scholarly journals Lipidic cubic phase serial millisecond crystallography using synchrotron radiation

IUCrJ ◽  
2015 ◽  
Vol 2 (2) ◽  
pp. 168-176 ◽  
Author(s):  
Przemyslaw Nogly ◽  
Daniel James ◽  
Dingjie Wang ◽  
Thomas A. White ◽  
Nadia Zatsepin ◽  
...  
Keyword(s):  
Room Temperature ◽  
Cubic Phase ◽  
Temperature Structure ◽  
Free Electron Lasers ◽  
X Ray ◽  
Cubic Phases ◽  
Lipidic Cubic Phase ◽  
Effective Delivery ◽  
Serial Femtosecond Crystallography ◽  
Delivery Medium

Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4 Å. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway.

scholarly journals Isoprenoid-chained lipid EROCOC17+4: a new matrix for membrane protein crystallization and a crystal delivery medium in serial femtosecond crystallography

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kentaro Ihara ◽  
Masakatsu Hato ◽  
Takanori Nakane ◽  
Keitaro Yamashita ◽  
Tomomi Kimura-Someya ◽  
...  
Keyword(s):  
Protein Crystallization ◽  
Water System ◽  
Cubic Phase ◽  
Marked Contrast ◽  
X Ray ◽  
X Ray Scattering ◽  
Membrane Protein Crystallization ◽  
Almost All ◽  
Serial Femtosecond Crystallography ◽  
Delivery Medium

Abstract In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase (LCP). However, almost all previous crystallization trials have used monoacylglycerols, with 1-(cis-9-octadecanoyl)-rac-glycerol (MO) being the most widely used lipid. We now report that EROCOC17+4 mixed with 10% (w/w) cholesterol (Fig. 1) serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A2A receptor (A2AR). The structures of EROCOC17+4-matrix grown A2AR crystals were determined at 2.0 Å resolution by serial synchrotron rotation crystallography at a cryogenic temperature, and at 1.8 Å by LCP-serial femtosecond crystallography, using an X-ray free-electron laser at 4 and 20 °C sample temperatures, and are comparable to the structure of the MO-matrix grown A2AR crystal (PDB ID: 4EIY). Moreover, X-ray scattering measurements indicated that the EROCOC17+4/water system did not form the crystalline LC phase at least down to − 20 °C, in marked contrast to the equilibrium MO/water system, which transforms into the crystalline LC phase below about 17 °C. As the LC phase formation within the LCP-matrix causes difficulties in protein crystallography experiments in meso, this feature of EROCOC17+4 will expand the utility of the in meso method.

scholarly journals Liquid sample delivery techniques for serial femtosecond crystallography

2014 ◽  
Vol 369 (1647) ◽  
pp. 20130337 ◽  
Author(s):  
Uwe Weierstall
Keyword(s):  
Flow Rate ◽  
Free Electron ◽  
Repetition Rate ◽  
Free Electron Laser ◽  
Room Temperature ◽  
Liquid Flow Rate ◽  
Free Electron Lasers ◽  
Liquid Sample ◽  
X Ray ◽  
Serial Femtosecond Crystallography

X-ray free-electron lasers overcome the problem of radiation damage in protein crystallography and allow structure determination from micro- and nanocrystals at room temperature. To ensure that consecutive X-ray pulses do not probe previously exposed crystals, the sample needs to be replaced with the X-ray repetition rate, which ranges from 120 Hz at warm linac-based free-electron lasers to 1 MHz at superconducting linacs. Liquid injectors are therefore an essential part of a serial femtosecond crystallography experiment at an X-ray free-electron laser. Here, we compare different techniques of injecting microcrystals in solution into the pulsed X-ray beam in vacuum. Sample waste due to mismatch of the liquid flow rate to the X-ray repetition rate can be addressed through various techniques.

