Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography.

Riboswitches are structural RNA elements that are generally located in the 5' untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of 'mix-and-inject' time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

X-ray lasers for structural and dynamic biology.

Research opportunities and techniques are reviewed for the application of hard x-ray pulsed free-electron lasers (XFEL) to structural biology. These include the imaging of protein nanocrystals, single particles such as viruses, pump--probe experiments for time-resolved nanocrystallography, and snapshot wide-angle x-ray scattering (WAXS) from molecules in solution. The use of femtosecond exposure times, rather than freezing of samples, as a means of minimizing radiation damage is shown to open up new opportunities for the molecular imaging of biochemical reactions at room temperature in solution. This is possible using a 'diffract-and-destroy' mode in which the incident pulse terminates before radiation damage begins. Methods for delivering hundreds of hydrated bioparticles per second (in random orientations) to a pulsed x-ray beam are described. New data analysis approaches are outlined for the correlated fluctuations in fast WAXS, for protein nanocrystals just a few molecules on a side, and for the continuous x-ray scattering from a single virus. Methods for determining the orientation of a molecule from its diffraction pattern are reviewed. Methods for the preparation of protein nanocrystals are also reviewed. New opportunities for solving the phase problem for XFEL data are outlined. A summary of the latest results is given, which now extend to atomic resolution for nanocrystals. Possibilities for time-resolved chemistry using fast WAXS (solution scattering) from mixtures is reviewed, toward the general goal of making molecular movies of biochemical processes.

X-ray diffraction from membrane protein nanocrystals.

Membrane proteins constitute > 30% of the proteins in an average cell, and yet the number of currently known structures of unique membrane proteins is < 300. To develop new concepts for membrane protein structure determination, we have explored the serial nanocrystallography method, in which fully hydrated protein nanocrystals are delivered to an x-ray beam within a liquid jet at room temperature. As a model system, we have collected x-ray powder diffraction data from the integral membrane protein Photosystem I, which consists of 36 subunits and 381 cofactors. Data were collected from crystals ranging in size from 100 nm to 2 µm. The results demonstrate that there are membrane protein crystals that contain < 100 unit cells (200 total molecules) and that 3D crystals of membrane proteins, which contain < 200 molecules, may be suitable for structural investigation. Serial nanocrystallography overcomes the problem of x-ray damage, which is currently one of the major limitations for x-ray structure determination of small crystals. By combining serial nanocrystallography with x-ray free-electron laser sources in the future, it may be possible to produce molecular-resolution electron-density maps using membrane protein crystals that contain only a few hundred or thousand unit cells.

New light on disordered ensembles: ab initio structure determination of one particle from scattering fluctuations of many copies.

We report on the first experimental ab initio reconstruction of an image of a single particle from fluctuations in the scattering from an ensemble of copies, randomly oriented about an axis. The method is applicable to identical particles frozen in space or time (as by snapshot diffraction from an x-ray free electron laser). These fluctuations enhance information obtainable from an experiment such as conventional small angle x-ray scattering.

In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors.

Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5'-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.

Diffraction and imaging from a beam of laser-aligned proteins: resolution limits.

The effect of the limited alignment of hydrated molecules is considered in a laser-aligned molecular beam, on diffraction patterns taken from the beam. Simulated patterns for a protein beam are inverted using the Fienup-Gerchberg-Saxton phasing algorithm, and the effect of limited alignment on the resolution of the resulting potential maps is studied. For a typical protein molecule (lysozyme) with anisotropic polarizability, it is found that up to 1 kW of continuous-wave near-infrared laser power (depending on dielectric constant), together with cooling to liquid-nitrogen temperatures, may be needed to produce sufficiently accurate alignment for direct observation of the secondary structure of proteins in the reconstructed potential or charge-density map. For a typical virus (TMV), a 50 W continuous-wave laser is adequate for subnanometre resolution at room temperature. The dependence of resolution on laser power, temperature, molecular size, shape and dielectric constant is analyzed.

