Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.

The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the “diffraction-before-destruction” approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.

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 atmosphere1. 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 developed2 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 studies3,4. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.

The morphology of micrometre−size particulate matter is of critical importance in fields ranging from toxicology to climate science, yet these properties are surprisingly difficult to measure in the particles' native environment. Electron microscopy requires collection of particles on a substrate; visible light scattering provides insufficient resolution; and X−ray synchrotron studies have been limited to ensembles of particles. Here we demonstrate an in situ method for imaging individual sub−micrometre particles to nanometre resolution in their native environment, using intense, coherent X−ray pulses from the Linac Coherent Light Source free−electron laser. We introduced individual aerosol particles into the pulsed X−ray beam, which is sufficiently intense that diffraction from individual particles can be measured for morphological analysis. At the same time, ion fragments ejected from the beam were analysed using mass spectrometry, to determine the composition of single aerosol particles. Our results show the extent of internal dilation symmetry of individual soot particles subject to non−equilibrium aggregation, and the surprisingly large variability in their fractal dimensions. More broadly, our methods can be extended to resolve both static and dynamic morphology of general ensembles of disordered particles. Such general morphology has implications in topics such as solvent accessibilities in proteins, vibrational energy transfer by the hydrodynamic interaction of amino acids, and large−scale production of nanoscale structures by flame synthesi

We report the first image of an intact, frozen hydrated eukaryotic cell using x-ray diffraction microscopy, or coherent x-ray diffraction imaging. By plunge freezing the specimen in liquid ethane and maintaining it below −170 °C, artifacts due to dehydration, ice crystallization, and radiation damage are greatly reduced. In this example, coherent diffraction data using 520 eV x rays were recorded and reconstructed to reveal a budding yeast cell at a resolution better than 25 nm. This demonstration represents an important step towards high resolution imaging of cells in their natural, hydrated state, without limitations imposed by x-ray optics.X-ray microscopes allow high resolution microscopy of intact, hydrated biological specimens with thicknesses of many micrometers, beyond the limit of biological electron microscopy [1][2][3]. Radiation damage precludes repeated imaging of live specimens [4], but this can be mitigated by working at liquid nitrogen temperature [5,6]. In addition, single view flash imaging of cells using ultrabright sources has been proposed [7,8] as a way of capturing the image before radiolytical and thermal damage become evident.In recent years, there has been much progress in developing zone plate microscopy for 3D imaging of frozen hydrated cells [9][10][11][12][13]. While there are demonstrations of x-ray optics with higher resolution [14][15][16], scientific applications using x-ray microscopes have mainly used Fresnel zone plate optics with 25-40 nm spatial resolution. These optics typically have a focusing efficiency in the 10% range [17] and the modulation transfer function for incoherent bright field imaging decreases the efficiency of utilization of higher spatial frequency information. As a result, while the practical advantages of lens-based microscopes will be the deciding factor for most studies, it is also worthwhile to consider alternative methods for high resolution x-ray imaging.X-ray diffraction microscopy (XDM), also called coherent x-ray diffraction imaging, was proposed by Sayre as an imaging method that dispenses with the technological limits of lens efficiency and resolution [18]. Instead, the far-field diffraction pattern of an isolated object illuminated by a coherent x-ray beam is recorded. If the object is finite, and the diffraction pattern is sampled finely enough, the object can be reconstructed from the measured diffraction intensities alone [19,20]. In this manner one is able to eliminate limitations due to the efficiency and finite numerical aperture of x-ray optics [21]. Following a first demonstration by Miao et *Chris.Jacobsen@stonybrook.edu. An important limitation applies to the demonstrations of x-ray diffraction microscopy of cells, chromosomes, and virions cited above: they have all involved dehydrated specimens at room temperature. Though Nishino et al. [27] have obtained a very exciting 3D XDM image of a dehydrated chromosome, they note significant resolution degradation due to accumulated radiation dose. In electron microscopy, stability a...

X-ray free-electron lasers deliver intense femtosecond pulses that promise to yield high resolution diffraction data of nanocrystals before the destruction of the sample by radiation damage. Diffraction intensities of lysozyme nanocrystals collected at the Linac Coherent Light Source using 2 keV photons were used for structure determination by molecular replacement and analyzed for radiation damage as a function of pulse length and fluence. Signatures of radiation damage are observed for pulses as short as 70 fs. Parametric scaling used in conventional crystallography does not account for the observed effects.

Diffractive imaging with free-electron lasers allows structure determination from ensembles of weakly scattering identical nanoparticles. The ultra-short, ultra-bright X-ray pulses provide snapshots of the randomly oriented particles frozen in time, and terminate before the onset of structural damage. As signal strength diminishes for small particles, the synthesis of a threedimensional diffraction volume requires simultaneous involvement of all data. Here we report the first application of a three-dimensional spatial frequency correlation analysis to carry out this synthesis from noisy single-particle femtosecond X-ray diffraction patterns of nearly identical samples in random and unknown orientations, collected at the Linac Coherent Light Source. Our demonstration uses unsupported test particles created via aerosol self-assembly, and composed of two polystyrene spheres of equal diameter. The correlation analysis avoids the need for orientation determination entirely. This method may be applied to the structural determination of biological macromolecules in solution.

Single-particle experiments using X-ray Free Electron Lasers produce more than 10 5 snapshots per hour, consisting of an admixture of blank shots (no particle intercepted), and exposures of one or more particles. Experimental data sets also often contain unintentional contamination with different species. We present an unsupervised method able to sort experimental snapshots without recourse to templates, specific noise models, or user-directed learning. The results show 90% agreement with manual classification. ©2011 Optical Society of America