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Ultrafast X-ray imaging on individual fragile specimens such as aerosols1 , metastable particles2 , superfluid quantum systems3 and live biospecimens4 provides high-resolution information that is inaccessible with conventional imaging techniques. Coherent X-ray diffractive imaging, however, suffers from intrinsic loss of phase, and therefore structure recovery is often complicated and not always uniquely defined4,5. Here, we introduce the method of in-flight holography, where we use nanoclusters as reference X-ray scatterers to encode relative phase information into diffraction patterns of a virus. The resulting hologram contains an unambiguous three-dimensional map of a virus and two nanoclusters with the highest lateral resolution so far achieved via single shot X-ray holography. Our approach unlocks the benefits of holography for ultrafast X-ray imaging of nanoscale, non-periodic systems and paves the way to direct observation of complex electron dynamics down to the attosecond timescale. Femtosecond X-ray Fourier holography imaging with record-high lateral resolution below 20 nm is demonstrated. Phase information is encoded into the interference of the diffraction patterns of a reference particle with a measurement sample.

Structural studies of biological macromolecules are severely limited by radiation damage. Traditional crystallography curbs the effects of damage by spreading damage over many copies of the molecule of interest in the crystal. X-ray lasers offer an additional opportunity for limiting damage by out-running damage processes with ultrashort and very intense X-ray pulses. Such pulses may allow the imaging of single molecules, clusters, or nanoparticles. Coherent flash imaging will also open up new avenues for structural studies on nano- and microcrystalline substances. This paper addresses the theoretical potentials and limitations of nanocrystallography with extremely intense coherent X-ray pulses. We use urea nanocrystals as a model for generic biological substances and simulate the primary and secondary ionization dynamics in the crystalline sample. The results establish conditions for ultrafast single-shot nanocrystallography diffraction experiments as a function of X-ray fluence, pulse duration, and the size of nanocrystals. Nanocrystallography using ultrafast X-ray pulses has the potential to open up a new route in protein crystallography to solve atomic structures of many systems that remain inaccessible using conventional X-ray sources.

We describe a camera to record coherent scattering patterns with a soft-x-ray free-electron laser (FEL). The camera consists of a laterally graded multilayer mirror, which reflects the diffraction pattern onto a CCD detector. The mirror acts as a bandpass filter for both the wavelength and the angle, which isolates the desired scattering pattern from nonsample scattering or incoherent emission from the sample. The mirror also solves the particular problem of the extreme intensity of the FEL pulses, which are focused to greater than 10^14 W/cm^2. The strong undiffracted pulse passes through a hole in the mirror and propagates onto a beam dump at a distance behind the instrument rather than interacting with a beam stop placed near the CCD. The camera concept is extendable for the full range of the fundamental wavelength of the free electron laser in Hamburg (FLASH) FEL (i.e., between 6 and 60 nm) and into the water window. We have fabricated and tested various multilayer mirrors for wavelengths of 32, 16, 13.5, and 4.5 nm. At the shorter wavelengths mirror roughness must be minimized to reduce scattering from the mirror. We have recorded over 30,000 diffraction patterns at the FLASH FEL with no observable mirror damage or degradation of performance.

Life is fueled by available energy. In fact, life is organized around the utilization of available energy, either from photons or from chemicals. The basic architecture of life can be understood from the following propositions: catalysts are able to control the path of chemical reactions and influence their rate by transient and reversible exchange of high-grade energy between themselves and reactants, while conserving the total energy. That, in turn, enables the synthesis of essential chemicals as well as the duplication of templates. We argue in this paper that energy utilization and evolution are emergent properties in life that are based on a small number of well-established laws of physics and chemistry governing catalysis. We propose that the relevant laws constitute a framework for biology on a level intermediate between quantum chemistry and cell biology.

Kinetic analyses led to the discovery that N-acetylated tripeptides with polar residues at P3 are inhibitors of porcine pancreatic elastase (PPE) that form unusually stable acyl-enzyme complexes. Peptides terminating in a C-terminal carboxylate were more potent than those terminating in a C-terminal amide, suggesting recognition by the oxy-anion hole is important in binding. X-ray diffraction data were recorded to 0.95-A resolution for an acyl-enzyme complex formed between PPE and N-acetyl-Asn-Pro-Ile-CO2H at approximately pH 5. The accuracy of the crystallographic coordinates allows structural issues concerning the mechanism of serine proteases to be addressed. Significantly, the ester bond of the acyl-enzyme showed a high level of planarity, suggesting geometric strain of the ester link is not important during catalysis. Several hydrogen atoms could be clearly identified and were included within the model. In keeping with a recent x-ray structure of subtilisin at 0.78 A (1), limited electron density is visible consistent with the putative location of a hydrogen atom approximately equidistant between the histidine and aspartate residues of the catalytic triad. Comparison of this high resolution crystal structure of the acyl-enzyme complex with that of native elastase at 1.1 A (2) showed that binding of the N-terminal part of the substrate can be accommodated with negligible structural rearrangements. In contrast, comparison with structures obtained as part of "time-resolved" studies on the reacting acyl-enzyme complex at >pH 7 (3) indicate small but significant structural differences, consistent with the proposed synchronization of ester hydrolysis and substrate release.

