scholarly journals Viscous medium-based crystal support in sample holder for fixed-target serial femtosecond crystallography

2020 ◽  
Author(s):  
Keondo Lee ◽  
Donghyeon Lee ◽  
Sangwon Baek ◽  
Jaehyun Park ◽  
Sang Jae Lee ◽  
...  
Keyword(s):  
Room Temperature ◽  
Polyimide Film ◽  
Glucose Isomerase ◽  
Viscous Medium ◽  
Sample Holder ◽  
Polyimide Films ◽  
Fixed Target ◽  
Time Resolved ◽  
Viscous Media ◽  
Serial Femtosecond Crystallography

AbstractSerial femtosecond crystallography (SFX) enables the determination of a room-temperature crystal structure of macromolecules without causing radiation damage, as well as provides time-resolved molecular dynamics data in pump-probe experiments. Fixed-target SFX (FT-SFX) can minimize sample consumption and physical effects to crystals during sample delivery. Various types of sample holders have been developed and applied in FT-SFX; however, no sample holder has been developed that can universally mount crystals of various sizes and shapes. Here, we introduce a viscous media-based crystal support in a sample holder for FT-SFX. Crystal samples were embedded in viscous media such as gelatin and agarose, which were enclosed in a polyimide film. In the vertically placed sample holder, the viscous medium stably supported crystals between the two polyimide films without crystal sinking due to gravity. Using this method, we performed FT-SFX experiments with glucose isomerase and lysozyme embedded in gelatin and agarose, respectively. The room-temperature crystal structures of glucose isomerase and lysozyme were successfully determined at 1.75 and 1.80 Å resolutions, respectively. Viscous media used in this experiment showed negligible background scattering in data processing. This method is useful for delivering crystal samples of various sizes and shapes in FT-SFX experiments.

scholarly journals Viscous-medium-based crystal support in a sample holder for fixed-target serial femtosecond crystallography

2020 ◽  
Vol 53 (4) ◽  
pp. 1051-1059 ◽  
Author(s):  
Keondo Lee ◽  
Donghyeon Lee ◽  
Sangwon Baek ◽  
Jaehyun Park ◽  
Sang Jae Lee ◽  
...  
Keyword(s):  
Room Temperature ◽  
Polyimide Film ◽  
Glucose Isomerase ◽  
Viscous Medium ◽  
Sample Holder ◽  
Fixed Target ◽  
Time Resolved ◽  
Efficient Data ◽  
Viscous Media ◽  
Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) enables the determination of the room-temperature crystal structure of macromolecules, as well as providing time-resolved molecular dynamics data in pump–probe experiments. Fixed-target SFX (FT-SFX) can minimize sample consumption and physical effects on crystals during sample delivery. In FT-SFX studies, having a sample holder that can stably fix crystal samples is one of the key elements required for efficient data collection. Hence, development of sample holders from new materials capable of supporting various crystal sizes and shapes may expand the applications of FT-SFX. Here, a viscous-media-based crystal support in a sample holder for FT-SFX is introduced. Crystal samples were embedded in viscous media, namely gelatin and agarose, which were enclosed in a polyimide film. In the vertically placed sample holder, 10–15%(w/v) viscous gelatin and 1–4%(w/v) agarose gel stably supported crystals between two polyimide films, thereby preventing the crystals from descending owing to gravity. Using this method, FT-SFX experiments were performed with glucose isomerase and lysozyme embedded in gelatin and agarose, respectively. The room-temperature crystal structures of glucose isomerase and lysozyme were successfully determined at 1.75 and 1.80 Å resolutions, respectively. The glucose isomerase and lysozyme diffraction analyses were not impeded by excessive background scattering from the viscous media. This method is useful for delivering crystal samples of various sizes and shapes in FT-SFX experiments.

scholarly journals Lard Injection Matrix for Serial Crystallography

2020 ◽  
Vol 21 (17) ◽  
pp. 5977
Author(s):  
Ki Hyun Nam
Keyword(s):  
Flow Rate ◽  
Room Temperature ◽  
Glucose Isomerase ◽  
Viscous Medium ◽  
Cubic Phase ◽  
Time Resolved ◽  
Crystal Sample ◽  
X Ray ◽  
Serial Crystallography ◽  
Delivery Medium

