Stable sample delivery in a viscous medium via a polyimide-based single-channel microfluidic chip for serial crystallography

2021 ◽  
Vol 54 (4) ◽  
Author(s):  
Ki Hyun Nam ◽  
Yunje Cho
Keyword(s):  
Crystal Structures ◽  
Flow Rate ◽  
Microfluidic Chip ◽  
Room Temperature ◽  
Single Channel ◽  
Viscous Medium ◽  
X Rays ◽  
Interaction Point ◽  
X Ray ◽  
Serial Crystallography

Serial crystallography (SX) provides room-temperature crystal structures with minimal radiation damage and facilitates the comprehension of molecular dynamics through time-resolved studies. In SX experiments, it is important to deliver a large number of crystal samples to the X-ray interaction point in a serial and stable manner. The advantage of crystal delivery in a viscous medium via a capillary is the ability to deliver all of the crystal samples to the X-ray interaction point at a low flow rate; however, the capillary often breaks during handling and high X-ray absorption can occur at low energy states. This study aimed to develop a stable system for sample delivery in a viscous medium via a polyimide-based single-channel microfluidic (PSM) chip for SX. Since this microfluidic chip comprises a polyimide film, it has high tensile strength and higher X-ray transmittance than a quartz capillary. The PSM chip was connected to a syringe containing the microcrystals embedded in viscous medium. The channel of the PSM chip was aligned to the X-ray path, and the viscous medium containing lysozyme crystals was stably delivered using a syringe pump at a flow rate of 100 nl min−1. Room-temperature lysozyme crystal structures were successfully determined at 1.85 Å resolution. This method would greatly facilitate sample delivery for SX experiments using synchrotron X-rays.

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 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.

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 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.

Dynamic orientational disorder in crystals of fluoroelpasolites, structural refinement of (NH4)3AlF6, (NH4)3TiOF5 and Rb2KTiOF5

2011 ◽  
Vol 67 (6) ◽  
pp. 447-454 ◽  
Author(s):  
Anatoly A. Udovenko ◽  
Natalia M. Laptash
Keyword(s):  
Crystal Structures ◽  
Room Temperature ◽  
Local Scale ◽  
X Rays ◽  
Structural Refinement ◽  
X Ray Diffraction ◽  
Dynamic Nature ◽  
Orientational Disorder ◽  
X Ray ◽  
Stretching Vibrations

Room-temperature crystal structures of triammonium hexafluoroaluminate, (NH4)3AlF6 (I), and triammonium oxopentafluorotitanate, (NH4)3TiOF5 (II), were refined, and the crystal structures of dirubidium potassium oxopentaflourotitanate, Rb2KTiOF5, at 297 K (III) and 218 K (IV) were determined using single-crystal X-ray diffraction techniques. In ammonium fluoroelpasolites [(I) and (II)], the ligand (O, F) atoms are located in the mixed 24e + 96j position of the Fm3m unit cell. The 24e position is occupied by the ligand atoms predominantly in (III) and fully in (IV). `Ordered' N1 and Rb atoms are tetrahedrally displaced from the 8c position into the 32f site, and the H atoms of the disordered ammonium group N2 are statistically distributed in the 96k and 32f positions. The Ti atoms in (II) and (IV) are shifted from the 4a position to 24e thus allowing identification of the O and F atoms in the octahedron on a local scale. The disorder in the crystals is of a dynamic nature. Unique Raman spectra of Rb2KTiOF5 under the laser beam of 1064 nm indicate fast octahedral reorientations resulting in physical equalizations of the Ti—O and Ti—F distances as well as in the appearance of totally synchronous Ti—O and Ti—F stretching vibrations at 750 cm−1. This phenomenon is assumed to also take place under X-rays.

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.

Distorted chiolite crystal structures of α-Na5M3F14 (M=Cr,Fe,Ga) studied by X-ray powder diffraction

2003 ◽  
Vol 18 (2) ◽  
pp. 128-134 ◽  
Author(s):  
A. Le Bail ◽  
A.-M. Mercier
Keyword(s):  
Crystal Structures ◽  
Powder Diffraction ◽  
Room Temperature ◽  
Rietveld Refinements ◽  
Space Group ◽  
X Ray ◽  
Precise Characterization

The crystal structures of the chiolite-related room temperature phases α-Na5M3F14 (MIII=Cr,Fe,Ga) are determined. For all of them, the space group is P21/n, Z=2; a=10.5096(3) Å, b=7.2253(2) Å, c=7.2713(2) Å, β=90.6753(7)° (M=Cr); a=10.4342(7) Å, b=7.3418(6) Å, c=7.4023(6) Å, β=90.799(5)° (M=Fe), and a=10.4052(1) Å, b=7.2251(1) Å, c=7.2689(1), β=90.6640(4)° (M=Ga). Rietveld refinements produce final RF factors 0.036, 0.033, and 0.035, and RWP factors, 0.125, 0.116, and 0.096, for MIII=Cr, Fe, and Ga, respectively. The MF6 polyhedra in the defective isolated perovskite-like layers deviate very few from perfect octahedra. Subtle octahedra tiltings lead to the symmetry decrease from the P4/mnc space group adopted by the Na5Al3F14 chiolite aristotype to the P21/n space group adopted by the title series. Facile twinning precluded till now the precise characterization of these compounds.

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