Excited-state geometries determined by time-resolved synchrotron diffraction are summarized with emphasis on their comparison with a series of theoretical results. The relative merits of monochromatic and polychromatic (Laue) techniques are discussed.
Definitive experimental results on the geometry of fleeting species are at the time of writing still limited to monochromatic data collection, but methods for modifications of the polychromatic Laue data to increase their accuracy and their suitability for pump–probe experiments have been implemented and are reviewed. In the monochromatic experiments summarized, excited-state conversion percentages are small when neat crystals are used, but are higher when photoactive species are embedded in an inert framework in supramolecular crystals. With polychromatic techniques and increasing source brightness, smaller samples down to tenths of a micrometre or less can be used, increasing homogeneity of exposure and the fractional population of the excited species. Experiments described include a series of transition metal complexes and a fully organic example involving excimer formation. In the final section, experimental findings are compared with those from theoretical calculations on the isolated species. Qualitative agreement is generally obtained, but the theoretical results are strongly dependent on the details of the calculation, indicating the need for further systematic analysis.
pump–probe experiments; time-resolved diffraction; excited-state molecular geometries; excimers
The excited state structure of [Cu(1)[(1,10-phenanthroline-N,N’) bis(triphenylphosphine)] cations in their crystalline [BF4] salt has been determined at both 180 and 90K by single-pulse time-resolved synchrotron experiments with the modified polychromatic Laue method. The two independent molecules in the crystal show distortions on MLCT excitation which differ in magnitude and direction, a difference attributed to a pronounced difference in the molecular environment of the two complexes. As the excited states differ, the decay of the emission is bi-exponential with two strongly different lifetimes, the longer lifetime, assigned to the more restricted molecule, becoming more prevalent as the temperature increases. Standard deviations in the current Laue study are very much lower than those achieved in a previous monochromatic study of a Cu(I) 2,9 dimethyl-phenanthroline substituted complex (J. Am. Chem. Soc.
2009, 131, 6566), but the magnitude of the shifts on excitation is similar, indicating that lattice restrictions dominate over the steric effect of the methyl substitution. Above all the study illustrates emphatically that molecules in solids have physical properties different from those of isolated molecules and that their properties depend on the specific molecular environment. This conclusion is relevant for the understanding of the properties of molecular solid state devices which are increasingly used in current technology.
Structure determination was successfully carried out using single Laue exposures from a group of lysozyme crystals. The Laue method may be a viable option for collection of one-shot-per-crystal data from microcrystals.
Crystal size is an important factor in determining the number of diffraction patterns which may be obtained from a protein crystal before severe radiation damage sets in. As crystal dimensions decrease this number is reduced, eventually falling to one, at which point a complete data set must be assembled using data from multiple crystals. When only a single exposure is to be collected from each crystal, the polychromatic Laue technique may be preferable to monochromatic methods owing to its simultaneous recording of a large number of fully recorded reflections per image. To assess the feasibility of solving structures using single Laue images from multiple crystals, data were collected using a ‘pink’ beam at the CHESS D1 station from groups of lysozyme crystals with dimensions of the order of 20–30 µm mounted on MicroMesh grids. Single-shot Laue data were used for structure determination by molecular replacement and correct solutions were obtained even when as few as five crystals were used.
Laue diffraction; microcrystallography; X-ray optics
A new method for determination of the orientation matrix of Laue X-ray data is presented. The method is based on matching of the patterns of central reciprocal lattice rows projected on a unit sphere centered on the origin of the reciprocal lattice of the Laue data set with the corresponding pattern of a monochromatic data set on the same material.
A new method for determination of the orientation matrix of Laue X-ray data is presented. The method is based on matching of the experimental patterns of central reciprocal lattice rows projected on a unit sphere centered on the origin of the reciprocal lattice with the corresponding pattern of a monochromatic data set on the same material. This technique is applied to the complete data set and thus eliminates problems often encountered when single frames with a limited number of peaks are to be used for orientation matrix determination. Application of the method to a series of Laue data sets on organometallic crystals is described. The corresponding program is available under a Mozilla Public License-like open-source license.
LaueUtil; Laue photocrystallography; orientation matrix determination; computer programs
A RATIO method for analysis of intensity changes in time-resolved pump–probe Laue diffraction experiments is described.
A RATIO method for analysis of intensity changes in time-resolved pump–probe Laue diffraction experiments is described. The method eliminates the need for scaling the data with a wavelength curve representing the spectral distribution of the source and removes the effect of possible anisotropic absorption. It does not require relative scaling of series of frames and removes errors due to all but very short term fluctuations in the synchrotron beam.