Serial crystallography (SX) using X-ray free electron laser or synchrotron X-ray allows for the determination of structures, at room temperature, with reduced radiation damage. Moreover, it allows for the study of structural dynamics of macromolecules using a time-resolved pump-probe, as well as mix-and-inject experiments. Delivering a crystal sample using a viscous medium decreases sample consumption by lowering the flow rate while being extruded from the injector or syringe as compared to a liquid jet injector. Since the environment of crystal samples varies, continuous development of the delivery medium is important for extended SX applications. Herein, I report the preparation and characterization of a lard-based sample delivery medium for SX. This material was obtained using heat treatment, and then the soluble impurities were removed through phase separation. The lard injection medium was highly stable and could be injected via a syringe needle extruded at room temperature with a flow rate < 200 nL/min. Serial millisecond crystallography experiments were performed using lard, and the room temperature structures of lysozyme and glucose isomerase embedded in lard at 1.75 and 1.80 Å, respectively, were determined. The lard medium showed X-ray background scattering similar or relatively lower than shortenings and lipidic cubic phase; therefore, it can be used as sample delivery medium in SX experiments.

scholarly journals Beef tallow injection matrix for serial crystallography

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Ki Hyun Nam
Keyword(s):  
Beef Tallow ◽  
Room Temperature ◽  
Heat Treating ◽  
Glucose Isomerase ◽  
Viscous Medium ◽  
Low Flow ◽  
Interaction Point ◽  
Time Resolved ◽  
Experimental Environment ◽  
Serial Crystallography

AbstractSerial crystallography (SX) enables the visualization of the time-resolved molecular dynamics of macromolecular structures at room temperature while minimizing radiation damage. In SX experiments, the delivery of a large number of crystals into an X-ray interaction point in a serial and stable manner is key. Sample delivery using viscous medium maintains the stable injection stream at low flow rates, markedly reducing sample consumption compared with that of a liquid jet injector and is widely applied in SX experiments with low repetition rates. As the sample properties and experimental environment can affect the stability of the injection stream of a viscous medium, it is important to develop sample delivery media with various characteristics to optimize the experimental environment. In this study, a beef tallow injection matrix possessing a higher melting temperature than previously reported fat-based shortening and lard media was introduced as a sample delivery medium and applied to SX. Beef tallow was prepared by heat treating fats from cattle, followed by the removal of soluble impurities from the extract by phase separation. Beef tallow exhibited a very stable injection stream at room temperature and a flow rate of < 10 nL/min. The room-temperature structures of lysozyme and glucose isomerase embedded in beef tallow were successfully determined at 1.55 and 1.60 Å, respectively. The background scattering of beef tallow was higher than that of previously reported fat-based shortening and lard media but negligible for data processing. In conclusion, the beef tallow matrix can be employed for sample delivery in SX experiments conducted at temperatures exceeding room temperature.

scholarly journals Polyimide mesh-based sample holder with irregular crystal mounting holes for fixed-target serial crystallography

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ki Hyun Nam ◽  
Jihan Kim ◽  
Yunje Cho
Keyword(s):  
Data Collection ◽  
Room Temperature ◽  
Picosecond Laser ◽  
Temperature Structure ◽  
Sample Holder ◽  
Fixed Target ◽  
Random Orientation ◽  
Physical Damage ◽  
Crystal Sample ◽  
Serial Crystallography

