scholarly journals Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments

2019 ◽  
Vol 75 (10) ◽  
pp. 937-946 ◽  
Author(s):  
Rebecka Andersson ◽  
Cecilia Safari ◽  
Petra Båth ◽  
Robert Bosman ◽  
Anastasya Shilova ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Protein Structures ◽  
Cubic Phase ◽  
Crystal Formation ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
Lipidic Cubic Phase ◽  
Serial Crystallography

Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.

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.

Temperature-Jump Time-Resolved X-ray Diffraction Study of Cubic−Cubic Phase-Transition Kinetics in Thermotropic Cubic Mesogen 1,2-Bis(4′-n-alkoxybenzoyl)hydrazines (BABH-n)

Langmuir ◽  
2010 ◽  
Vol 26 (14) ◽  
pp. 11605-11608 ◽  
Author(s):  
Hiroyuki Mori ◽  
Shoichi Kutsumizu ◽  
Kazuya Saito ◽  
Katsuhiro Yamamoto ◽  
Shinichi Sakurai ◽  
...  
Keyword(s):  
Phase Transition ◽  
Diffraction Study ◽  
Temperature Jump ◽  
Cubic Phase ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
Jump Time ◽  
Phase Transition Kinetics

scholarly journals Viscous hydrophilic injection matrices for serial crystallography

IUCrJ ◽  
2017 ◽  
Vol 4 (4) ◽  
pp. 400-410 ◽  
Author(s):  
Gabriela Kovácsová ◽  
Marie Luise Grünbein ◽  
Marco Kloos ◽  
Thomas R. M. Barends ◽  
Ramona Schlesinger ◽  
...  
Keyword(s):  
Protein Crystallization ◽  
High Viscosity ◽  
Stream Velocity ◽  
Cubic Phase ◽  
Time Resolved ◽  
Flow Rates ◽  
Block Polymer ◽  
X Ray ◽  
Crystallization Conditions ◽  
Serial Crystallography

Serial (femtosecond) crystallography at synchrotron and X-ray free-electron laser (XFEL) sources distributes the absorbed radiation dose over all crystals used for data collection and therefore allows measurement of radiation damage prone systems, including the use of microcrystals for room-temperature measurements. Serial crystallography relies on fast and efficient exchange of crystals upon X-ray exposure, which can be achieved using a variety of methods, including various injection techniques. The latter vary significantly in their flow rates – gas dynamic virtual nozzle based injectors provide very thin fast-flowing jets, whereas high-viscosity extrusion injectors produce much thicker streams with flow rates two to three orders of magnitude lower. High-viscosity extrusion results in much lower sample consumption, as its sample delivery speed is commensurate both with typical XFEL repetition rates and with data acquisition rates at synchrotron sources. An obvious viscous injection medium is lipidic cubic phase (LCP) as it is used forin mesomembrane protein crystallization. However, LCP has limited compatibility with many crystallization conditions. While a few other viscous media have been described in the literature, there is an ongoing need to identify additional injection media for crystal embedding. Critical attributes are reliable injection properties and a broad chemical compatibility to accommodate samples as heterogeneous and sensitive as protein crystals. Here, the use of two novel hydrogels as viscous injection matrices is described, namely sodium carboxymethyl cellulose and the thermo-reversible block polymer Pluronic F-127. Both are compatible with various crystallization conditions and yield acceptable X-ray background. The stability and velocity of the extruded stream were also analysed and the dependence of the stream velocity on the flow rate was measured. In contrast with previously characterized injection media, both new matrices afford very stable adjustable streams suitable for time-resolved measurements.

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 Solving a new R2lox protein structure by microcrystal electron diffraction

2019 ◽  
Vol 5 (8) ◽  
pp. eaax4621 ◽  
Author(s):  
Hongyi Xu ◽  
Hugo Lebrette ◽  
Max T. B. Clabbers ◽  
Jingjing Zhao ◽  
Julia J. Griese ◽  
...  
Keyword(s):  
Protein Structure ◽  
Electron Diffraction ◽  
Model Building ◽  
Protein Structures ◽  
X Ray Diffraction ◽  
X Ray ◽  
X Ray Crystallography ◽  
Potential Map ◽  
Metal Cofactor ◽  
And Function

