| 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. |
- Fornell, Anna, et al.
(författare)
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A microfluidic platform for SAXS measurements of liquid samples
- 2022
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Konferensbidrag (refereegranskat)abstract
- Small-angle X-ray scattering (SAXS) is a technique that can measure the size and shape of small particles such as proteins and nanoparticles using X-rays. At MAX IV, we are developing a microfluidic sample delivery platform to measure liquid samples containing proteins under flow using SAXS. One of the main advantages of using microfluidics is that the sample is continuously flowing, thus minimizing the risk of radiation damage as the sample is continuously refreshed. Other advantages include low sample volume and the possibility to study dynamic processes, e.g. mixing. To obtain good SAXS signals, the X-ray properties of the chip material are essential. The microfluidic chip must have low attenuation of X-rays, low background scattering, and high resistance to X-ray-induced damage, and preferably be low cost and easy to fabricate. In this work, we have evaluated the performance of two different polymer microfluidic chips for SAXS measurements.
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| 3. |
- Fornell, Anna, et al.
(författare)
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AdaptoCell : Microfluidics at MAX IV Laboratory
- 2022
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Ingår i: 25th Swedish Conference on Macromolecular Structure and Function.
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Konferensbidrag (refereegranskat)abstract
- The AdaptoCell project at MAX IV has developed a microfluidic sample delivery platform for academic and industrial users to enable studies of protein samples in solution and in microcrystals underflow. The platform is compatible with various X-ray techniques and has so far been integrated onto two beamlines at MAX IV: the CoSAXS beamline for small angle X-ray scattering studies and the Balder beamline for X-ray absorption spectroscopy studies. Initial implementation of the platform for serial crystallography sample delivery is ongoing and will be integrated onto the BioMAX and MicroMAX beamlines once commissioned. With this platform, we aim to meet the demand from our user community for studying proteins at physiologically relevant temperatures and give the ability to follow dynamical processes in situ as well as decreasing sample volumes and radiation damage.To determine the optimized flow rates and components for mixing etc. using different microfluidic chips, a dedicated off(beam)line test station with a microscope has been established at the Biolab. The Biolab also provides a number of characterization techniques, such as Dynamic Light Scattering, UV-Vis spectrophotometry, for quality control of the samples; as well as an anaerobic chamber for preparation and characterization of metalloproteins. The microfluidic flows are controlled via syringe pumps or a pressure-driven system. Channel design varies, depending on the needs of the experiment, from straight channel, cross-junction to herringbone micromixers etc. On-chip mixing of buffers with different viscosity, pH, ion strength and protein concentrations has been demonstrated successful and will be presented.
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| 4. |
- 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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| 5. |
- 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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| 6. |
- 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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| 7. |
- Ursby, Thomas, et al.
(författare)
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BioMAX the first macromolecular crystallography beamline at MAX IV Laboratory
- 2020
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Ingår i: Journal of Synchrotron Radiation. - Chichester : Wiley-Blackwell. - 0909-0495 .- 1600-5775. ; 27, s. 1415-1429
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Tidskriftsartikel (refereegranskat)abstract
- BioMAX is the first macromolecular crystallography beamline at the MAX IV Laboratory 3 GeV storage ring, which is the first operational multi-bend achromat storage ring. Due to the low-emittance storage ring, BioMAX has a parallel, high-intensity X-ray beam, even when focused down to 20 μm × 5 μm using the bendable focusing mirrors. The beam is tunable in the energy range 5-25 keV using the in-vacuum undulator and the horizontally deflecting double-crystal monochromator. BioMAX is equipped with an MD3 diffractometer, an ISARA high-capacity sample changer and an EIGER 16M hybrid pixel detector. Data collection at BioMAX is controlled using the newly developed MXCuBE3 graphical user interface, and sample tracking is handled by ISPyB. The computing infrastructure includes data storage and processing both at MAX IV and the Lund University supercomputing center LUNARC. With state-of-the-art instrumentation, a high degree of automation, a user-friendly control system interface and remote operation, BioMAX provides an excellent facility for most macromolecular crystallography experiments. Serial crystallography using either a high-viscosity extruder injector or the MD3 as a fixed-target scanner is already implemented. The serial crystallography activities at MAX IV Laboratory will be further developed at the microfocus beamline MicroMAX, when it comes into operation in 2022. MicroMAX will have a 1 μm × 1 μm beam focus and a flux up to 1015 photons s with main applications in serial crystallography, room-temperature structure determinations and time-resolved experiments.
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