Short and intense x-ray pulses may be used for atomic-resolution diffraction imaging of single biological molecules. Radiation damage and a low signal-to-noise ratio impose stringent pulse ...requirements. In this Letter, we describe methods for decreasing the damage and improving the signal by encapsulating the molecule in a sacrificial layer (tamper) that reduces atomic motion and by postprocessing the pulse-averaged diffraction pattern to correct for ionization damage. Simulations show that these methods greatly improve the image quality.
Numerical simulations of the current and future pulse intensity distributions at selected locations along the Far Experimental Hall, the hard X‐ray section of the Linac Coherent Light Source (LCLS), ...are provided. Estimates are given for the pulse fluence, energy and size in and out of focus, taking into account effects due to the experimentally measured divergence of the X‐ray beam, and measured figure errors of all X‐ray optics in the beam path. Out‐of‐focus results are validated by comparison with experimental data. Previous work is expanded on, providing quantitatively correct predictions of the pulse intensity distribution. Numerical estimates in focus are particularly important given that the latter cannot be measured with direct imaging techniques due to detector damage. Finally, novel numerical estimates of improvements to the pulse intensity distribution expected as part of the on‐going upgrade of the LCLS X‐ray transport system are provided. We suggest how the new generation of X‐ray optics to be installed would outperform the old one, satisfying the tight requirements imposed by X‐ray free‐electron laser facilities.
Numerical simulations of the current and future pulse intensity distributions at selected locations along the hard X‐ray section of the Linac Coherent Light Source are provided. Estimates are given for the pulse fluence, energy and size in and out of focus, taking into account effects due to the experimentally measured divergences of the X‐ray beam, and measured figure errors of all X‐ray optics in the beam path.
While Black’s equation for electromigration (EM) in interconnects with
n
= 1 is rigorously based on the principles of electrotransport,
n
> 1 is more commonly observed empirically. This deviation ...is usually attributed to Joule heating. An alternative explanation is suggested by the recent discovery of EM plasticity. To examine this possibility, we have retested samples that had been previously subjected to a predamaging phase of high temperature and current densities to determine whether the loss of median time to failure (MTF) is retained. We find that the predamaged samples exhibit MTFs that are permanently reduced, which is a characteristic of EM plasticity.
SPEDEN is a computer program that reconstructs the electron density of single particles from their X‐ray diffraction patterns, using a single‐particle adaptation of the holographic method in ...crystallography Szöke, Szöke & Somoza (1997). Acta Cryst. A53, 291–313. The method, like its parent, is unique because it does not rely on `back' transformation from the diffraction pattern into real space and on interpolation within measured data. It is designed to deal successfully with sparse, irregular, incomplete and noisy data. It is also designed to use prior information for ensuring sensible results and for reliable convergence. This article describes the theoretical basis for the reconstruction algorithm, its implementation, and quantitative results of tests on synthetic and experimentally obtained data. The program could be used for determining the structures of radiation‐tolerant samples and, eventually, of large biological molecular structures without the need for crystallization.
Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II ...(PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth's oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the 'dangler' Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.
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.
X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary ...increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.
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.
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