The advent of the X-ray free-electron laser (XFEL) has made it possible to record diffraction snapshots of biological entities injected into the X-ray beam before the onset of radiation damage. Algorithmic means must then be used to determine the snapshot orientations and thence the three-dimensional structure of the object. Existing Bayesian approaches are limited in reconstruction resolution typically to 1/10 of the object diameter, with the computational expense increasing as the eighth power of the ratio of diameter to resolution. We present an approach capable of exploiting object symmetries to recover three-dimensional structure to high resolution, and thus reconstruct the structure of the satellite tobacco necrosis virus to atomic level. Our approach offers the highest reconstruction resolution for XFEL snapshots to date and provides a potentially powerful alternative route for analysis of data from crystalline and nano-crystalline objects.
macromolecular assemblies; symmetry; X-ray lasers; manifold embedding; dimensionality reduction
A complete set of structure factors has been extracted from hundreds of thousands of femtosecond X-ray diffraction patterns from randomly oriented Photosystem I membrane protein nanocrystals, using the Monte Carlo method of intensity integration. The data, collected at the Linac Coherent Light Source, are compared with conventional single-crystal data collected at a synchrotron source, and the quality of each data set was found to be similar.
A complete set of structure factors has been extracted from hundreds of thousands of femtosecond single-shot X-ray microdiffraction patterns taken from randomly oriented nanocrystals. The method of Monte Carlo integration over crystallite size and orientation was applied to experimental data from Photosystem I nanocrystals. This arrives at structure factors from many partial reflections without prior knowledge of the particle-size distribution. The data were collected at the Linac Coherent Light Source (the first hard-X-ray laser user facility), to which was fitted a hydrated protein nanocrystal injector jet, according to the method of serial crystallography. The data are single ‘still’ diffraction snapshots, each from a different nanocrystal with sizes ranging between 100 nm and 2 µm, so the angular width of Bragg peaks was dominated by crystal-size effects. These results were compared with single-crystal data recorded from large crystals of Photosystem I at the Advanced Light Source and the quality of the data was found to be similar. The implications for improving the efficiency of data collection by allowing the use of very small crystals, for radiation-damage reduction and for time-resolved diffraction studies at room temperature are discussed.
nanocrystals; femtosecond diffraction; free-electron lasers; Monte Carlo methods; protein microdiffraction
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.
Emerging X-ray free-electron lasers with femtosecond pulse duration enable single-shot snapshot imaging almost free from sample damage by outrunning major radiation damage processes. In bioimaging, it is essential to keep the sample close to its natural state. Conventional high-resolution imaging, however, suffers from severe radiation damage that hinders live cell imaging. Here we present a method for capturing snapshots of live cells kept in a micro-liquid enclosure array by X-ray laser diffraction. We place living Microbacterium lacticum cells in an enclosure array and successively expose each enclosure to a single X-ray laser pulse from the SPring-8 Angstrom Compact Free-Electron Laser. The enclosure itself works as a guard slit and allows us to record a coherent diffraction pattern from a weakly-scattering submicrometre-sized cell with a clear fringe extending up to a 28-nm full-period resolution. The reconstructed image reveals living whole-cell structures without any staining, which helps advance understanding of intracellular phenomena.
Live cell imaging at high resolution is very challenging because cells die upon prolonged radiation exposure. Kimura et al. overcome this problem by using pulsed coherent X-ray diffraction to image live microbacterium in a nanofabricated liquid enclosure at resolution far exceeding optical methods.
Bragg diffraction achieved from two-dimensional protein crystals using femtosecond X-ray laser snapshots is presented.
X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.
two-dimensional protein crystal; femtosecond crystallography; single layer X-ray diffraction; membrane protein
X-ray free-electron laser crystallography relies on the collection of still-shot diffraction patterns. New methods are developed for optimal modeling of the crystals’ orientations and mosaic block properties.
X-ray diffraction patterns from still crystals are inherently difficult to process because the crystal orientation is not uniquely determined by measuring the Bragg spot positions. Only one of the three rotational degrees of freedom is directly coupled to spot positions; the other two rotations move Bragg spots in and out of the reflecting condition but do not change the direction of the diffracted rays. This hinders the ability to recover accurate structure factors from experiments that are dependent on single-shot exposures, such as femtosecond diffract-and-destroy protocols at X-ray free-electron lasers (XFELs). Here, additional methods are introduced to optimally model the diffraction. The best orientation is obtained by requiring, for the brightest observed spots, that each reciprocal-lattice point be placed into the exact reflecting condition implied by Bragg’s law with a minimal rotation. This approach reduces the experimental uncertainties in noisy XFEL data, improving the crystallographic R factors and sharpening anomalous differences that are near the level of the noise.
