pulsed mode using pink beam, deposited dose 4*10(7) photons/µm(2) or 4.2*10(4) Gy) is sufficient for the analysis of this protein's electron dynamics and catalytic mechanism at room temperature. Reported time frames are expected to be representative for other metalloproteins. The described instrumentation, based on the short working distance dispersive spectrometer, and experimental methodology is broadly applicable to time-resolved x-ray emission analysis at synchrotron and x-ray free-electron laser light sources.

Synchrotron X-ray micro-diffraction experiments were carried out on Nephila clavipes (NC) and Argiope aurantia (AA) major (MA) and minor ampullate (MiA) fibers that make up dragline spider silk. The diffraction patterns show a semi-crystalline structure with β-poly(l-alanine) nanocrystallites embedded in a partially oriented amorphous matrix. A superlattice reflection 'S' diffraction ring is observed, which corresponds to a crystalline component larger in size and is poorly oriented, when compared to the β-poly(l-alanine) nanocrystallites that are commonly observed in dragline spider silks. Crystallite size, crystallinity and orientation about the fiber axis have been determined from the wide-angle X-ray diffraction (WAXD) patterns. In both NC and AA, the MiA silks are found to be more highly crystalline, when compared with the corresponding MA silks. Detailed analysis on the amorphous matrix shows considerable differences in the degree of order of the oriented amorphous component between the different silks studied and may play a crucial role in determining the mechanical properties of the silks.

The two Flag/MaSp 2 silk proteins produced recombinantly were based on the basic consensus repeat of the dragline silk spidroin 2 protein (MaSp 2) from the Nephila clavipes orb weaving spider. However, the proline-containing pentapeptides juxtaposed to the polyalanine segments resembled those found in the flagelliform silk protein (Flag) composing the web spiral: (GPGGX(1) GPGGX(2))(2) with X(1) /X(2) = A/A or Y/S. Fibers were formed from protein films in aqueous solutions or extruded from resolubilized protein dopes in organic conditions when the Flag motif was (GPGGX(1) GPGGX(2))(2) with X(1) /X(2) = Y/S or A/A, respectively. Post-fiber processing involved similar drawing ratios (2-2.5×) before or after water-treatment. Structural (ssNMR and XRD) and morphological (SEM) changes in the fibers were compared to the mechanical properties of the fibers at each step. Nuclear magnetic resonance indicated that the fraction of β-sheet nanocrystals in the polyalanine regions formed upon extrusion, increased during stretching, and was maximized after water-treatment. X-ray diffraction showed that nanocrystallite orientation parallel to the fiber axis increased the ultimate strength and initial stiffness of the fibers. Water furthered nanocrystal orientation and three-dimensional growth while plasticizing the amorphous regions, thus producing tougher fibers due to increased extensibility. These fibers were highly hygroscopic and had similar internal network organization, thus similar range of mechanical properties that depended on their diameters. The overall structure of the consensus repeat of the silk-like protein dictated the mechanical properties of the fibers while protein molecular weight limited these same properties. Subtle structural motif re-design impacted protein self-assembly mechanisms and requirements for fiber formation., (Copyright © 2011 Wiley Periodicals, Inc.)

BioCARS, a NIH-supported national user facility for macromolecular time-resolved X-ray crystallography at the Advanced Photon Source (APS), has recently completed commissioning of an upgraded undulator-based beamline optimized for single-shot laser-pump X-ray-probe measurements with time resolution as short as 100 ps. The source consists of two in-line undulators with periods of 23 and 27 mm that together provide high-flux pink-beam capability at 12 keV as well as first-harmonic coverage from 6.8 to 19 keV. A high-heat-load chopper reduces the average power load on downstream components, thereby preserving the surface figure of a Kirkpatrick-Baez mirror system capable of focusing the X-ray beam to a spot size of 90 µm horizontal by 20 µm vertical. A high-speed chopper isolates single X-ray pulses at 1 kHz in both hybrid and 24-bunch modes of the APS storage ring. In hybrid mode each isolated X-ray pulse delivers up to ~4 × 10(10) photons to the sample, thereby achieving a time-averaged flux approaching that of fourth-generation X-FEL sources. A new high-power picosecond laser system delivers pulses tunable over the wavelength range 450-2000 nm. These pulses are synchronized to the storage-ring RF clock with long-term stability better than 10 ps RMS. Monochromatic experimental capability with Biosafety Level 3 certification has been retained.

A new method of performing Mössbauer spectroscopy with synchrotron radiation is demonstrated that involves using a high-speed periodic shutter near the focal spot of a microfocused X-ray beam. This fast microshuttering technique operates without a high-resolution monochromator and has the potential to produce much higher signal rates. It also offers orders of magnitude more suppression of unwanted electronic charge scattering. Measurement results are shown that prove the principle of the method and improvements are discussed to deliver a very pure beam of Mössbauer photons (E/ΔE ≃ 10(12)) with previously unavailable spectral brightness. Such a source will allow both Mössbauer spectroscopy in the energy domain with the many advantageous characteristics of synchrotron radiation and new opportunities for measurements using X-rays with ultra-high energy resolution.

