| 1. |
- Bjelčić, Monika, et al.
(författare)
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Anaerobic fixed-target serial crystallography using sandwiched silicon nitride membranes
- 2023
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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 79:Pt 11, s. 1018-1025
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Tidskriftsartikel (refereegranskat)abstract
- In recent years, the emergence of serial crystallography, initially pioneered at X-ray free-electron lasers (XFELs), has sparked a growing interest in collecting macromolecular crystallographic data at room temperature. Various fixed-target serial crystallography techniques have been developed, ranging from commercially available chips to in-house designs implemented at different synchrotron facilities. Nevertheless, there is currently no commercially available chip (known to the authors) specifically designed for the direct handling of oxygen-sensitive samples. This study presents a methodology employing silicon nitride chips arranged in a 'sandwich' configuration, enabling reliable room-temperature data collection from oxygen-sensitive samples. The method involves the utilization of a custom-made 3D-printed assembling tool and a MX sample holder. To validate the effectiveness of the proposed method, deoxyhemoglobin and methemoglobin samples were investigated using the BioMAX X-ray macromolecular crystallography beamline, the Balder X-ray absorption spectroscopy beamline and UV-Vis absorption spectroscopy.
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| 2. |
- Ghosh, Swagatha, et al.
(författare)
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A simple goniometer-compatible flow cell for serial synchrotron X-ray crystallography
- 2023
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Ingår i: Journal of Applied Crystallography. - 0021-8898. ; 56:Pt 2, s. 449-460
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Tidskriftsartikel (refereegranskat)abstract
- Serial femtosecond crystallography was initially developed for room-temperature X-ray diffraction studies of macromolecules at X-ray free electron lasers. When combined with tools that initiate biological reactions within microcrystals, time-resolved serial crystallography allows the study of structural changes that occur during an enzyme catalytic reaction. Serial synchrotron X-ray crystallography (SSX), which extends serial crystallography methods to synchrotron radiation sources, is expanding the scientific community using serial diffraction methods. This report presents a simple flow cell that can be used to deliver microcrystals across an X-ray beam during SSX studies. This device consists of an X-ray transparent glass capillary mounted on a goniometer-compatible 3D-printed support and is connected to a syringe pump via lightweight tubing. This flow cell is easily mounted and aligned, and it is disposable so can be rapidly replaced when blocked. This system was demonstrated by collecting SSX data at MAX IV Laboratory from microcrystals of the integral membrane protein cytochrome c oxidase from Thermus thermophilus, from which an X-ray structure was determined to 2.12 Å resolution. This simple SSX platform may help to lower entry barriers for non-expert users of SSX.
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| 3. |
- Leonarski, Filip, et al.
(författare)
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Kilohertz serial crystallography with the JUNGFRAU detector at a fourth-generation synchrotron source
- 2023
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Ingår i: IUCrJ. - 2052-2525. ; 10:Pt 6, s. 729-737
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Tidskriftsartikel (refereegranskat)abstract
- Serial and time-resolved macromolecular crystallography are on the rise. However, beam time at X-ray free-electron lasers is limited and most thirdgeneration synchrotron-based macromolecular crystallography beamlines do not offer the necessary infrastructure yet. Here, a new setup is demonstrated, based on the JUNGFRAU detector and Jungfraujoch data-acquisition system, that enables collection of kilohertz serial crystallography data at fourthgeneration synchrotrons. More importantly, it is shown that this setup is capable of collecting multiple-time-point time-resolved protein dynamics at kilohertz rates, allowing the probing of microsecond to second dynamics at synchrotrons in a fraction of the time needed previously. A high-quality complete X-ray dataset was obtained within 1 min from lysozyme microcrystals, and the dynamics of the light-driven sodium-pump membrane protein KR2 with a time resolution of 1 ms could be demonstrated. To make the setup more accessible for researchers, downstream data handling and analysis will be automated to allow on-the-fly spot finding and indexing, as well as data processing.
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| 4. |
- Monika, Bjelčić
(författare)
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Implementation and development of serial synchrotron crystallography at MAX IV
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Over the past decade, remarkable advancements in femtosecond X-ray free-electron lasers (XFELs) have brought about a profound transformation in structural biology. These XFELs have opened up exciting opportunities for conducting high-time resolution, room-temperature studies on protein structures and dynamics. This cutting-edge methodology involves exposing thousands of crystals to X-ray beams in random orientations at room temperature. These innovations have spurred the emergence of serial crystallography techniques, which have gained traction on more adaptable and readily accessible microfocus beamlines in synchrotron facilities. The primary focus of this thesis revolves around the development and implementation of serial synchrotron crystallography for proteins at the macromolecular beamlines BioMAX and MicroMAX, located at MAX IV Laboratory in Sweden. \newline This dissertation encompasses the development of two innovative sample delivery devices (Serial-X and AdaptoCell), incorporation of various fixed-target methods and preparation for TR-SSX. In Paper I, we introduced a novel approach for collecting data from oxygen-sensitive samples using fixed-target, complemented by the introduction of 3-D printed accessories. Paper III demonstrates the successful application of the methodology outlined in Paper I to determine the structure of CYP3A4. Paper II delves into the creation of the Serial-X flow cell, designed for efficient delivery of viscous samples, a tool that has now found utility on numerous beamlines worldwide. Lastly, in Paper IV, we present the first room-temperature serial synchrotron crystallography structure of spinach RuBisCo, along with the prepatory steps for a time-resolved SSX study of RuBisCO at BioMAX.
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