X-ray free-electron lasers; single-shot exposures
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 recorded1-3. 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 Source4. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes5. 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 processes6. 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.
A new method for the treatment of partial reflections from X-ray snapshots is implemented in the program package nXDS, which yields intensity data of almost the same quality as those obtained by the classical rotation method.
A functional expression is introduced that relates scattered X-ray intensities from a still or a rotation snapshot to the corresponding structure-factor amplitudes. The new approach was implemented in the program nXDS for processing monochromatic diffraction images recorded by a multi-segment detector where each exposure could come from a different crystal. For images containing indexable spots, the intensities of the expected reflections and their variances are obtained by profile fitting after mapping the contributing pixel contents to the Ewald sphere. The varying intensity decline owing to the angular distance of the reflection from the surface of the Ewald sphere is estimated using a Gaussian rocking curve. This decline is dubbed ‘Ewald offset correction’, which is well defined even for still images. Together with an image-scaling factor and other corrections, an explicit expression is defined that predicts each recorded intensity from its corresponding structure-factor amplitude. All diffraction parameters, scaling and correction factors are improved by post-refinement. The ambiguous case of a lower point group than the lattice symmetry is resolved by a method reminiscent of the technique of ‘selective breeding’. It selects the indexing alternative for each image that yields, on average, the highest correlation with intensities from all other images. Processing a test set of rotation images by XDS and treating the same images by nXDS as snapshots of crystals in random orientations yields data of comparable quality, clearly indicating an anomalous signal from Se atoms.
Although electron cryo-microscopy (cryo-EM) single-particle analysis has become an important tool for structural biology of large and flexible macro-molecular assemblies, the technique has not yet reached its full potential. Besides fundamental limits imposed by radiation damage, poor detectors and beam-induced sample movement have been shown to degrade attainable resolutions. A new generation of direct electron detectors may ameliorate both effects. Apart from exhibiting improved signal-to-noise performance, these cameras are also fast enough to follow particle movements during electron irradiation. Here, we assess the potentials of this technology for cryo-EM structure determination. Using a newly developed statistical movie processing approach to compensate for beam-induced movement, we show that ribosome reconstructions with unprecedented resolutions may be calculated from almost two orders of magnitude fewer particles than used previously. Therefore, this methodology may expand the scope of high-resolution cryo-EM to a broad range of biological specimens.
Determining the structure of proteins and other biomolecules at the atomic level is central to understanding many aspects of biology. X-ray crystallography is the best-known technique for structural biology but, as the name suggests, it works only with samples that can be crystallized. Electron cryo-microscopy (cryo-EM) could, potentially, be used to determine the atomic structures of biomolecules that cannot be crystallized, but at present the resolution that can be achieved with this approach is sufficient only for imaging certain types of viruses.
In cryo-EM, a solution of the biomolecule of interest is frozen in a thin layer of ice, and this layer is imaged in an electron microscope. By combining images of many identical biomolecules in many different orientations, it is possible to work backwards and determine their 3D structure. However, in order to determine this structure at high resolution, it is necessary to make repeated measurements to reduce high levels of noise in the images.
Cryo-EM images are usually recorded on a photographic film or a CCD (charge-coupled device) camera. However, photographic film is unsuitable for high-throughput methods because it has to be handled manually, while the efficiency of CCD cameras is limited because the electrons have to be converted into visible light to be detected. Digital cameras that can detect electrons directly have become available recently, and these are more efficient than both film and CCD cameras. They are also much faster, which means that it is possible to record videos of the sample during the time (typically ∼1 s) it is being exposed to the electron beam. Processing these videos could then—in theory—compensate for any movements of the biomolecules that are induced by the electron beam. Along with radiation damage caused by the electrons, these beam-induced movements have been a major limitation on the resolution that can be achieved with cryo-EM.
Bai et al. demonstrate the potential of direct-electron detectors in cryo-EM by determining the structures of two ribosomes. Using a novel statistical algorithm to accurately follow the movements of the ribosomes during the time they are exposed to the electron beam, they are able to compensate for these movements, and this makes it possible to determine the structures of the ribosomes with near-atomic precision. Moreover, the resolution they achieve with just ∼30,000 ribosomes is better than that previously achieved with more than a million ribosomes, allowing small details inside the ribosome – such as ß-strands and bulky amino-acid side chains – to be resolved with cryo-EM for the first time. The work of Bai et al. could, therefore, allow researchers to use cryo-EM to determine the structure of many more biomolecules with atomic precision.