Laue diffraction; time-resolved diffraction; ratio method; data reduction
The accuracy achieved in single-pulse pump-probe Laue experiments at beamline 14-ID at APS is estimated to be 3–4%.
The accuracy that can be achieved in single-pulse pump-probe Laue experiments is discussed. It is shown that with careful tuning of the experimental conditions a reproducibility of the intensity ratios of equivalent intensities obtained in different measurements of 3–4% can be achieved. The single-pulse experiments maximize the time resolution that can be achieved and, unlike stroboscopic techniques in which the pump-probe cycle is rapidly repeated, minimize the temperature increase due to the laser exposure of the sample.
single-pulse diffraction; accuracy; Laue method; RATIO method; photo-crystallography
Time-resolved structural studies of proteins have undergone several significant developments during the last decade. Recent developments using time-resolved X-ray methods, such as time-resolved Laue diffraction, low-temperature intermediate trapping, time-resolved wide-angle X-ray scattering and time-resolved X-ray absorption spectroscopy, are reviewed.
Proteins undergo conformational changes during their biological function. As such, a high-resolution structure of a protein’s resting conformation provides a starting point for elucidating its reaction mechanism, but provides no direct information concerning the protein’s conformational dynamics. Several X-ray methods have been developed to elucidate those conformational changes that occur during a protein’s reaction, including time-resolved Laue diffraction and intermediate trapping studies on three-dimensional protein crystals, and time-resolved wide-angle X-ray scattering and X-ray absorption studies on proteins in the solution phase. This review emphasizes the scope and limitations of these complementary experimental approaches when seeking to understand protein conformational dynamics. These methods are illustrated using a limited set of examples including myoglobin and haemoglobin in complex with carbon monoxide, the simple light-driven proton pump bacteriorhodopsin, and the superoxide scavenger superoxide reductase. In conclusion, likely future developments of these methods at synchrotron X-ray sources and the potential impact of emerging X-ray free-electron laser facilities are speculated upon.
time-resolved diffraction; structural biology; protein structural dynamics; Laue diffraction; kinetic crystallography; WAXS; XAS
Neutron diffraction data of hydrogenated recombinant urate oxidase enzyme (Rasburicase), complexed with a purine-type inhibitor 8-azaxanthin, was collected to 2.1 Å resolution from a crystal grown in D2O by careful control and optimization of crystallization conditions via knowledge of the phase diagram. Deuterium atoms were clearly seen in the neutron-scattering density map.
Crystallization and preliminary neutron diffraction measurements of rasburicase, a recombinant urate oxidase enzyme expressed by a genetically modified Saccharomyces cerevisiae strain, complexed with a purine-type inhibitor (8-azaxanthin) are reported. Neutron Laue diffraction data were collected to 2.1 Å resolution using the LADI instrument from a crystal (grown in D2O) with volume 1.8 mm3. The aim of this neutron diffraction study is to determine the protonation states of the inhibitor and residues within the active site. This will lead to improved comprehension of the enzymatic mechanism of this important enzyme, which is used as a protein drug to reduce toxic uric acid accumulation during chemotherapy. This paper illustrates the high quality of the neutron diffraction data collected, which are suitable for high-resolution structural analysis. In comparison with other neutron protein crystallography studies to date in which a hydrogenated protein has been used, the volume of the crystal was relatively small and yet the data still extend to high resolution. Furthermore, urate oxidase has one of the largest primitive unit-cell volumes (space group I222, unit-cell parameters a = 80, b = 96, c = 106 Å) and molecular weights (135 kDa for the homotetramer) so far successfully studied with neutrons.
urate oxidase; heavy water; phase diagram; neutron Laue diffraction
A 2.0 Å resolution neutron diffraction data set has been collected from a D2O-soaked γ-chymotrypsin crystal at low pH on the Institute Laue–Langevin LADI-III beamline.
The crystal preparation and preliminary neutron diffraction analysis of γ-chymotrypsin are presented. Large hydrogenated crystals of γ-chymotrypsin were exchanged into deuterated buffer via vapor diffusion in a capillary and neutron Laue diffraction data were collected from the resulting crystal to 2.0 Å resolution on the LADI-III diffractometer at the Institut Laue–Langevin (ILL) at room temperature. The neutron structure of a well studied protein such as γ-chymotrypsin, which is also amenable to ultrahigh-resolution X-ray crystallography, represents the first step in developing a model system for the study of H atoms in protein crystals.