Tomographic diffractive imaging of monolayer crystals at atomic resolution with onedimensional compact support.

A solution to the phase problem for diffraction by two-dimensional crystalline slabs is described, based on the application of a compact support normal to the slab. Specifically we apply the iterative Gerchberg-Saxton-Fienup algorithm to simulated three-dimensional transmission electron diffraction data from monolayer organic crystals. We find that oversampling normal to the monolayer alone does not solve the phase problem in this geometry in general. However, based on simulations for a crystalline monolayer (tetracyanoethylene), we find that convergence is obtained if phases are supplied from a few high-resolution electron microscope images recorded at small tilts to the beam direction. Since current cryomicroscopy methods required a large number of images to phase tomographic diffraction data, this method should greatly reduce the labor involved in data acquisition and analysis in cryo-electron microscopy of organic thin crystals by avoiding the need to record images at high tilt angles. We discuss also the use of laser tweezers as a method of supporting nanoparticles in TEM for diffractive imaging.

Coherent X-ray diffractive imaging: applications and limitations.

The inversion of a diffraction pattern offers aberration-free diffraction-limited 3D images without the resolution and depth-of-field limitations of lens-based tomographic systems, the only limitation being radiation damage. We review our experimental results, discuss the fundamental limits of this technique and future plans.

Experimental lensless soft-X-ray imaging using iterative algorithms: phasing diffuse scattering.

Images of randomly placed two-dimensional arrays of gold balls have been reconstructed from their soft-X-ray transmission diffraction patterns. An iterative hybrid input-output (HiO) algorithm was used to solve the phase problem for the continuous distribution of diffuse X-ray scattering. Knowledge of the approximate size of the clusters was required. The images compare well with scanning electron microscope (SEM) images of the same sample. The use of micrometre-sized silicon nitride window supports is suggested, and absorption filters have been used to allow collection of low spatial frequencies often obscured by a beam stop. This method of phasing diffuse scattering may have application to scattering from individual inorganic nanostructures or single macromolecules.

Phasing diffuse scattering. Application of the SIR2002 algorithm to the non-crystallographic phase problem.

A new phasing algorithm has been used to determine the phases of diffuse elastic X-ray scattering from a non-periodic array of gold balls of 50 nm diameter. Two-dimensional real-space images, showing the charge-density distribution of the balls, have been reconstructed at 50 nm resolution from transmission diffraction patterns recorded at 550 eV energy. The reconstructed image fits well with a scanning-electron-microscope (SEM) image of the same sample. The algorithm, which uses only the density modification portion of the SIR2002 program, is compared with the results obtained via the Gerchberg-Saxton-Fienup HiO algorithm. The new algorithm requires no knowledge of the object's boundary and proceeds from low to high resolution. In this way, the relationship between density modification in crystallography and the HiO algorithm used in signal and image processing is elucidated.

Coherence and sampling requirements for diffractive imaging.

Coherent Diffractive Imaging (CDI) allows images to be reconstructed from diffraction patterns by solving the non-crystallographic phase problem for isolated nanostructures. We show that the Shannon sampling of diffraction intensities needed in CDI requires a coherence width about twice the lateral dimensions of the object, and that the linear number of detector pixels fixes the energy spread needed in the beam. The Shannon sampling, defined by the transform of the periodically repeated autocorrelation of the object, is related to Bragg scattering from an equivalent crystal, and shown to be consistent with the sampling of Young's fringes established by scattering from extreme points in the object. The results are relevant to the design of diffraction cameras for CDI and plans for femotosecond X-ray diffraction from individual proteins.

Image reconstruction from electron and X-ray diffraction patterns using iterative algorithms: experiment and simulation.

