| 1. |
- Wells, Daniel J., et al.
(författare)
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Observations of phase changes in monoolein during high viscous injection
- 2022
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Ingår i: Journal of Synchrotron Radiation. - : International Union Of Crystallography. - 0909-0495 .- 1600-5775. ; 29:3, s. 602-614
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Tidskriftsartikel (refereegranskat)abstract
- Serial crystallography of membrane proteins often employs high-viscosity injectors (HVIs) to deliver micrometre-sized crystals to the X-ray beam. Typically, the carrier medium is a lipidic cubic phase (LCP) media, which can also be used to nucleate and grow the crystals. However, despite the fact that the LCP is widely used with HVIs, the potential impact of the injection process on the LCP structure has not been reported and hence is not yet well understood. The self-assembled structure of the LCP can be affected by pressure, dehydration and temperature changes, all of which occur during continuous flow injection. These changes to the LCP structure may in turn impact the results of X-ray diffraction measurements from membrane protein crystals. To investigate the influence of HVIs on the structure of the LCP we conducted a study of the phase changes in monoolein/water and monoolein/buffer mixtures during continuous flow injection, at both atmospheric pressure and under vacuum. The reservoir pressure in the HVI was tracked to determine if there is any correlation with the phase behaviour of the LCP. The results indicated that, even though the reservoir pressure underwent (at times) significant variation, this did not appear to correlate with observed phase changes in the sample stream or correspond to shifts in the LCP lattice parameter. During vacuum injection, there was a three-way coexistence of the gyroid cubic phase, diamond cubic phase and lamellar phase. During injection at atmospheric pressure, the coexistence of a cubic phase and lamellar phase in the monoolein/water mixtures was also observed. The degree to which the lamellar phase is formed was found to be strongly dependent on the co-flowing gas conditions used to stabilize the LCP stream. A combination of laboratory-based optical polarization microscopy and simulation studies was used to investigate these observations.
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| 2. |
- Ekeberg, Tomas, 1983-, et al.
(författare)
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Observation of a single protein by ultrafast X-ray diffraction
- 2024
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Ingår i: Light. - : Springer Nature. - 2095-5545 .- 2047-7538. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- The idea of using ultrashort X-ray pulses to obtain images of single proteins frozen in time has fascinated and inspired many. It was one of the arguments for building X-ray free-electron lasers. According to theory, the extremely intense pulses provide sufficient signal to dispense with using crystals as an amplifier, and the ultrashort pulse duration permits capturing the diffraction data before the sample inevitably explodes. This was first demonstrated on biological samples a decade ago on the giant mimivirus. Since then, a large collaboration has been pushing the limit of the smallest sample that can be imaged. The ability to capture snapshots on the timescale of atomic vibrations, while keeping the sample at room temperature, may allow probing the entire conformational phase space of macromolecules. Here we show the first observation of an X-ray diffraction pattern from a single protein, that of Escherichia coli GroEL which at 14 nm in diameter is the smallest biological sample ever imaged by X-rays, and demonstrate that the concept of diffraction before destruction extends to single proteins. From the pattern, it is possible to determine the approximate orientation of the protein. Our experiment demonstrates the feasibility of ultrafast imaging of single proteins, opening the way to single-molecule time-resolved studies on the femtosecond timescale.
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| 3. |
- Esmaeildoost, Niloofar, et al.
(författare)
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Anomalous temperature dependence of the experimental x-ray structure factor of supercooled water
- 2021
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Ingår i: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 155:21, s. 214501-214501
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Tidskriftsartikel (refereegranskat)abstract
- The structural changes of water upon deep supercooling were studied through wide-angle x-ray scattering at SwissFEL. The experimental setup had a momentum transfer range of 4.5 Å-1, which covered the principal doublet of the x-ray structure factor of water. The oxygen-oxygen structure factor was obtained for temperatures down to 228.5 ± 0.6 K. Similar to previous studies, the second diffraction peak increased strongly in amplitude as the structural change accelerated toward a local tetrahedral structure upon deep supercooling. We also observed an anomalous trend for the second peak position of the oxygen-oxygen structure factor (q2). We found that q2 exhibits an unprecedented positive partial derivative with respect to temperature for temperatures below 236 K. Based on Fourier inversion of our experimental data combined with reference data, we propose that the anomalous q2 shift originates from that a repeat spacing in the tetrahedral network, associated with all peaks in the oxygen-oxygen pair-correlation function, gives rise to a less dense local ordering that resembles that of low-density amorphous ice. The findings are consistent with that liquid water consists of a pentamer-based hydrogen-bonded network with low density upon deep supercooling.