Electron Microscopy; Direct electron detectors; Image processing; T. thermophilus; ribosome; Bayesian; S. cerevisiae
We describe a new generation of algorithms capable of mapping the structure and conformations of macromolecules and their complexes from large ensembles of heterogeneous snapshots, and demonstrate the feasibility of determining both discrete and continuous macromolecular conformational spectra. These algorithms naturally incorporate conformational heterogeneity without resort to sorting and classification, or prior knowledge of the type of heterogeneity present. They are applicable to single-particle diffraction and image datasets produced by X-ray lasers and cryo-electron microscopy, respectively, and particularly suitable for systems not easily amenable to purification or crystallization.
macromolecular structure; macromolecular conformations; X-ray lasers; cryo-electron microscopy; manifold embedding; dimensionality reduction
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.
X-ray free-electron lasers (X-FELs) produce X-ray pulses with extremely brilliant peak intensity and ultrashort pulse duration. It has been proposed that radiation damage can be “outrun” by using an ultra intense and short X-FEL pulse that passes a biological sample before the onset of significant radiation damage. The concept of “diffraction-before-destruction” has been demonstrated recently at the Linac Coherent Light Source, the first operational hard X-ray FEL, for protein nanocrystals and giant virus particles. The continuous diffraction patterns from single particles allow solving the classical “phase problem” by the oversampling method with iterative algorithms. If enough data are collected from many identical copies of a (biological) particle, its three-dimensional structure can be reconstructed. We review the current status and future prospects of serial femtosecond crystallography (SFX) and single-particle coherent diffraction imaging (CDI) with X-FELs.
Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only a hundred unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet brought fully-hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses (~ 70 fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. During the initial experiments at the AMO beamline using 6.9-Å wavelength, Bragg peaks were recorded to 8.5-Å resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage [Chapman H.N., et al. Femtosecond X-ray protein nanocrystallography, Nature 470 (2011) 73–81]. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins.
membrane proteins; structure determination; femtosecond nanocrystallography; protein nanocrystals; X-ray crystallography; XFEL
Schemes for X-ray imaging single protein molecules using new x-ray sources, like x-ray free electron lasers (XFELs), require processing many frames of data that are obtained by taking temporally short snapshots of identical molecules, each with a random and unknown orientation. Due to the small size of the molecules and short exposure times, average signal levels of much less than 1 photon/pixel/frame are expected, much too low to be processed using standard methods. One approach to process the data is to use statistical methods developed in the EMC algorithm (Loh & Elser, Phys. Rev. E, 2009) which processes the data set as a whole. In this paper we apply this method to a real-space tomographic reconstruction using sparse frames of data (below 10−2 photons/pixel/frame) obtained by performing x-ray transmission measurements of a low-contrast, randomly-oriented object. This extends the work by Philipp et al. (Optics Express, 2012) to three dimensions and is one step closer to the single molecule reconstruction problem.
(000.2190) Experimental physics; (040.7480) X-rays, soft x-rays, extreme ultraviolet (EUV); (100.6950) Tomographic image processing; (110.4155) Multiframe image processing; (110.4280) Noise in imaging systems; (110.6955) Tomographic imaging; (110.7440) X-ray imaging; (340.7440) X-ray imaging
The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a “focused electron tomography reconstruction” (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single “snapshot” molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.
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.
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.
(170.7160) Ultrafast technology; (170.7440) X-ray imaging; (140.3450) Laser-induced chemistry; (140.7090) Ultrafast lasers; (170.0170) Medical optics and biotechnology
A code with an algorithm for high-speed classification of X-ray diffraction patterns has been developed. Results obtained for a set of 1 × 106 simulated diffraction patterns are also reported.
Single-particle coherent X-ray diffraction imaging using an X-ray free-electron laser has the potential to reveal the three-dimensional structure of a biological supra-molecule at sub-nanometer resolution. In order to realise this method, it is necessary to analyze as many as 1 × 106 noisy X-ray diffraction patterns, each for an unknown random target orientation. To cope with the severe quantum noise, patterns need to be classified according to their similarities and average similar patterns to improve the signal-to-noise ratio. A high-speed scalable scheme has been developed to carry out classification on the K computer, a 10PFLOPS supercomputer at RIKEN Advanced Institute for Computational Science. It is designed to work on the real-time basis with the experimental diffraction pattern collection at the X-ray free-electron laser facility SACLA so that the result of classification can be feedback for optimizing experimental parameters during the experiment. The present status of our effort developing the system and also a result of application to a set of simulated diffraction patterns is reported. About 1 × 106 diffraction patterns were successfully classificatied by running 255 separate 1 h jobs in 385-node mode.
