Serial femtosecond crystallography using ultrashort pulses from x-ray free electron lasers (XFELs) enables studies of the light-triggered dynamics of biomolecuies. We used microcrystals of ...photoactive yellow protein (a bacterial blue light photoreceptor) as a model system and obtained high-resolution, time-resolved difference electron density maps of excellent quality with strong features; these allowed the determination of structures of reaction intermediates to a resolution of 1.6 angstroms. Our results open the way to the study of reversible and nonreversible biological reactions on time scales as short as femtoseconds under conditions that maximize the extent of reaction initiation throughout the crystal.
An iterated projection algorithm (N-Phaser) is developed that reconstructs a scattering potential from N-beam multiple Bragg scattered intensities. The method may be used to eliminate multiple ...scattering artifacts from electron diffraction data, solving the phase problem and increasing the thicknesses of samples used in materials science, solid-state chemistry, and small molecule crystallography. For high-energy transmission electron diffraction, we show that the algorithm recovers accurate complex structure factors from a wide range of thicknesses, orientations, and relativistic beam energies, and does not require known thickness or atomic-resolution data if sufficient multiple scattering occurs. Extensions to Cryo-electron microscopy and Micro-electron diffraction are suggested.
Toward the end of his career, Zewail developed strong interest in fast electron spectroscopy and
imaging, a field to which he made important contributions toward his aim of making
molecular movies ...free of radiation damage. We therefore compare here the atomistic
mechanisms leading to destruction of protein samples in diffract-and-destroy
experiments for the cases of high-energy electron beam irradiation and X-ray laser pulses. The damage
processes and their time-scales are compared and relevant elastic, inelastic, and
photoelectron cross sections are given. Inelastic mean-free paths for ejected electrons at
very low energies in insulators are compared with the bioparticle size. The dose rate and
structural damage rate for electrons are found to be much lower, allowing longer pulses,
reduced beam
current, and Coulomb interactions for the formation of smaller probes. High-angle
electron
scattering from the nucleus, which has no parallel in the X-ray case, tracks the slowly
moving nuclei during the explosion, just as the gain of the XFEL (X-ray free-electron laser) has
no parallel in the electron case. Despite reduced damage and much larger elastic
scattering cross sections in the electron case, leading to not
dissimilar elastic
scattering rates (when account is taken of the greatly increased
incident XFEL fluence), progress for single-particle electron diffraction is seen to
depend on the effort to reduce emittance growth due to Coulomb interactions, and so allow
formation of intense sub-micron beams no larger than a virus.
► Femtosecond nanocrystallography is based on Free Electron X-ray Lasers ► The diffract-before-destroy principle overcomes damage problem in crystallography ► Nanocrystals are delivered at Room ...temperature in stream by new type of gas-focusing injector ► Coherently illuminated nanocrystals provide interference effects solving the phase problem ► The method opens new avenues for time-resolved studies showing proteins at work.
The invention of free electron X-ray lasers has opened a new era for membrane protein structure determination with the recent first proof-of-principle of the new concept of femtosecond nanocrystallography. Structure determination is based on thousands of diffraction snapshots that are collected on a fully hydrated stream of nanocrystals. This review provides a summary of the method and describes how femtosecond X-ray crystallography overcomes the radiation-damage problem in X-ray crystallography, avoids the need for growth and freezing of large single crystals while offering a new method for direct digital phase determination by making use of the fully coherent nature of the X-ray beam. We briefly review the possibilities for time-resolved crystallography, and the potential for making ‘molecular movies’ of membrane proteins at work.
Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl
into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ...ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.
Advances in the development of free-electron lasers offer the realistic prospect of nanoscale imaging on the timescale of atomic motions. We identify X-ray Fourier-transform holography as a promising ...but, so far, inefficient scheme to do this. We show that a uniformly redundant array placed next to the sample, multiplies the efficiency of X-ray Fourier transform holography by more than three orders of magnitude, approaching that of a perfect lens, and provides holographic images with both amplitude- and phase-contrast information. The experiments reported here demonstrate this concept by imaging a nano-fabricated object at a synchrotron source, and a bacterial cell with a soft-X-ray free-electron laser, where illumination by a single 15-fs pulse was successfully used in producing the holographic image. As X-ray lasers move to shorter wavelengths we expect to obtain higher spatial resolution ultrafast movies of transient states of matter.
Coherent x-ray diffraction microscopy is a method of imaging nonperiodic isolated objects at resolutions limited, in principle, by only the wavelength and largest scattering angles recorded. We ...demonstrate x-ray diffraction imaging with high resolution in all three dimensions, as determined by a quantitative analysis of the reconstructed volume images. These images are retrieved from the three-dimensional diffraction data using no a priori knowledge about the shape or composition of the object, which has never before been demonstrated on a nonperiodic object. We also construct two-dimensional images of thick objects with greatly increased depth of focus (without loss of transverse spatial resolution). These methods can be used to image biological and materials science samples at high resolution with x-ray undulator radiation and establishes the techniques to be used in atomic-resolution ultrafast imaging at x-ray free-electron laser sources.
Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser ...(XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5-20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A
adenosine receptor (A
AR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites
and
enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A
AR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A
AR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5-20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. These developments will enable structure determination from smaller and/or weakly diffracting microcrystals.
X-ray diffraction patterns may be obtained from individual submicron protein nanocrystals using a femtosecond pulse from a free-electron X-ray laser. Many "single-shot" patterns are read out every ...second from a stream of nanocrystals lying in random orientations. The short pulse terminates before significant atomic (or electronic) motion commences, minimizing radiation damage. Simulated patterns for Photosystem I nanocrystals are used to develop a method for recovering structure factors from tens of thousands of snapshot patterns from nanocrystals varying in size, shape and orientation. We determine the number of shots needed for a required accuracy in structure factor measurement and resolution, and investigate the convergence of our Monte-Carlo integration method.
Bifunctional μ- and δ-opioid receptor (OR) ligands are potential therapeutic alternatives, with diminished side effects, to alkaloid opiate analgesics. We solved the structure of human δ-OR bound to ...the bifunctional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. The observed receptor-peptide interactions are critical for understanding of the pharmacological profiles of opioid peptides and for development of improved analgesics.
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