AbstractThe serial crystallography (SX) technique enables the determination of the room-temperature structure of a macromolecule while causing minimal radiation damage, as well as the visualization of the molecular dynamics by time-resolved studies. The fixed-target (FT) scanning approach is one method for SX sample delivery that minimizes sample consumption and minimizes physical damage to crystals during data collection. Settling of the crystals on the sample holder in random orientation is important for complete three dimensional data collection. To increase the random orientation of crystals on the sample holder, we developed a polyimide mesh-based sample holder with irregular crystal mounting holes for FT-SX. The polyimide mesh was fabricated using a picosecond laser. Each hole in the polyimide mesh has irregularly shaped holes because of laser thermal damage, which may cause more crystals to settle at random orientations compared to regular shaped sample holders. A crystal sample was spread onto a polyimide-mesh, and a polyimide film was added to both sides to prevent dehydration. Using this sample holder, FT-SX was performed at synchrotron and determined the room-temperature lysozyme structure at 1.65 Å. The polyimide mesh with irregularly shaped holes will allow for expanded applications in sample delivery for FT-SX experiments.

Sample delivery using viscous media, a syringe and a syringe pump for serial crystallography

2019 ◽  
Vol 26 (5) ◽  
pp. 1815-1819 ◽  
Author(s):  
Suk-Youl Park ◽  
Ki Hyun Nam
Keyword(s):  
Crystal Structures ◽  
Room Temperature ◽  
Viscous Medium ◽  
Cubic Phase ◽  
Syringe Pump ◽  
Beam Position ◽  
Crystal Sample ◽  
Injector Nozzle ◽  
Viscous Media ◽  
Serial Crystallography

Sample delivery using injectors is widely used in serial crystallography (SX) and has significantly contributed to the determination of crystal structures at room temperature. However, sophisticated injector nozzle fabrication methods and sample delivery operations have made it difficult for ordinary users to access the SX research. Herein, a simple and easily accessible sample delivery method for SX experiments is introduced, that uses a viscous medium, commercially available syringe and syringe pump. The syringe containing the lysozyme crystals embedded in lipidic cubic phase (LCP) or polyacrylamide (PAM) delivery media was connected to a needle having an inner diameter of 168 µm, after which it was installed on a syringe pump. By driving the syringe pump, the syringe plunger was pushed and the crystal sample was delivered to the X-ray beam position in a stable manner. Using this system, the room-temperature crystal structures of lysozyme embedded in LCP and PAM at 1.56 Å and 1.75 Å, respectively, were determined. This straightforward syringe pump-based sample delivery system can be utilized in SX.

scholarly journals Polysaccharide-Based Injection Matrix for Serial Crystallography

2020 ◽  
Vol 21 (9) ◽  
pp. 3332 ◽  
Author(s):  
Ki Hyun Nam
Keyword(s):  
Room Temperature ◽  
Glucose Isomerase ◽  
Viscous Medium ◽  
Specific Protein ◽  
Wheat Starch ◽  
X Ray ◽  
Crystallization Solution ◽  
Serial Crystallography ◽  
X Ray Background

Serial crystallography (SX) provides an opportunity to observe the molecular dynamics of macromolecular structures at room temperature via pump-probe studies. The delivery of crystals embedded in a viscous medium via an injector or syringe is widely performed in synchrotrons or X-ray free-electron laser facilities with low repetition rates. Various viscous media have been developed; however, there are cases in which the delivery material undesirably interacts chemically or biologically with specific protein samples, or changes the stability of the injection stream, depending on the crystallization solution. Therefore, continued discovery and characterization of new delivery media is necessary for expanding future SX applications. Here, the preparation and characterization of new polysaccharide (wheat starch (WS) and alginate)-based sample delivery media are introduced for SX. Crystals embedded in a WS or alginate injection medium showed a stable injection stream at a flow rate of < 200 nL/min and low-level X-ray background scattering similar to other hydrogels. Using these media, serial millisecond crystallography (SMX) was performed, and the room temperature crystal structures of glucose isomerase and lysozyme were determined at 1.9–2.0 Å resolutions. WS and alginate will allow an expanded application of sample delivery media in SX experiments.

scholarly journals Nylon mesh-based sample holder for fixed-target serial femtosecond crystallography

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Donghyeon Lee ◽  
Sangwon Baek ◽  
Jaehyun Park ◽  
Keondo Lee ◽  
Jangwoo Kim ◽  
...  
Keyword(s):  
Sample Holder ◽  
Fixed Target ◽  
Nylon Mesh ◽  
Serial Femtosecond Crystallography

scholarly journals Fixed-Target Serial Synchrotron Crystallography Using Nylon Mesh and Enclosed Film-Based Sample Holder