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| 4. |
- Esmaeildoost, Niloofar, et al.
(författare)
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Heterogeneous Ice Growth in Micron-Sized Water Droplets Due to Spontaneous Freezing
- 2022
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Ingår i: Crystals. - : MDPI AG. - 2073-4352. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Understanding how ice nucleates and grows into larger crystals is of crucial importance for many research fields. The purpose of this study was to shed light on the phase and structure of ice once a nucleus is formed inside a metastable water droplet. Wide-angle X-ray scattering (WAXS) was performed on micron-sized droplets evaporatively cooled to temperatures where homogeneous nucleation occurs. We found that for our weak hits ice grows more cubic compared to the strong hits that are completely hexagonal. Due to efficient heat removal caused by evaporation, we propose that the cubicity of ice at the vicinity of the droplet’s surface is higher than for ice formed within the bulk of the droplet. Moreover, the Bragg peaks were classified based on their geometrical shapes and positions in reciprocal space, which showed that ice grows heterogeneously with a significant population of peaks indicative of truncation rods and crystal defects. Frequent occurrences of the (100) reflection with extended in-planar structure suggested that large planar ice crystals form at the droplet surface, then fracture into smaller domains to accommodate to the curvature of the droplets. Planar faulting due to misaligned domains would explain the increased cubicity close to the droplet surface.
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| 5. |
- Esmaeildoost, Niloofar
(författare)
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Optical and x-ray studies of ice growth in water
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The key purpose of this thesis is to study the structure of metastable water and its transformation into ice upon deep supercooling using x-ray scattering as well as optical microscopy. In all experiments, micrometer-sized water droplets were evaporatively cooled in vacuum and probed either by x-rays or optical illumination. In addition to these, an infrared (IR) heating pulse was employed in one of the experiments to introduce a temperature jump in water droplets and achieve ultrafast calorimetry, which can measure specific heat capacity upon supercooling. The second peak of the structure factor presented a maximum at 236 K. The anomalous decrease in peak positions below 236 K was related to a repeat spacing in the tetrahedral network, associated with the intermediaterange correlations in water. The decrease in temperature makes the paircorrelation function change in a similar manner to that of low-density amorphous ice (LDA), meaning that the structure moves towards a less dense local ordering. This is in consistency with a low-density pentamer-bonded tetrahedral network that shifts continuously towards an LDA structure as it cools down. The x-ray scattering data showed that there is a maximum in the specific heat capacity of water at about 229 K and it increases from 88 J/mol/K at 244 K to 218 J/mol/K at 229 K upon cooling. Homogeneous ice nucleation showed that there is a mechanism of freezing based on the rates at which different frozen stages, i.e., partially frozen, liquid extrusion and fractured droplets, are seen in the microscopic images. Experimental nucleation data at temperatures as low as ∼230 K resulted in a nucleation fitting curve that shows a slower nucleation rate increase upon supercooling. Using self-diffusion data that was experimentally measured through wide-angle x-ray scattering and ultrafast calorimetry, we can assess the interfacial energy as a function of temperature. This resulted in a minimum in interfacial energy at around 236 K. Moreover, within the droplet, ice tends to form different structures after it has nucleated based on where in the droplet it is growing. It was observed that for crystals inside the bulk and close to the center of the droplets, ice crystallizes with hexagonal structure whereas on the surface it crystallizes with stacking-disorder containing a considerable amount of cubic structure. This can come from the fact that planar growth of crystals at the surface breaks down into a faulty structure that needs to accommodate the curvature of the droplet’s surface.
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| 6. |
- Hammarström, Björn, et al.
(författare)
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Acoustic Focusing of Protein Crystals for In-Line Monitoring and Up-Concentration during Serial Crystallography
- 2022
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 94:37, s. 12645-12656
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Tidskriftsartikel (refereegranskat)abstract
- Serial femtosecond crystallography (SFX) has become one of the standard techniques at X-ray free-electron lasers (XFELs) to obtain high-resolution structural information from microcrystals of proteins. Nevertheless, reliable sample delivery is still often limiting data collection, as microcrystals can clog both field- and flow-focusing nozzles despite in-line filters. In this study, we developed acoustic 2D focusing of protein microcrystals in capillaries that enables real-time online characterization of crystal size and shape in the sample delivery line after the in-line filter. We used a piezoelectric actuator to create a standing wave perpendicular to the crystal flow, which focused lysozyme microcrystals into a single line inside a silica capillary so that they can be imaged using a high-speed camera. We characterized the acoustic contrast factor, focus size, and the coaxial flow lines and developed a splitting union that enables up-concentration to at least a factor of five. The focus size, flow rates, and geometry may enable an upper limit of up-concentration as high as 200 fold. The novel feedback and concentration control could be implemented for serial crystallography at synchrotrons with minor modifications. It will also aid the development of improved sample delivery systems that will increase SFX data collection rates at XFELs, with potential applications to many proteins that can only be purified and crystallized in small amounts.