X-ray free-electron laser; K computer; single-particle coherent diffraction imaging; classification of diffraction patterns; big-data analysis
Single-particle imaging experiments of biomolecules at x-ray free-electron lasers (XFELs) require processing hundreds of thousands of images that contain very few x-rays. Each low-fluence image of the diffraction pattern is produced by a single, randomly oriented particle, such as a protein. We demonstrate the feasibility of recovering structural information at these extremes using low-fluence images of a randomly oriented 2D x-ray mask. Successful reconstruction is obtained with images averaging only 2.5 photons per frame, where it seems doubtful there could be information about the state of rotation, let alone the image contrast. This is accomplished with an expectation maximization algorithm that processes the low-fluence data in aggregate, and without any prior knowledge of the object or its orientation. The versatility of the method promises, more generally, to redefine what measurement scenarios can provide useful signal.
(040.7480) X-rays, soft x-rays, extreme ultraviolet (EUV); (110.7440) X-ray imaging; (000.2190) Experimental physics; (110.3055) Information theoretical analysis; (110.4155) Multiframe image processing; (040.0040) Detectors
Serial femtosecond X-ray (SFX) diffraction extending beyond 6 Å resolution using T. thermophilus 30S ribosomal subunit crystals is reported.
High-resolution ribosome structures determined by X-ray crystallography have provided important insights into the mechanism of translation. Such studies have thus far relied on large ribosome crystals kept at cryogenic temperatures to reduce radiation damage. Here, the application of serial femtosecond X-ray crystallography (SFX) using an X-ray free-electron laser (XFEL) to obtain diffraction data from ribosome microcrystals in liquid suspension at ambient temperature is described. 30S ribosomal subunit microcrystals diffracted to beyond 6 Å resolution, demonstrating the feasibility of using SFX for ribosome structural studies. The ability to collect diffraction data at near-physiological temperatures promises to provide fundamental insights into the structural dynamics of the ribosome and its functional complexes.
30S ribosomal subunit; serial femtosecond X-ray crystallography; X-ray free-electron laser; ribosome
X-ray free-electron lasers have enabled new approaches to the structural determination of protein crystals that are too small or radiation-sensitive for conventional analysis1. For sufficiently short pulses, diffraction is collected before significant changes occur to the sample, and it has been predicted that pulses as short as 10 fs may be required to acquire atomic-resolution structural information1–4. Here, we describe a mechanism unique to ultrafast, ultra-intense X-ray experiments that allows structural information to be collected from crystalline samples using high radiation doses without the requirement for the pulse to terminate before the onset of sample damage. Instead, the diffracted X-rays are gated by a rapid loss of crystalline periodicity, producing apparent pulse lengths significantly shorter than the duration of the incident pulse. The shortest apparent pulse lengths occur at the highest resolution, and our measurements indicate that current X-ray free-electron laser technology5 should enable structural determination from submicrometre protein crystals with atomic resolution.
The aim of this study was to design, develop, and optimize respirable tacrolimus microparticles and nanoparticles and multifunctional tacrolimus lung surfactant mimic particles for targeted dry powder inhalation delivery as a pulmonary nanomedicine. Particles were rationally designed and produced at different pump rates by advanced spray-drying particle engineering design from organic solution in closed mode. In addition, multifunctional tacrolimus lung surfactant mimic dry powder particles were prepared by co-dissolving tacrolimus and lung surfactant mimic phospholipids in methanol, followed by advanced co-spray-drying particle engineering design technology in closed mode. The lung surfactant mimic phospholipids were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-[phosphor-rac-1-glycerol]. Laser diffraction particle sizing indicated that the particle size distributions were suitable for pulmonary delivery, whereas scanning electron microscopy imaging indicated that these particles had both optimal particle morphology and surface morphology. Increasing the pump rate percent of tacrolimus solution resulted in a larger particle size. X-ray powder diffraction patterns and differential scanning calorimetry thermograms indicated that spray drying produced particles with higher amounts of amorphous phase. X-ray powder diffraction and differential scanning calorimetry also confirmed the preservation of the phospholipid bilayer structure in the solid state for all engineered respirable particles. Furthermore, it was observed in hot-stage micrographs that raw tacrolimus displayed a liquid crystal transition following the main phase transition, which is consistent with its interfacial properties. Water vapor uptake and lyotropic phase transitions in the solid state at varying levels of relative humidity were determined by gravimetric vapor sorption technique. Water content in the various powders was very low and well within the levels necessary for dry powder inhalation, as quantified by Karl Fisher coulometric titration. Conclusively, advanced spray-drying particle engineering design from organic solution in closed mode was successfully used to design and optimize solid-state particles in the respirable size range necessary for targeted pulmonary delivery, particularly for the deep lung. These particles were dry, stable, and had optimal properties for dry powder inhalation as a novel pulmonary nanomedicine.
