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
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.
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.
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
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.
An expectation maximization algorithm is implemented to resolve the indexing ambiguity which arises when merging data from many crystals in protein crystallography, especially in cases where partial reflections are recorded in serial femtosecond crystallography (SFX) at XFELs.
Crystallographic auto-indexing algorithms provide crystal orientations and unit-cell parameters and assign Miller indices based on the geometric relations between the Bragg peaks observed in diffraction patterns. However, if the Bravais symmetry is higher than the space-group symmetry, there will be multiple indexing options that are geometrically equivalent, and hence many ways to merge diffraction intensities from protein nanocrystals. Structure factor magnitudes from full reflections are required to resolve this ambiguity but only partial reflections are available from each XFEL shot, which must be merged to obtain full reflections from these ‘stills’. To resolve this chicken-and-egg problem, an expectation maximization algorithm is described that iteratively constructs a model from the intensities recorded in the diffraction patterns as the indexing ambiguity is being resolved. The reconstructed model is then used to guide the resolution of the indexing ambiguity as feedback for the next iteration. Using both simulated and experimental data collected at an X-ray laser for photosystem I in the P63 space group (which supports a merohedral twinning indexing ambiguity), the method is validated.
indexing ambiguity; serial femtosecond crystallography (SFX); XFELs; protein crystallography; expectation maximization algorithm
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
Femtosecond X-ray crystallography allows structural analysis of a difficult-to-crystallize fusion protein that is a potential component of a candidate HIV-1 subunit vaccine.
CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.
X-ray crystallography; femtosecond nanocrystallography; HIV-1; gp41; membrane-proximal region; cholera toxin B subunit; crystallization; free-electron lasers
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.
The shape transforms of nanocrystals with incomplete unit cells are studied using computer simulations. Structure-factor phases can be retrieved from the molecular transforms after removing the modulating shape transform terms.
X-ray free electron lasers are used in measuring diffraction patterns from nanocrystals in the ‘diffract-before-destroy’ mode by outrunning radiation damage. The finite-sized nanocrystals provide an opportunity to recover intensity between Bragg spots by removing the modulating function that depends on crystal shape, i.e. the transform of the crystal shape. This shape-transform dividing-out scheme for solving the phase problem has been tested using simulated examples with cubic crystals. It provides a phasing method which does not require atomic resolution data, chemical modification to the sample, or modelling based on the protein databases. It is common to find multiple structural units (e.g. molecules, in symmetry-related positions) within a single unit cell, therefore incomplete unit cells (e.g. one additional molecule) can be observed at surface layers of crystals. In this work, the effects of such incomplete unit cells on the ‘dividing-out’ phasing algorithm are investigated using 2D crystals within the projection approximation. It is found that the incomplete unit cells do not hinder the recovery of the scattering pattern from a single unit cell (after dividing out the shape transforms from data merged from many nanocrystals of different sizes), assuming that certain unit-cell types are preferred. The results also suggest that the dynamic range of the data is a critical issue to be resolved in order to apply the shape transform method practically.
shape transform; nanocrystallography; X-ray free electron lasers; phasing
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
Intense femtosecond X-ray pulses produced at the Linac Coherent Light Source (LCLS) were used for simultaneous X-ray diffraction (XRD) and X-ray emission spectroscopy (XES) of microcrystals of Photosystem II (PS II) at room temperature. This method probes the overall protein structure and the electronic structure of the Mn4CaO5 cluster in the oxygen-evolving complex of PS II. XRD data are presented from both the dark state (S1) and the first illuminated state (S2) of PS II. Our simultaneous XRD/XES study shows that the PS II crystals are intact during our measurements at the LCLS, not only with respect to the structure of PS II, but also with regard to the electronic structure of the highly radiation sensitive Mn4CaO5 cluster, opening new directions for future dynamics studies.
An ultrasensitive Medipix2 detector allowed the collection of rotation electron-diffraction data from single three-dimensional protein nanocrystals for the first time. The data could be analysed using the standard X-ray crystallography programs MOSFLM and SCALA.
When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e− Å−2), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electron-diffraction data from a single ∼100 nm thick protein crystal. A highly parallel 200 keV electron beam (λ = 0.025 Å) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4°. This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLM and SCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins.
electron diffraction; electron microscopy; Medipix2; MOSFLM; nanocrystals
We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1–1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. This proof of principle paves the way for the implementation of a new technique, which we name ‘MicroED’, that may have wide applicability in structural biology.
X-ray crystallography has been used to work out the atomic structure of a large number of proteins. In a typical X-ray crystallography experiment, a beam of X-rays is directed at a protein crystal, which scatters some of the X-ray photons to produce a diffraction pattern. The crystal is then rotated through a small angle and another diffraction pattern is recorded. Finally, after this process has been repeated enough times, it is possible to work backwards from the diffraction patterns to figure out the structure of the protein.