We report the in situ observation from diffraction data of the conversion of a gas hydrate with the structure II (sII) lattice to one with the structure I (sI) lattice. Initially, the in situ formation, dissociation, and reactivity of argon gas clathrate hydrate was investigated by time-of-flight neutron powder diffraction at temperatures ranging from 230 to 263 K and pressures up to 5000 psi (34.5 MPa). These samples were prepared from deuterated ice crystals and transformed to hydrate by pressurizing the system with argon gas. Complete transformation from D(2)O ice to sII Ar hydrate was observed as the sample temperature was slowly increased through the D(2)O ice melting point. The transformation of sII argon hydrate to sI hydrate was achieved by removing excess Ar gas and exposing the hydrate to liquid CO(2) by pressurizing the Ar hydrate with CO(2). Results suggest the sI hydrate formed from CO(2) exchange in argon sII hydrate is a mixed Ar/CO(2) hydrate. The proposed exchange mechanism is consistent with clathrate hydrate being an equilibrium system in which guest molecules are exchanging between encapsulated molecules in the solid hydrate and free molecules in the surrounding gas or liquid phase.

The variation of the EPR spectra with degree of deuteration of the partially deuterated Tutton salt ammonium hexaaquacopper(II) sulfate, (NH4)2[Cu(H2O)6](SO4)2, has been measured at 293 K. The measurements indicate that the structure changes quite abruptly from that of the pure hydrogenous salt to that of the fully deuterated salt at approximately 50% deuteration. The structure of a crystal in which approximately 42% of the hydrogen atoms were replaced by deuterium was elucidated at 15 K by single-crystal time-of-flight neutron diffraction. The hexaaquacopper(II) complex exhibits an orthorhombically distorted, tetragonally elongated octahedral coordination geometry (Cu-O bond distances of 2.281(1), 2.007(1), and 1.975(1) A). The structure is very similar to that reported for the undeuterated salt at 9.6 K, and markedly different from that of the fully deuterated compound at 15 K, which has similar Cu-O bond lengths but with the directions of the long and intermediate bonds interchanged. There is no evidence for disorder or partial switching of the Cu-O bond directions. This is consistent with the temperature dependence of the EPR spectrum of the approximately 42% deuterated compound, which indicates a thermal equilibrium between the two structural forms close to room temperature similar to that reported for the undeuterated compound, but complete reversion to the low-temperature phase on cooling to 5 K. The possible influence of deuteration upon the hydrogen-bonding distances and the bearing of this upon the structural modifications of the compound are discussed.

Fusion of the elements, and alkali-metal halide when appropriate, in stoichiometric amounts in Ta containers followed by slow cooling results in high yields of the title compounds. X-ray structures refined for rhombohedral Cs(8)Ga(11) (R&thremacr;c, Z = 6, a = 9.9962(5) Å, c = 50.839(6) Å) and Cs(8)Ga(11)Cl (R&thremacr;&thremacr;c, Z = 6, a = 10.0111(7) Å, c = 50.504(6) Å) reveal isolated clusters of pentacapped, trigonal prismatic gallium anions, Ga(11)(7)(-), the former compound being isostructural with K(8)In(11) and A(8)Tl(11) (A = K, Rb, Cs). The clusters are arranged in pseudo-ccp layers separated by double layers of cesium atoms. The halide in Cs(8)Ga(11)Cl is bound in a preformed cavity within the cesium double layers where it is surrounded by eight cations. Of the nine examples reported for A(8)Tr(11)X, three chlorides occur in systems in which the binary A(8)Tr(11) do not form, Rb-Ga, Rb-In, and Cs-In. These halides are the first examples of Tr(11)(7)(-) compounds that are valence phases and do not contain an extra alkali-metal cation and electron. Magnetic susceptibility data indicate an apparently localized electron in paramagnetic Cs(8)Ga(11) and diamagnetism for Cs(8)Ga(11)Cl.

Synthesis of the phase formerly reported as Ba(5)Ga(6) succeeds only in the presence of hydrogen. The heavy atom structure of Ba(5)Ga(6)H(2) has been redetermined by single-crystal X-ray diffraction (trigonal P3c1, Z = 2, a = 7.7698(2) Å, c = 14.3902(7) Å), and the hydrogen positions have been elucidated by time-of-flight neutron powder diffraction. The unit cell contains isolated slightly distorted octahedra Ga(6)(8)(-) with barium cations over all edges. Hydride is bound in two types of barium tetrahedra [d(Ba-H) = 2.61-2.62 Å]. The stoichiometry is appropriate for a Zintl phase: (Ba(2+))(5)Ga(6)(8)(-)(H(-))(2).