The hybrid input-output iterative algorithm, which solves the phase problem for scattering from non-periodic objects, is reviewed for application to X-ray and electron diffraction data. Desirable convex constraints, including the sign of the scattering potential for electrons, and compact support, are discussed. The cases of complex and real exit-face wavefunctions, strong and weak phase objects, various supports, and the use of coherent focussed radiation are reviewed. Reconstruction of general complex objects requires accurate knowledge of the support, which should consist of two holes or a triangle in an opaque mask. The support boundaries should be as sharp as possible. Strong phase objects without absorption can be recovered if the support consists of one hole, is accurately known and has sufficiently sharp boundaries. Real and weak phase objects with absorption can be recovered without accurate knowledge of the support area if the support boundaries are sufficiently sharp and the support consists of one or more holes. A sign constraint on the scattering potential is used to recover weak phase objects. The experimental realization of theoretically desirable support conditions is discussed. A two-stage method of finding the support for complex objects is proposed. Experimental results from applying the Gerchberg-Saxton-Fienup HiO-algorithm to coherent electron diffraction patterns are presented, using specially made e-beam lithographed support structures. Images with a resolution of about 5 nm are thus recovered from the intensities alone in coherent electron diffraction patterns from non-periodic objects. Limitations of the present experiments are identified and suggestions made for development of both X-ray and electron work.

Transmission Electron Diffraction at 200 eV and Damage Thresholds below the Carbon K Edge.

Transmission electron diffraction patterns from ultra-thin aromatic and aliphatic organic films at beam energies of 200 eV-1 keV have been recorded in a custom low energy electron transmission (LEET) chamber. A significant reduction of the molecular damage cross-section, measured by fading of diffraction spots, was found for thin films of the aromatic perylene when the beam energy was reduced from 400 to 200 eV. The corresponding measurements for the aliphatic tetracontane showed a smaller "threshold energy" and the differences are discussed. Electron beam damage from other aromatic materials has also been studied at low energy. Comparison of the carbon K shell ionization cross-section and the measured damage cross-sections show that carbon K-shell ionization is strongly correlated with the damage observed in aromatics at beam energies higher than 284 eV. Calculation of the minimum number of unit cells needed for imaging a single molecule, and comparison of calculated elastic with measured damage cross-sections both indicate new possibilities for imaging biomolecules with low energy electrons.

Injector for scattering measurements on fully solvated biospecies.

We describe a liquid jet injector system developed to deliver fully solvated microscopic target species into a probe beam under either vacuum or ambient conditions. The injector was designed specifically for x-ray scattering studies of biological nanospecies using x-ray free electron lasers and third generation synchrotrons, but is of interest to any application in which microscopic samples must be delivered in a fully solvated state and with microscopic precision. By utilizing a gas dynamic virtual nozzle (GDVN) to generate a sample-containing liquid jet of diameter ranging from 300 nm to 20 µm, the injector avoids the clogging problems associated in this size range with conventional Rayleigh jets. A differential pumping system incorporated into the injector shields the experimental chamber from the gas load of the GDVN, making the injector compatible with high vacuum systems. The injector houses a fiber-optically coupled pump laser to illuminate the jet for pump-probe experiments and a hermetically sealed microscope to observe the liquid jet for diagnostics and alignment during operation. This injector system has now been used during several experimental runs at the Linac Coherent Light Source. Recent refinements in GDVN design are also presented.

Towards ETEM serial crystallography: Electron diffraction from liquid jets.

A sufficiently thin column of liquid was produced to permit penetration with a 200 keV electron beam as evidenced by the observation of diffraction rings due to the intermolecular spacing of the liquid samples. For liquid thickness below 800 nm, the diffraction rings became visible above the inelastic background. Studies were carried out in the environmental chamber of a transmission electron microscope using water and isopropanol.

SEM imaging of liquid jets.

We present a technique for the study of liquid jets in an environmental scanning electron microscope (ESEM). By using a two-fluid stream consisting of a water inner core and a co-flowing outer gas sheath, we are able to produce liquid streams of sufficiently low flow rate to be compatible with ESEM vacuum requirements. We have recorded ESEM images of water jets down to 700 nm diameter. Details of the jet structure, such as the point of jet breakup and size and shape of the jet cone, can be measured with ESEM to far greater accuracy than with optical microscopy. ESEM imaging of liquid jets offers a valuable research tool for the study of aerosol production, combustion processes, ink-jet generation, and many other attributes of micro- and nanojet systems.