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 Double-flow focused liquid injector for efficient serial femtosecond crystallography

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Dominik Oberthuer ◽  
Juraj Knoška ◽  
Max O. Wiedorn ◽  
Kenneth R. Beyerlein ◽  
David A. Bushnell ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Room Temperature ◽  
Temperature Structure ◽  
Flow Focusing ◽  
X Ray ◽  
Jet Stability ◽  
Efficient Delivery ◽  
Structural Details ◽  
Liquid Injector ◽  
Serial Femtosecond Crystallography

Abstract Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices.

scholarly journals Native phasing of x-ray free-electron laser data for a G protein–coupled receptor

2016 ◽  
Vol 2 (9) ◽  
pp. e1600292 ◽  
Author(s):  
Alexander Batyuk ◽  
Lorenzo Galli ◽  
Andrii Ishchenko ◽  
Gye Won Han ◽  
Cornelius Gati ◽  
...  
Keyword(s):  
Free Electron ◽  
Room Temperature ◽  
De Novo ◽  
Ultrashort Pulses ◽  
Temperature Structure ◽  
X Ray ◽  
Anomalous Signal ◽  
G Protein Coupled ◽  
Serial Femtosecond Crystallography ◽  
General Method

Serial femtosecond crystallography (SFX) takes advantage of extremely bright and ultrashort pulses produced by x-ray free-electron lasers (XFELs), allowing for the collection of high-resolution diffraction intensities from micrometer-sized crystals at room temperature with minimal radiation damage, using the principle of “diffraction-before-destruction.” However, de novo structure factor phase determination using XFELs has been difficult so far. We demonstrate the ability to solve the crystallographic phase problem for SFX data collected with an XFEL using the anomalous signal from native sulfur atoms, leading to a bias-free room temperature structure of the human A2A adenosine receptor at 1.9 Å resolution. The advancement was made possible by recent improvements in SFX data analysis and the design of injectors and delivery media for streaming hydrated microcrystals. This general method should accelerate structural studies of novel difficult-to-crystallize macromolecules and their complexes.

scholarly journals Harnessing the power of an X-ray laser for serial crystallography of membrane proteins crystallized in lipidic cubic phase

IUCrJ ◽  
2020 ◽  
Vol 7 (6) ◽  
pp. 976-984
Author(s):  
Ming-Yue Lee ◽  
James Geiger ◽  
Andrii Ishchenko ◽  
Gye Won Han ◽  
Anton Barty ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Dynamic Range ◽  
High Dynamic Range ◽  
Cubic Phase ◽  
Experimental Setup ◽  
Temperature Structure ◽  
Helium Atmosphere ◽  
X Ray ◽  
Lipidic Cubic Phase ◽  
Time Required

Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase; however, like most techniques, it has limitations. Here we attempt to address some of these limitations related to the use of a vacuum chamber and the need for attenuation of the XFEL beam, in order to further improve the efficiency of this method. Using an optimized SFX experimental setup in a helium atmosphere, the room-temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Å resolution is determined and compared with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, the capability of utilizing high XFEL beam transmissions is demonstrated, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete dataset. The experimental setup presented herein can be applied to future SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.

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 Serial femtosecond crystallography of soluble proteins in lipidic cubic phase

IUCrJ ◽  
2015 ◽  
Vol 2 (5) ◽  
pp. 545-551 ◽  
Author(s):  
Raimund Fromme ◽  
Andrii Ishchenko ◽  
Markus Metz ◽  
Shatabdi Roy Chowdhury ◽  
Shibom Basu ◽  
...  
Keyword(s):  
Data Collection ◽  
Protein Structure Determination ◽  
Soluble Proteins ◽  
Cubic Phase ◽  
Data Sets ◽  
Carrier Medium ◽  
X Ray ◽  
Membrane Mimetic ◽  
Lipidic Cubic Phase ◽  
Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is demonstrated that LCP can also be used as a suitable carrier medium for microcrystals of soluble proteins, enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals delivered by liquid injectors. High-quality LCP–SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein.

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