γ-chymotrypsin; neutron diffraction
The effect of the X-ray dose on room-temperature time-resolved Laue data is discussed.
Protein X-ray structures are determined with ionizing radiation that damages the protein at high X-ray doses. As a result, diffraction patterns deteriorate with the increased absorbed dose. Several strategies such as sample freezing or scavenging of X-ray-generated free radicals are currently employed to minimize this damage. However, little is known about how the absorbed X-ray dose affects time-resolved Laue data collected at physiological temperatures where the protein is fully functional in the crystal, and how the kinetic analysis of such data depends on the absorbed dose. Here, direct evidence for the impact of radiation damage on the function of a protein is presented using time-resolved macromolecular crystallography. The effect of radiation damage on the kinetic analysis of time-resolved X-ray data is also explored.
radiation damage; X-ray dose; room temperature; time-resolved crystallography; Laue crystallography
Neutron crystallography has had an important, but relatively small role in structural biology over the years. In this review of recently determined neutron structures, a theme emerges of a field currently expanding beyond its traditional boundaries, to address larger and more complex problems, with smaller samples and shorter data collection times, and employing more sophisticated structure determination and refinement methods. The origin of this transformation can be found in a number of advances including first, the development of neutron image-plates and quasi-Laue methods at nuclear reactor neutron sources and the development of time-of-flight Laue methods and electronic detectors at spallation neutron sources; second, new facilities and methods for sample perdeuteration and crystallization; third, new approaches and computational tools for structure determination.
The polychromatic Laue technique has been applied in 100 ps delay synchrotron pump–probe experiments of the triplet excited state of a Rh(I) dinuclear complex. The observed contraction of the Rh–Rh distance of 0.154 (13) Å is less than predicted by a series of theoretical calculations, a difference attributed to the constraining effect of the crystal lattice.
X-ray free electron lasers (XFELs) are potentially revolutionary X-ray sources because of their very short pulse duration, extreme peak brilliance and high spatial coherence, features that distinguish them from today’s synchrotron sources. We review recent time-resolved Laue diffraction and time-resolved wide angle X-ray scattering (WAXS) studies at synchrotron sources, and initial static studies at XFELs. XFELs have the potential to transform the field of time-resolved structural biology, yet many challenges arise in devising and adapting hardware, experimental design and data analysis strategies to exploit their unusual properties. Despite these challenges, we are confident that XFEL sources are poised to shed new light on ultrafast protein reaction dynamics.
Preliminary neutron crystallographic data from the sweet protein thaumatin have been recorded using the LADI-III diffractometer at the Institut Laue Langevin (ILL). The results illustrate the feasibility of a full neutron structural analysis aimed at further understanding the molecular basis of the perception of sweet taste. Such an analysis will exploit the use of perdeuterated thaumatin.
A preliminary neutron crystallographic study of the sweet protein thaumatin is presented. Large hydrogenated crystals were prepared in deuterated crystallization buffer using the gel-acupuncture method. Data were collected to a resolution of 2 Å on the LADI-III diffractometer at the Institut Laue Langevin (ILL). The results demonstrate the feasibility of a full neutron crystallographic analysis of this structure aimed at providing relevant information on the location of H atoms, the distribution of charge on the protein surface and localized water in the structure. This information will be of interest for understanding the specificity of thaumatin–receptor interactions and will contribute to further understanding of the molecular mechanisms underlying the perception of taste.
thaumatin; neutron diffraction; sweet proteins
Preliminary neutron crystallographic data from the serine protease proteinase K have been recorded using the LADI-III diffractometer at the Institut Laue–Langevin. The results illustrate the feasibility of a full neutron structural analysis aimed at further understanding the catalytic mechanism of proteinase K.
A preliminary neutron crystallographic study of the proteolytic enzyme proteinase K is presented. Large hydrogenated crystals were prepared in deuterated crystallization buffer using the vapor-diffusion method. Data were collected to a resolution of 2.3 Å on the LADI-III diffractometer at the Institut Laue–Langevin (ILL) in 2.5 d. The results demonstrate the feasibility of a full neutron crystallographic analysis of this structure with the aim of providing relevant information on the location of H atoms, particularly at the active site. This information will contribute to further understanding of the molecular mechanisms underlying the catalytic activity of proteinase K and to an enriched understanding of the subtilisin clan of serine proteases.
neutron diffraction; proteinase K; serine protease
Twinned crystals of humaan MD-2 are transformed into single crystals with cryoprotectant optimization.
