Stable sample delivery in viscous media via a capillary for serial crystallography

2020 ◽  
Vol 53 (1) ◽  
pp. 45-50 ◽  
Author(s):  
Ki Hyun Nam
Keyword(s):  
Room Temperature ◽  
Capillary Tube ◽  
Glucose Isomerase ◽  
Low Flow ◽  
Innovative Technology ◽  
Delivery Method ◽  
Crystal Sample ◽  
X Ray ◽  
Serial Crystallography ◽  
Delivery Medium

Serial crystallography (SX) is an innovative technology in structural biology that enables the visualization of the molecular dynamics of macromolecules at room temperature. SX experiments always require a considerable amount of effort to deliver a crystal sample to the X-ray interaction point continuously and reliably. Here, a sample-delivery method using a capillary and a delivery medium is introduced. The crystals embedded in the delivery medium can pass through the capillary tube, which is aligned with the X-ray beam, at very low flow rates without requiring elaborate delivery techniques, drastically reducing sample consumption. In serial millisecond crystallography using a viscous medium via a capillary, crystals of lysozyme embedded in agarose, which produce an unstable injection stream at atmospheric pressure, and crystals of glucose isomerase embedded in gelatin, which is known to be problematic for open-extruder operation, were stably delivered at a flow rate of 100 nl min−1. The room-temperature crystal structures of lysozyme and glucose isomerase were successfully determined at 1.85 and 1.70 Å resolutions, respectively. This simple but highly efficient sample-delivery method can allow researchers to deliver crystals precisely to an X-ray beam in SX experiments.

scholarly journals Sample delivery using capillary and delivery medium for serial crystallography

2019 ◽  
Author(s):  
Ki Hyun Nam
Keyword(s):  
Capillary Tube ◽  
Low Flow ◽  
Innovative Technology ◽  
X Rays ◽  
Delivery Method ◽  
Interaction Point ◽  
Crystal Sample ◽  
X Ray ◽  
Serial Crystallography ◽  
Delivery Medium

AbstractSerial crystallography (SX) is an innovative technology in structural biology that enables the visualization of molecular dynamics of macromolecules at room temperature. SX experiments always require a considerable amount of effort to deliver a crystal sample to the X-ray interaction point continuously and reliably. Here, a sample delivery method using a capillary and a delivery medium is introduced. The crystals embedded in the delivery medium can pass through the capillary tube, which is aligned with the X-ray beam, at very low flow rates without requiring elaborate delivery techniques and drastically reducing sample consumption. This simple but highly efficient sample delivery method can allow researchers to deliver crystals precisely to X-rays in SX 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 Shortening injection matrix for serial crystallography

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ki Hyun Nam
Keyword(s):  
Crystal Structures ◽  
Room Temperature ◽  
Low Cost ◽  
Glucose Isomerase ◽  
Low Flow ◽  
Cubic Phase ◽  
Interaction Point ◽  
X Ray ◽  
Serial Crystallography ◽  
Delivery Medium

AbstractSerial crystallography allows crystal structures to be determined at room temperature through the steady delivery of crystals to the X-ray interaction point. Viscous delivery media are advantageous because they afford efficient sample delivery from an injector or syringe at a low flow rate. Hydrophobic delivery media, such as lipidic cubic phase (LCP) or grease, provide a stable injection stream and are widely used. The development of new hydrophobic delivery materials can expand opportunities for future SX studies with various samples. Here, I introduce fat-based shortening as a delivery medium for SX experiments. This material is commercially available at low cost and is straightforward to handle because its phase (i.e., solid or liquid) can be controlled by temperature. Shortening was extruded from a syringe needle in a stable injection stream even below 200 nl/min. X-ray exposed shortening produced several background scattering rings, which have similar or lower intensities than those of LCP and contribute negligibly to data processing. Serial millisecond crystallography was performed using two shortening delivery media, and the room temperature crystal structures of lysozyme and glucose isomerase were successfully determined at resolutions of 1.5–2.0 Å. Therefore, shortening can be used as a sample delivery medium in SX experiments.

scholarly journals Shortening injection matrix for serial crystallography

2019 ◽  
Author(s):  
Ki Hyun Nam
Keyword(s):  
Crystal Structures ◽  
Room Temperature ◽  
Low Cost ◽  
Glucose Isomerase ◽  
Low Flow ◽  
Cubic Phase ◽  
Interaction Point ◽  
X Ray ◽  
Serial Crystallography ◽  
Delivery Medium

AbstractSerial crystallography (SX) allows crystal structures to be observed at room temperature through the steady delivery of crystals to the X-ray interaction point. Viscous delivery media are advantageous because they afford efficient sample delivery from an injector or syringe at a low flow rate. Hydrophobic delivery media, such as lipidic cubic phase (LCP) or grease, provide a very stable injection stream and are widely used. The development of new hydrophobic delivery materials can expand opportunities for future SX studies with various samples. Here, I introduce fat-based shortening as a delivery medium for SX experiments. This material is commercially available at low cost and is straightforward to handle because its phase (i.e., solid or liquid) can be controlled by temperature. Shortening was extruded from a syringe needle in a very stable injection stream even below 200 nl/min. X-ray exposed shortening produced several background scattering rings, which have similar or lower intensities than those of LCP and contribute negligibly to data processing. Serial millisecond crystallography was performed using two shortening delivery media, and the room temperature crystal structures of lysozyme and glucose isomerase were successfully determined at resolutions of 1.5–2.0 Å. Therefore, shortening can be used as a sample delivery medium in SX experiments.

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 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.

Plug-and-play polymer microfluidic chips for hydrated, room temperature, fixed-target serial crystallography

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Deepshika Gilbile ◽  
Megan L. Shelby ◽  
Artem Y. Lyubimov ◽  
Jennifer L. Wierman ◽  
Diana C. F. Monteiro ◽  
...  
Keyword(s):  
Room Temperature ◽  
Microfluidic Chips ◽  
Fixed Target ◽  
Plug And Play ◽  
X Ray ◽  
X Ray Crystallography ◽  
Serial Crystallography

This work presents our development of versatile, inexpensive, and robust polymer microfluidic chips for routine and reliable room temperature serial X-ray crystallography measurements.

scholarly journals Approach of Serial Crystallography

Crystals ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 854
Author(s):  
Ki Hyun Nam
Keyword(s):  
Radiation Damage ◽  
Room Temperature ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Past ◽  
Wide Range ◽  
Experimental Limitation ◽  
Serial Crystallography ◽  
Collection Strategies

Radiation damage and cryogenic sample environment are an experimental limitation observed in the traditional X-ray crystallography technique. However, the serial crystallography (SX) technique not only helps to determine structures at room temperature with minimal radiation damage, but it is also a useful tool for profound understanding of macromolecules. Moreover, it is a new tool for time-resolved studies. Over the past 10 years, various sample delivery techniques and data collection strategies have been developed in the SX field. It also has a wide range of applications in instruments ranging from the X-ray free electron laser (XFEL) facility to synchrotrons. The importance of the various approaches in terms of the experimental techniques and a brief review of the research carried out in the field of SX has been highlighted in this editorial.

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