Studies on the catalytic mechanism and inhibition of serine proteases are widely used as paradigms for teaching enzyme catalysis. Ground-breaking work on the structures of chymotrypsin and subtilisin led to the idea of a conserved catalytic triad formed by the active site Ser, His and Asp residues. An oxyanion hole, consisting of the peptide amide of the active site serine and a neighbouring glycine, was identified, and hydrogen bonding in the oxyanion hole was suggested to stabilize the two proposed tetrahedral intermediates on the catalytic pathway. Here we show electron density changes consistent with the formation of a tetrahedral intermediate during the hydrolysis of an acyl–enzyme complex formed between a natural heptapeptide and elastase. No electron density for an enzyme–product complex was observed. The structures also suggest a mechanism for the synchronization of hydrolysis and peptide release triggered by the conversion of the sp2hybridized carbonyl carbon to an sp3carbon in the tetrahedral intermediate. This affects the location of the peptide in the active site cleft, triggering the collapse of a hydrogen bonding network between the peptide and the β-sheet of the active site.

The heme ligation in the isolated cdomain of Paracoccus pantotrophuscytochromecd1nitrite reductase has been characterized in both oxidation states in solution by NMR spectroscopy. In the reduced form, the heme ligands are His69-Met106, and the tertiary structure around the cheme is similar to that found in reduced crystals of intact cytochromecd1nitrite reductase. In the oxidized state, however, the structure of the isolated cdomain is different from the structure seen in oxidized crystals of intact cytochrome cd1, where the cheme ligands are His69-His17. An equilibrium mixture of heme ligands is present in isolated oxidized cdomain. Two-dimensional exchange NMR spectroscopy shows that the dominant species has His69-Met106ligation, similar to reduced cdomains. This form is in equilibrium with a high-spin form in which Met106has left the heme iron. Melting studies show that the midpoint of unfolding of the isolatedcdomain is 320.9 ± 1.2 K in the oxidized and 357.7 ± 0.6 K in the reduced form. The thermally denatured forms are high-spin in both oxidation states. The results reveal how redox changes modulate conformational plasticity around the cheme and show the first key steps in the mechanism that lead to ligand switching in the holoenzyme. This process is not solely a function of the properties of the cdomain. The role of thed1heme in guiding His17to thecheme in the oxidized holoenzyme is discussed.

Time-resolved structural studies on biomolecular function are coming of age. Focus has shifted from studies on 'systems of opportunities' to a more problem-oriented approach, addressing significant questions in biology and chemistry. An important step in this direction has been the use of physical and chemical trapping methods to capture and then freeze reaction intermediates in crystals. Subsequent monochromatic data collection at cryogenic temperatures can produce high resolution structures of otherwise elusive intermediates. The combination of diffraction methods with spectroscopic techniques provides a means to directly correlate electronic transitions with structural transitions in the sample, eliminating much of the guesswork from experiments. Studies on cytochrome P450, isopenicillin N synthase, cytochrome cd1nitrite reductase, copper amine oxidase and bacteriorhodopsin were selected as examples, and the results are discussed.

The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25 % of total soluble protein in Escherichia coli and purified by an efficient large-scale procedure. Purified protein catalysed the conversions of penicillins N and G to deacetoxycephems. Gel filtration and light scattering studies showed that in solution monomeric apo-DAOCS is in equilibrium with a trimeric form from which it crystallizes. DAOCS was crystallized ±Fe(II) and/or 2-oxoglutarate using the hanging drop method. Crystals diffracted to beyond 1.3 A˚ resolution and belonged to the R3 space group (unit cell dimensions: a = b = 106.4 A˚, c = 71.2 A˚; α = β=90 °, γ = 120 ° (in the hexagonal setting)). Despite the structure revealing that Met180 is located close to the reactive oxidizing centre of DAOCS, there was no functional difference between the wild-type and selenomethionine derivatives. X-ray absorption spectroscopic studies in solution generally supported the iron co-ordination chemistry defined by the crystal structures. The Fe K-edge positions of 7121.2 and 7121.4 eV for DAOCS alone and with 2-oxoglutarate were both consistent with the presence of Fe(II). For Fe(II) in DAOCS the best fit to the Extended X-ray Absorption Fine Structure (EXAFS) associated with the Fe K-edge was found with two His imidazolate groups at 1.96 A˚, three nitrogen or oxygen atoms at 2.11 A˚ and one other light atom at 2.04 A˚. For the Fe(II) in the DAOCS-2-oxoglutarate complex the EXAFS spectrum was successfully interpreted by backscattering from two His residues (Fe-N at 1.99 A˚), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 A˚, another O atom at 2.08 A˚ and one at 2.03 A˚. Analysis of the X-ray crystal structural data suggests a binding mode for the penicillin N substrate and possible roles for the C terminus in stabilising the enzyme and ordering the reaction mechanism.