The crystals used for X-ray crystallography must be large to withstand the damage caused by repeated exposure to the X-ray beam. However, some proteins do not form crystals at all, and others only form small crystals. It is possible to overcome this problem by using extremely short pulses of X-rays, but this requires a very large number of small crystals and ultrashort X-ray pulses are only available at a handful of research centers around the world. There is, therefore, a need for other approaches that can determine the structure of proteins that only form small crystals.
Electron crystallography is similar to X-ray crystallography in that a protein crystal scatters a beam to produce a diffraction pattern. However, the interactions between the electrons in the beam and the crystal are much stronger than those between the X-ray photons and the crystal. This means that meaningful amounts of data can be collected from much smaller crystals. However, it is normally only possible to collect one diffraction pattern from each crystal because of beam induced damage. Researchers have developed methods to merge the diffraction patterns produced by hundreds of small crystals, but to date these techniques have only worked with very thin two-dimensional crystals that contain only one layer of the protein of interest.
Now Shi et al. report a new approach to electron crystallography that works with very small three-dimensional crystals. Called MicroED, this technique involves placing the crystal in a transmission electron cryo-microscope, which is a fairly standard piece of equipment in many laboratories. The normal ‘low-dose’ electron beam in one of these microscopes would normally damage the crystal after a single diffraction pattern had been collected. However, Shi et al. realized that it was possible to obtain diffraction patterns without severely damaging the crystal if they dramatically reduced the normal low-dose electron beam. By reducing the electron dose by a factor of 200, it was possible to collect up to 90 diffraction patterns from the same, very small, three-dimensional crystal, and then—similar to what happens in X-ray crystallography—work backwards to figure out the structure of the protein. Shi et al. demonstrated the feasibility of the MicroED approach by using it to determine the structure of lysozyme, which is widely used as a test protein in crystallography, with a resolution of 2.9 Å. This proof-of principle study paves the way for crystallographers to study protein that cannot be studied with existing techniques.
electron crystallography; electron diffraction; electron cryomicroscopy (cryo-EM); microED; protein structure; microcrystals; None
In situ microzone X-ray diffraction analysis of natural teeth is presented. From our experiment, layer orientation and continuous crystal variations in teeth could be conveniently studied using fast online measurements by high-resolution X-ray microdiffraction equipment.
The main component of natural teeth was determined many years ago as calcium phosphate, mostly in the form of hydroxyapatite with different crystallites. In the past, the method used in tooth crystal investigation has been mainly powder X-ray diffraction analysis, but this method has its drawbacks, i.e. the destruction of the natural tooth structure and the difficulty in examining the preferred orientation in different layers of the tooth. During the last century, microzone X-ray diffraction on the tooth surface was carried out, but, as the technology was less sophisticated, the results obtained were not very detailed. The newly developed microdiffraction equipment permits analysis of the microzone of teeth in situ. To test this new microdiffraction equipment, microdiffraction analysis of one natural healthy deciduous molar tooth and one carious deciduous molar tooth has been performed, using a Bruker D8 instrument. Phase analysis of the two teeth was performed; the crystal size at six test points in the natural healthy tooth was calculated by reflection (211), and the crystal preferred orientation of reflection (300) and reflection (002) at six test points in the natural healthy tooth were compared. The results showed that the tooth was a kind of biological mixed crystal composed of several crystal phases, the main crystal phase being hydroxyapatite. The crystal size grew larger going from the dentin to the enamel. The crystal preferred orientation mainly existed in the enamel, especially in the reflection (002). From our experiment, layer orientation and continuous crystal variations in teeth could be conveniently studied using fast online measurements by high-resolution X-ray microdiffraction equipment.
X-ray microdiffraction; enamel; caries; texture; crystal
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.
An algorithm is described that calculates the most likely primitive unit cell given a set of randomly oriented electron-diffraction patterns with unknown angular relationships.
Unit-cell determination is the first step towards the structure solution of an unknown crystal form. Standard procedures for unit-cell determination cannot cope with data collections that consist of single diffraction patterns of multiple crystals, each with an unknown orientation. However, for beam-sensitive nanocrystals these are often the only data that can be obtained. An algorithm for unit-cell determination that uses randomly oriented electron-diffraction patterns with unknown angular relationships is presented here. The algorithm determined the unit cells of mineral, pharmaceutical and protein nanocrystals in orthorhombic high- and low-symmetry space groups, allowing (well oriented) patterns to be indexed.