Dose, exposure time and resolution in serial X-ray crystallography.

The resolution of X-ray diffraction microscopy is limited by the maximum dose that can be delivered prior to sample damage. In the proposed serial crystallography method, the damage problem is addressed by distributing the total dose over many identical hydrated macromolecules running continuously in a single-file train across a continuous X-ray beam, and resolution is then limited only by the available molecular and X-ray fluxes and molecular alignment. Orientation of the diffracting molecules is achieved by laser alignment. The incident X-ray fluence (energy/area) is evaluated that is required to obtain a given resolution from (i) an analytical model, giving the count rate at the maximum scattering angle for a model protein, (ii) explicit simulation of diffraction patterns for a GroEL-GroES protein complex, and (iii) the spatial frequency cut-off of the transfer function following iterative solution of the phase problem, and reconstruction of an electron density map in the projection approximation. These calculations include counting shot noise and multiple starts of the phasing algorithm. The results indicate counting time and the number of proteins needed within the beam at any instant for a given resolution and X-ray flux. An inverse fourth-power dependence of exposure time on resolution is confirmed, with important implications for all coherent X-ray imaging. It is found that multiple single-file protein beams will be needed for sub-nanometer resolution on current third-generation synchrotrons, but not on fourth-generation designs, where reconstruction of secondary protein structure at a resolution of 7 A should be possible with relatively short exposures.

Tomographic femtosecond x-ray diffractive imaging.

A method is proposed for obtaining three simultaneous projections of a target from a single radiation pulse, which also allows the relative orientation of successive targets to be determined. The method has application to femtosecond x-ray diffraction, and does not require solution of the phase problem. We show that the principal axes of a compact charge-density distribution can be obtained from projections of its autocorrelation function, which is directly accessible in diffraction experiments. The results may have more general application to time resolved tomographic pump-probe experiments and time-series imaging.

Powder diffraction from a continuous microjet of submicrometer protein crystals.

Atomic-resolution structures from small proteins have recently been determined from high-quality powder diffraction patterns using a combination of stereochemical restraints and Rietveld refinement [Von Dreele (2007), J. Appl. Cryst. 40, 133-143; Margiolaki et al. (2007), J. Am. Chem. Soc. 129, 11865-11871]. While powder diffraction data have been obtained from batch samples of small crystal-suspensions, which are exposed to X-rays for long periods of time and undergo significant radiation damage, the proof-of-concept that protein powder diffraction data from nanocrystals of a membrane protein can be obtained using a continuous microjet is shown. This flow-focusing aerojet has been developed to deliver a solution of hydrated protein nanocrystals to an X-ray beam for diffraction analysis. This method requires neither the crushing of larger polycrystalline samples nor any techniques to avoid radiation damage such as cryocooling. Apparatus to record protein powder diffraction in this manner has been commissioned, and in this paper the first powder diffraction patterns from a membrane protein, photosystem I, with crystallite sizes of less than 500 nm are presented. These preliminary patterns show the lowest-order reflections, which agree quantitatively with theoretical calculations of the powder profile. The results also serve to test our aerojet injector system, with future application to femtosecond diffraction in free-electron X-ray laser schemes, and for serial crystallography using a single-file beam of aligned hydrated molecules.

Three-dimensional coherent x-ray diffraction imaging of a ceramic nanofoam: determination of structural deformation mechanisms.

Ultralow density polymers, metals, and ceramic nanofoams are valued for their high strength-to-weight ratio, high surface area, and insulating properties ascribed to their structural geometry. We obtain the labrynthine internal structure of a tantalum oxide nanofoam by x-ray diffractive imaging. Finite-element analysis from the structure reveals mechanical properties consistent with bulk samples and with a diffusion-limited cluster aggregation model, while excess mass on the nodes discounts the dangling fragments hypothesis of percolation theory.