Twinning of crystals causes overlapping of two or more reciprocal lattice points, and hence structure amplitudes for a single crystalline domain are hardly obtained from X-ray diffraction intensities. MD-2 protein forms a stable complex with Toll-like receptor 4 and recognizes bacterial lipopolysaccharide (LPS). Excessive immune responses activated by LPS cause septic shocks. Saccharide-trimmed human MD-2 crystallizes in the tetragonal form with apparent Laue symmetry of 4/mmm, and diffraction intensities from these crystals indicate crystal twinning. The crystal consists of two different domains, A and B. The c
A axis of domain A coincides with the c
B axis of domain B with a smaller lattice, and the a
A axis corresponds to the (a
B + b
B) axis. This twinning severely imposes difficulty in structure determination. Through optimization of cryoprotectant, domain A was thoroughly transformed into domain B. The crystal containing only domain B is in space group P41212 with one MD-2 molecule in the asymmetric unit. The structure of this form of MD-2 as well as its complex with antiendotoxic lipid IVa was successfully determined using the multiple isomorphous replacement method.
crystal twinning; innate immunity; endotoxin
The LADI-III diffractometer at the Institut Laue–Langevin has been used to carry out the first neutron crystallographic study of a DNA oligonucleotide in the A conformation. The crystal size was 0.06 mm3, the smallest ever used successfully for a study of this type. The results provide evidence of unexpected base protonation and illustrate the opportunities that now exist for nucleic acid crystallography using both hydrogenated and perdeuterated oligonucleotides.
The LADI-III diffractometer at the Institut Laue–Langevin has been used to carry out a preliminary neutron crystallographic study of the self-complementary DNA oligonucleotide d(AGGGGCCCCT)2 in the A conformation. The results demonstrate the viability of a full neutron crystallographic analysis with the aim of providing enhanced information on the ion–water networks that are known to be important in stabilizing A-DNA. This is the first account of a single-crystal neutron diffraction study of A-DNA. The study was carried out with the smallest crystal used to date for a neutron crystallographic study of a biological macromolecule.
neutron crystallography; A-DNA; LADI-III
A soft X-ray angle-resolved photoemission system applicable to 100 µm crystals has been developed.
A system for angle-resolved photoemission spectroscopy (ARPES) of small single crystals with sizes down to 100 µm has been developed. Soft X-ray synchrotron radiation with a spot size of ∼40 µm × 65 µm at the sample position is used for the excitation. Using this system an ARPES measurement has been performed on a Si crystal of size 120 µm × 100 µm × 80 µm. The crystal was properly oriented on a sample stage by measuring the Laue spots. The crystal was cleaved in situ with a microcleaver at 100 K. The cleaved surface was adjusted to the beam spot using an optical microscope. Consequently, clear band dispersions along the Γ–X direction reflecting the bulk electronic states were observed with a photon energy of 879 eV.
angle-resolved photoemission spectroscopy (ARPES); soft X-ray; small crystal; microcleaving; micropositioning
Photosynthetic reaction centers from Blastochloris viridis possess Tyr-L162 located mid-way between the special pair chlorophyll (P) and the heme (heme3). While mutation of the tyrosine does not affect the kinetics of electron transfer from heme3 to P, recent time-resolved Laue diffraction studies reported displacement of Tyr-L162 in response to the formation of the photo-oxidized P+•, implying a possible tyrosine deprotonation event. pKa values for Tyr-L162 were calculated using the corresponding crystal structures. Movement of deprotonated Tyr-L162 toward Thr-M185 was observed in P+• formation. It was associated with rearrangement of the H-bond network that proceeds to P via Thr-M185 and His-L168.
Relating three-dimensional fold to function is a central challenge in RNA structural biology. Toward this goal, X-ray crystallography has long been considered the “gold standard” for structure determinations at atomic resolution, although NMR spectroscopy has become a powerhouse in this arena as well. In the area of dynamics, NMR remains the dominant technique to probe the magnitude and timescales of molecular motion. Although the latter area remains largely unassailable by conventional crystallographic methods, inroads have been made on proteins using Laue radiation on timescales of ms to ns. Proposed ‘fourth generation’ radiation sources, such as free-electron X-ray lasers, promise ps- to fs-timescale resolution, and credible evidence is emerging that supports the feasibility of single molecule imaging. At present however, the preponderance of RNA structural information has been derived from timescale and motion insensitive crystallographic techniques. Importantly, developments in computing, automation and high-flux synchrotron sources have propelled the rapidity of ‘conventional’ RNA crystal structure determinations to timeframes of hours once a suitable set of phases is obtained. With a sufficient number of crystal structures, it is possible to create a structural ensemble that can provide insight into global and local molecular motion characteristics that are relevant to biological function. Here we describe techniques to explore conformational changes in the hairpin ribozyme, a representative non-protein-coding RNA catalyst. The approaches discussed include: (i) construct choice and design using prior knowledge to improve X-ray diffraction; (ii) recognition of long-range conformational changes; and (iii) use of single-base or single-atom changes to create ensembles. The methods are broadly applicable to other RNA systems.