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| 7. |
- Holmes, Susannah, et al.
(författare)
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Megahertz pulse trains enable multi-hit serial femtosecond crystallography experiments at X-ray free electron lasers
- 2022
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by <1 mu s, yield up to 2.1 angstrom resolution structures. Comparisons between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in individual single crystals, a technique we refer to as multi-hit SFX. Free-electron lasers are capable of high repetition rates and it is assumed that protein crystals often do not survive the first X-ray pulse. Here the authors address these issues with a demonstration of multi-hit serial crystallography in which multiple FEL pulses interact with the sample without destroying it.
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| 8. |
- Kim, Kyung Hwan, et al.
(författare)
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Anisotropic X-Ray Scattering of Transiently Oriented Water
- 2020
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Ingår i: Physical Review Letters. - : American Physical Society (APS). - 0031-9007 .- 1079-7114. ; 125:7
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Tidskriftsartikel (refereegranskat)abstract
- We study the structural dynamics of liquid water by time-resolved anisotropic x-ray scattering under the optical Kerr effect condition. In this way, we can separate the anisotropic scattering decay of 160 fs from the delayed temperature increase of similar to 0.1 K occurring at 1 ps and quantify transient changes in the O-O pair distribution function. Polarizable molecular dynamics simulations reproduce well the experiment, indicating transient alignment of molecules along the electric field, which shortens the nearest-neighbor distances. In addition, analysis of the simulated water local structure provides evidence that two hypothesized fluctuating water configurations exhibit different polarizability.
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| 9. |
- Kim, Seonmyeong, et al.
(författare)
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Observing ice structure of micron-sized vapor-deposited ice with an x-ray free-electron laser
- 2023
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Ingår i: Structural Dynamics. - : AIP Publishing. - 2329-7778. ; 10:4
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Tidskriftsartikel (refereegranskat)abstract
- The direct observation of the structure of micrometer-sized vapor-deposited ice is performed at Pohang Accelerator Laboratory x-ray free electron laser (PAL-XFEL). The formation of micrometer-sized ice crystals and their structure is important in various fields, including atmospheric science, cryobiology, and astrophysics, but understanding the structure of micrometer-sized ice crystals remains challenging due to the lack of direct observation. Using intense x-ray diffraction from PAL-XFEL, we could observe the structure of micrometer-sized vapor-deposited ice below 150 K with a thickness of 2-50 μm grown in an ultrahigh vacuum chamber. The structure of the ice grown comprises cubic and hexagonal sequences that are randomly arranged to produce a stacking-disordered ice. We observed that ice with a high cubicity of more than 80% was transformed to partially oriented hexagonal ice when the thickness of the ice deposition grew beyond 5 μm. This suggests that precise temperature control and clean deposition conditions allow μm-thick ice films with high cubicity to be grown on hydrophilic Si3N4 membranes. The low influence of impurities could enable in situ diffraction experiments of ice nucleation and growth from interfacial layers to bulk ice.
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| 10. |
- Kördel, Mikael
(författare)
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Biological Laboratory X-Ray Microscopy
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Soft x-ray microscopy in the water window (? ≈ 2.3 − 4.3 nm) is a powerful technique for high-resolution biological imaging. The strong natural contrast between carbon-based structures and water allows visualization of hydrated and unstained samples, while providing enough transmission through up to ∼ 10 μm of organic matter. Furthermore, the full potential of this technique can be exploited by performing computed tomography, thus obtaining a complete 3D image of the object.Routine short-exposure water-window microscopy of whole cells and tissue is currently performed at synchrotron-radiation facilities around the world, but with a limited accessibility to the wider research community. For this reason, laboratory-based systems have been developed, which are now reaching maturity. The benefits compared to the synchrotron-based instruments include easier integration with complementary methods in the home laboratory, in addition to the increased access that allows for the often time-consuming optimization of experimental parameters as well as longitudinal studies.This Thesis presents recent developments of the Stockholm laboratory x-ray microscope as well as several biological applications. Work has been done on improving the mechanical and thermal stability of the microscope, resulting in a resolution of 25 nm (half period) in images of test targets. The biological applications were enabled by a significantly increased x-ray flux through the system as well as an improved operational stability. This work demonstrates 10-second exposure imaging of whole cryofixed cells, imaging of viral infections in cells, and 20 minutes total exposure cryotomography.
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