electron diffraction; nanocrystals; unit-cell determination; unknown orientation
With the use of highly coherent femtosecond X-ray pulses from a free-electron laser, it is possible to record protein nanocrystal diffraction patterns with far more information than is present in conventional crystallographic diffraction data. It has been suggested that diffraction phases may be retrieved from such data via iterative algorithms, without the use of a priori information and without restrictions on resolution. Here, we investigate the extension of this approach to nanocrystals with edge terminations that produce partial unit cells, and hence cannot be described by a common repeating unit cell. In this situation, the phase problem described in previous work must be reformulated. We demonstrate an approximate solution to this phase problem for crystals with random edge terminations.
protein crystallography; coherent diffractive imaging; free-electron laser
A low flow rate liquid microjet method for delivery of hydrated protein crystals to X-ray lasers is presented. Linac Coherent Light Source data demonstrates serial femtosecond protein crystallography with micrograms, a reduction of sample consumption by orders of magnitude.
An electrospun liquid microjet has been developed that delivers protein microcrystal suspensions at flow rates of 0.14–3.1 µl min−1 to perform serial femtosecond crystallography (SFX) studies with X-ray lasers. Thermolysin microcrystals flowed at 0.17 µl min−1 and diffracted to beyond 4 Å resolution, producing 14 000 indexable diffraction patterns, or four per second, from 140 µg of protein. Nanoflow electrospinning extends SFX to biological samples that necessitate minimal sample consumption.
serial femtosecond crystallography; nanoflow electrospinning
Strategies are described for optimizing the signal-to-noise of diffraction data, and for combining data from multiple crystals. One challenge that must be overcome is the non-random orientation of crystals with respect to one another and with respect to the surface that supports them.
X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltd in situ micro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from ∼100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face that preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. High-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.
in situ X-ray data collection; crystallography; acoustic droplet ejection; serial crystallography
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
Terbium fluoride nanocrystals, covered by a shell, composed of cerium fluoride were synthesized by a co-precipitation method. Their complex structure was formed spontaneously during the synthesis. The surface of these core/shell nanocrystals was additionally modified by silica. The properties of TbF3@CeF3 and TbF3@CeF3@SiO2 nanocrystals, formed in this way, were investigated. Spectroscopic studies showed that the differences between these two groups of products resulted from the presence of the SiO2 shell. X-ray diffraction patterns confirmed the trigonal crystal structure of TbF3@CeF3 nanocrystals. High resolution transmission electron microscopy in connection with energy-dispersive X-ray spectroscopy showed a complex structure of the formed nanocrystals. Crystallized as small discs, ‘the products’, with an average diameter around 10 nm, showed an increase in the concentration of Tb3+ ions from surface to the core of nanocrystals. In addition to photo-physical analyses, cytotoxicity studies were performed on HSkMEC (Human Skin Microvascular Endothelial Cells) and B16F0 mouse melanoma cancer cells. The cytotoxicity of the nanomaterials was neutral for the investigated cells with no toxic or antiproliferative effect in the cell cultures, either for normal or for cancer cells. This fact makes the obtained nanocrystals good candidates for biological applications and further modifications of the SiO2 shell.
Nanoparticles; Core/shell; Silica; Luminescence; Rare earth fluorides; Cytotoxicity
The room-temperature structure of lysozyme is determined using 40000 individual diffraction patterns from micro-crystals flowing in liquid suspension across a synchrotron microfocus beamline.
A new approach for collecting data from many hundreds of thousands of microcrystals using X-ray pulses from a free-electron laser has recently been developed. Referred to as serial crystallography, diffraction patterns are recorded at a constant rate as a suspension of protein crystals flows across the path of an X-ray beam. Events that by chance contain single-crystal diffraction patterns are retained, then indexed and merged to form a three-dimensional set of reflection intensities for structure determination. This approach relies upon several innovations: an intense X-ray beam; a fast detector system; a means to rapidly flow a suspension of crystals across the X-ray beam; and the computational infrastructure to process the large volume of data. Originally conceived for radiation-damage-free measurements with ultrafast X-ray pulses, the same methods can be employed with synchrotron radiation. As in powder diffraction, the averaging of thousands of observations per Bragg peak may improve the ratio of signal to noise of low-dose exposures. Here, it is shown that this paradigm can be implemented for room-temperature data collection using synchrotron radiation and exposure times of less than 3 ms. Using lysozyme microcrystals as a model system, over 40 000 single-crystal diffraction patterns were obtained and merged to produce a structural model that could be refined to 2.1 Å resolution. The resulting electron density is in excellent agreement with that obtained using standard X-ray data collection techniques. With further improvements the method is well suited for even shorter exposures at future and upgraded synchrotron radiation facilities that may deliver beams with 1000 times higher brightness than they currently produce.
serial crystallography; room-temperature protein crystallography; radiation damage; CrystFEL; microfocus beamline