RNA crystallography; ribozyme; crystallization; RNA structure; crystallographic ensembles; alternate conformation; long-range motion; fold and function; difference Fourier; non-protein-coding RNA
High-resolution crystallographic studies of the hydration of the coenzyme cob(II)alamin have provided hydrogen-bond parameters of unprecedented accuracy for a biomacromolecule.
The hydration of the coenzyme cob(II)alamin has been studied using high-resolution monochromatic neutron crystallographic data collected at room temperature to a resolution of 0.92 Å on the original D19 diffractometer with a prototype 4° × 64° detector at the high-flux reactor neutron source run by the Institute Laue–Langevin. The resulting structure provides hydrogen-bonding parameters for the hydration of biomacromolecules to unprecedented accuracy. These experimental parameters will be used to define more accurate force fields for biomacromolecular structure refinement. The presence of a hydrophobic bowl motif surrounded by flexible side chains with terminal functional groups may be significant for the efficient scavenging of ligands. The feasibility of extending the resolution of this structure to ultrahigh resolution was investigated by collecting time-of-flight neutron crystallographic data during commissioning of the TOPAZ diffractometer with a prototype array of 14 modular 2° × 21° detectors at the Spallation Neutron Source run by Oak Ridge National Laboratory.
cob(II)alamin; neutron crystallography; hydration; hydrogen bonding; high resolution; D19; TOPAZ
A summary of how to run the data-reduction programs in the CCP4 suite.
This paper presents an overview of how to run the CCP4 programs for data reduction (SCALA, POINTLESS and CTRUNCATE) through the CCP4 graphical interface ccp4i and points out some issues that need to be considered, together with a few examples. It covers determination of the point-group symmetry of the diffraction data (the Laue group), which is required for the subsequent scaling step, examination of systematic absences, which in many cases will allow inference of the space group, putting multiple data sets on a common indexing system when there are alternatives, the scaling step itself, which produces a large set of data-quality indicators, estimation of |F| from intensity and finally examination of intensity statistics to detect crystal pathologies such as twinning. An appendix outlines the scoring schemes used by the program POINTLESS to assign probabilities to possible Laue and space groups.
CCP4; data reduction; data scaling; software
The currently largest perfect-crystal neutron interferometer with six beam splitters and two interference loops offers novel applications in neutron interferometry. The two additional lamellas can be used for quantitative measurements of a phase shift due to crystal diffraction in the vicinity of a Bragg condition. The arising phase, referred to as “Laue phase,” reveals an extreme angular sensitivity, which allows the detection of beam deflections of the order of 10−6 s of arc. Furthermore, a precise measurement of the Laue phase at different reflections might constitute an interesting opportunity for the extraction of fundamental quantities like the neutron–electron scattering length, gravitational short-range interactions in the sub-micron range and the Debye Waller factor. For that purpose several harmonics can be utilized at the interferometer instrument ILL-S18.
Perfect crystal interferometer; Dynamical diffraction; Beam deflection; High angular resolution; Short-range interaction; Coriolis force
The crystallization and preliminary crystallographic analysis of a para-nitrophenol 4-monooxygenase PnpA from Pseudomonas putida DLL-E4 are presented.
Para-nitrophenol 4-monooxygenase (PnpA) plays an important role in bacterial degradation of para-nitrophenol by oxidative release of the nitro group from the aromatic ring to form p-benzoquinone. In order to understand the structural basis of the function of this enzyme, PnpA was cloned, expressed in Escherichia coli and purified. PnpA was crystallized by the hanging-drop vapour-diffusion technique with PEG 4000 as precipitant. The PnpA crystals belonged to space group P212121, with unit-cell parameters a = 54.47, b = 77.56, c = 209.17 Å, and diffracted to 2.24 Å resolution.
para-nitrophenol 4-monooxygenase; Pseudomonas putida DLL-E4
We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 μs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.