Crystals ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 803 ◽  
Author(s):  
Suk-Youl Park ◽  
Hyeongju Choi ◽  
Cheolsoo Eo ◽  
Yunje Cho ◽  
Ki Hyun Nam
Keyword(s):  
Crystal Structures ◽  
Room Temperature ◽  
Sample Holder ◽  
Macromolecular Crystallography ◽  
Fixed Target ◽  
Special Equipment ◽  
Crystal Sample ◽  
X Ray ◽  
Raster Scanning ◽  
Nylon Mesh

Serial crystallography (SX) technique using synchrotron X-ray allows the visualization of room-temperature crystal structures with low-dose data collection as well as time-resolved molecular dynamics. In an SX experiment, delivery of numerous crystals for X-ray interaction, in a serial manner, is very important. Fixed-target scanning approach has the advantage of dramatically minimizing sample consumption as well as any physical damage to crystal sample, compared to other sample delivery methods. Here, we introduce the simple approach of fixed-target serial synchrotron crystallography (FT-SSX) using nylon mesh and enclosed film (NAM)-based sample holder. The NAM-based sample holder consisted of X-ray-transparent nylon-mesh and polyimide film, attached to a magnetic base. This sample holder was mounted to a goniometer head on macromolecular crystallography beamline, and translated along vertical and horizontal directions for raster scanning by the goniometer. Diffraction data were collected in two raster scanning approaches: (i) 100 ms X-ray exposure and 0.011° oscillation at each scan point and (ii) 500 ms X-ray exposure and 0.222° oscillation at each scan point. Using this approach, we determined the room-temperature crystal structures of lysozyme and glucose isomerase at 1.5–2.0 Å resolution. The sample holder produced negligible X-ray background scattering for data processing. Therefore, the new approach provided an opportunity to perform FT-SSX with high accessibility using macromolecular crystallography beamlines at synchrotron without any special equipment.

scholarly journals Serial X-ray Crystallography

Crystals ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 99
Author(s):  
Ki Hyun Nam
Keyword(s):  
Room Temperature ◽  
X Rays ◽  
Time Resolved ◽  
Pump Probe ◽  
X Ray ◽  
X Ray Crystallography ◽  
Target Molecules ◽  
Serial Crystallography ◽  
Pump Probe Experiment ◽  
Serial Femtosecond Crystallography

Serial crystallography (SX) is an emerging technique to determine macromolecules at room temperature. SX with a pump–probe experiment provides the time-resolved dynamics of target molecules. SX has developed rapidly over the past decade as a technique that not only provides room-temperature structures with biomolecules, but also has the ability to time-resolve their molecular dynamics. The serial femtosecond crystallography (SFX) technique using an X-ray free electron laser (XFEL) has now been extended to serial synchrotron crystallography (SSX) using synchrotron X-rays. The development of a variety of sample delivery techniques and data processing programs is currently accelerating SX research, thereby increasing the research scope. In this editorial, I briefly review some of the experimental techniques that have contributed to advances in the field of SX research and recent major research achievements. This Special Issue will contribute to the field of SX research.

Photophysical properties of N719 and Z907 dyes, benchmark sensitizers for dye-sensitized solar cells, at room and low temperature

2021 ◽  
Vol 23 (10) ◽  
pp. 6182-6189
Author(s):  
Dariusz M. Niedzwiedzki
Keyword(s):  
Solar Cells ◽  
Low Temperature ◽  
Optical Spectroscopy ◽  
Room Temperature ◽  
Photophysical Properties ◽  
Dye Sensitized Solar Cells ◽  
Time Resolved ◽  
Dye Sensitized ◽  
Sensitized Solar Cells

Photophysical properties of N719 and Z907, benchmark Ru-dyes used as sensitizers in dye-sensitized solar cells, were studied by static and time-resolved optical spectroscopy at room temperature and 160 K.

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