dry powder inhaler (DPI); pulmonary nanomedicine; lung transplant; immunosuppression; lung surfactant; phospholipid colloidal self-assemblies; solid-state particle engineering design; organic solution advanced spray drying
A new algorithm is developed for reconstructing the high-resolution three-dimensional diffraction intensity function of a globular biological macromolecule from many quantum-noise-limited two-dimensional X-ray laser diffraction patterns, each for an unknown orientation. The structural resolution is expressed as a function of the incident X-ray intensity and quantities characterizing the target molecule.
A new two-step algorithm is developed for reconstructing the three-dimensional diffraction intensity of a globular biological macromolecule from many experimentally measured quantum-noise-limited two-dimensional X-ray laser diffraction patterns, each for an unknown orientation. The first step is classification of the two-dimensional patterns into groups according to the similarity of direction of the incident X-rays with respect to the molecule and an averaging within each group to reduce the noise. The second step is detection of common intersecting circles between the signal-enhanced two-dimensional patterns to identify their mutual location in the three-dimensional wavenumber space. The newly developed algorithm enables one to detect a signal for classification in noisy experimental photon-count data with as low as ∼0.1 photons per effective pixel. The wavenumber of such a limiting pixel determines the attainable structural resolution. From this fact, the resolution limit due to the quantum noise attainable by this new method of analysis as well as two important experimental parameters, the number of two-dimensional patterns to be measured (the load for the detector) and the number of pairs of two-dimensional patterns to be analysed (the load for the computer), are derived as a function of the incident X-ray intensity and quantities characterizing the target molecule.
biological macromolecules; classification of two-dimensional diffraction patterns; common intersecting circles; attainable structural resolution
Special methods are required to interpret sparse diffraction patterns collected from peptide crystals at X-ray free-electron lasers. Bragg spots can be indexed from composite-image powder rings, with crystal orientations then deduced from a very limited number of spot positions.
Still diffraction patterns from peptide nanocrystals with small unit cells are challenging to index using conventional methods owing to the limited number of spots and the lack of crystal orientation information for individual images. New indexing algorithms have been developed as part of the Computational Crystallography Toolbox (cctbx) to overcome these challenges. Accurate unit-cell information derived from an aggregate data set from thousands of diffraction patterns can be used to determine a crystal orientation matrix for individual images with as few as five reflections. These algorithms are potentially applicable not only to amyloid peptides but also to any set of diffraction patterns with sparse properties, such as low-resolution virus structures or high-throughput screening of still images captured by raster-scanning at synchrotron sources. As a proof of concept for this technique, successful integration of X-ray free-electron laser (XFEL) data to 2.5 Å resolution for the amyloid segment GNNQQNY from the Sup35 yeast prion is presented.
XFEL; Sup35 yeast prion; indexing methods; crystallography
X-ray free-electron lasers have opened up the possibility of structure determination of protein crystals at room temperature, free of radiation damage. The femtosecond-duration pulses of these sources enable diffraction signals to be collected from samples at doses of 1000 MGy or higher. The sample is vaporized by the intense pulse, but not before the scattering that gives rise to the diffraction pattern takes place. Consequently, only a single flash diffraction pattern can be recorded from a crystal, giving rise to the method of serial crystallography where tens of thousands of patterns are collected from individual crystals that flow across the beam and the patterns are indexed and aggregated into a set of structure factors. The high-dose tolerance and the many-crystal averaging approach allow data to be collected from much smaller crystals than have been examined at synchrotron radiation facilities, even from radiation-sensitive samples. Here, we review the interaction of intense femtosecond X-ray pulses with materials and discuss the implications for structure determination. We identify various dose regimes and conclude that the strongest achievable signals for a given sample are attained at the highest possible dose rates, from highest possible pulse intensities.
protein crystallography; radiation damage; X-ray lasers