Various enzymes use semi-stable ferryl intermediates and free radicals during their catalytic cycle, amongst them haem catalases. Structures for two transient intermediates (compounds I and II) of the NADPH-dependent catalase from Proteus mirabilis (PMC) have been determined by time-resolved X-ray crystallography and single crystal microspectrophotometry. The results show the formation and transformation of the ferryl group in the haem, and the unexpected binding of an anion during this reaction at a site distant from the haem.

Mass spectrometric screening reveals that an unmodified natural heptapeptide—human β-casomorphin-7, an internal sequence of human β-casein that possesses opioid-like activity—reacts with porcine pancreatic elastase to form an unusually stable acyl-enzyme complex at low pH. X-ray crystallographic analysis (to 1.9 Å resolution) at pH 5 shows continuous electron density linking the C-terminal isoleucine of β-casomorphin-7 to Ser 195 through an ester bond. The structure reveals a well defined water molecule (Wat 317), equidistant between the carbon of the ester carbonyl and Nε2 of His 57. Deprotonation of Wat 317 will produce a hydroxide ion positioned to attack the ester carbonyl through the favoured Bürgi-Dunitz trajectory.

Attempts to use X-ray crystallography to extract three-dimensional information on transient phenomena in crystals have been hampered primarily by long data collection times. Here we report on the first difference Fourier map obtained from Laue diffraction1–8photographs of a protein crystal, glycogen phosphorylase b. Data collection time was 3 s using the high-intensity white X-radiation generated on the wiggler magnet9of the Daresbury Synchrotron Radiation Source (SRS), but data acquisition in the millisecond-submillisecond range is possible. The method presented here uses a simple difference technique and was designed to analyse structural changes relative to a known starting structure. The combination of this approach with cine techniques allows the recording of three-dimensional motion pictures at atomic resolution and opens up new areas in structural biology and chemistry.

Cytochrome c₅₅₁ peroxidase is a periplasmic enzyme expressed in Pseudomas aeruginosa at low oxygen tensions. The glycosylated enzyme has been purified to homogeneity and crystallized by vapour diffusion techniques using polyethylene glycol 2000 as the precipitant in the presence of isopropanol. The crystals belong to the trigonal space group P3₁21 or P3₂21 with unit cell dimensions of a = b = 113·8 Å, c = 72·0 Å. They are suitable for X-ray analysis and diffract to d_min = 2·5 Å. There is one peroxidase molecule in the crystallographic asymmetric unit. Copyright 1993, 1999 Academic Press

Recombinant isopencillin N synthase from Cephalosporium acremoniumwas expressed in Escherichia coliand the protein was purified. After nearly 5000 crystallization trials, the apo enzyme was crystallized by the hanging drop vapour diffusion technique, using polyethylene glycol and lithium sulphate as precipitants. Two crystals forms have been obtained with either octahedral or elongated prismatic habits. The larger octahedral crystals (0·1 mm over-all dimensions) belong to space group I4 with unit cell dimensions of a= b= 124·7 Å, c= 156·9 Å, and diffract X-rays to about 3·5 Å resolution at synchrotrons. The crystallographic asymmetric unit contains a dimer.

Single crystals of cytochrome cd₁ nitrite reductase from Thiosphaera pantotropha have been grown in the presence of ammonium sulphate by vapour diffusion techniques. Crystals are suitable for X-ray analysis and diffract to d_min = 1·5 Å. They belong to space group P2₁ with unit cell dimensions of a = 107·8 Å, b = 61·9 Å, c = 101·2 Å, β = 112·5°. The crystallographic asymmetric unit contains a dimer. Copyright 1993, 1999 Academic Press

Results of coherent diffractive imaging experiments performed with soft X-rays (1-2 keV) at the Linac Coherent Light Source are presented. Both organic and inorganic nano-sized objects were injected into the XFEL beam as an aerosol focused with an aerodynamic lens. The high intensity and femtosecond duration of X-ray pulses produced by the Linac Coherent Light Source allow structural information to be recorded by X-ray diffraction before the particle is destroyed. Images were formed by using iterative methods to phase single shot diffraction patterns. Strategies for improving the reconstruction methods have been developed. This technique opens up exciting opportunities for biological imaging, allowing structure determination without freezing, staining or crystallization.