Microcrystal electron diffraction (MicroED) has recently shown potential for structural biology. It enables the study of biomolecules from micrometer-sized 3D crystals that are too small to be studied by conventional x-ray crystallography. However, to date, MicroED has only been applied to redetermine protein structures that had already been solved previously by x-ray diffraction. Here, we present the first new protein structure—an R2lox enzyme—solved using MicroED. The structure was phased by molecular replacement using a search model of 35% sequence identity. The resulting electrostatic scattering potential map at 3.0-Å resolution was of sufficient quality to allow accurate model building and refinement. The dinuclear metal cofactor could be located in the map and was modeled as a heterodinuclear Mn/Fe center based on previous studies. Our results demonstrate that MicroED has the potential to become a widely applicable tool for revealing novel insights into protein structure and function.

scholarly journals Evaluation of microflow configurations for scale inhibition and serial X-ray diffraction analysis of crystallization processes

Lab on a Chip ◽  
2020 ◽  
Vol 20 (16) ◽  
pp. 2954-2964
Author(s):  
Mark A. Levenstein ◽  
Yi-Yeoun Kim ◽  
Liam Hunter ◽  
Clara Anduix-Canto ◽  
Carlos González Niño ◽  
...  
Keyword(s):  
Crystal Growth ◽  
Diffraction Analysis ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
Scale Inhibition ◽  
Serial Crystallography

Exploration of crystal growth in microchannels led to effective scale inhibition, enabling time-resolved injector-based serial crystallography-on-a-chip.

scholarly journals Nanosecond pump–probe device for time-resolved serial femtosecond crystallography developed at SACLA

2017 ◽  
Vol 24 (5) ◽  
pp. 1086-1091 ◽  
Author(s):  
Minoru Kubo ◽  
Eriko Nango ◽  
Kensuke Tono ◽  
Tetsunari Kimura ◽  
Shigeki Owada ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Free Electron ◽  
Good Choice ◽  
Cubic Phase ◽  
Liquid Droplets ◽  
Optical Pump ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
Serial Femtosecond Crystallography

X-ray free-electron lasers (XFELs) have opened new opportunities for time-resolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TR-SFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.

scholarly journals Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 µm size X-ray synchrotron beam

2009 ◽  
Vol 6 (suppl_5) ◽  
Author(s):  
Vadim Cherezov ◽  
Michael A. Hanson ◽  
Mark T. Griffith ◽  
Mark C. Hilgart ◽  
Ruslan Sanishvili ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
G Protein Coupled Receptors ◽  
Cubic Phase ◽  
High Quality ◽  
Crystal Sample ◽  
X Ray ◽  
Synchrotron Beam ◽  
Lipidic Cubic Phase ◽  
Diffraction Patterns ◽  
G Protein Coupled

Crystallization of human membrane proteins in lipidic cubic phase often results in very small but highly ordered crystals. Advent of the sub-10 µm minibeam at the APS GM/CA CAT has enabled the collection of high quality diffraction data from such microcrystals. Herein we describe the challenges and solutions related to growing, manipulating and collecting data from optically invisible microcrystals embedded in an opaque frozen in meso material. Of critical importance is the use of the intense and small synchrotron beam to raster through and locate the crystal sample in an efficient and reliable manner. The resulting diffraction patterns have a significant reduction in background, with strong intensity and improvement in diffraction resolution compared with larger beam sizes. Three high-resolution structures of human G protein-coupled receptors serve as evidence of the utility of these techniques that will likely be useful for future structural determination efforts. We anticipate that further innovations of the technologies applied to microcrystallography will enable the solving of structures of ever more challenging targets.

The combustion synthesis of the ferroelectric material, BaTiO3, studied by time-resolved X-ray diffraction

1999 ◽  
Vol 14 (2) ◽  
pp. 111-113 ◽  
Author(s):  
E. M. Larson ◽  
Joe Wong ◽  
J. B. Holt ◽  
P. A. Waide ◽  
B. Rupp
Keyword(s):  
Combustion Synthesis ◽  
Ferroelectric Material ◽  
Bragg Diffraction ◽  
Cubic Phase ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
Tetragonal Form ◽  
Diffraction Peaks ◽  
The Common

The combustion synthesis of the common ferroelectric material, BaTiO3, was developed using the stoichiometry: BaO2+0.2 Ti+0.8 TiO2→BaTiO3+0.3 O2. An adiabatic temperature, Tad, of the reaction was calculated from known thermodynamic data to be 1917 °C. Real time chemical changes in the formation of BaTiO3 during the reaction have been monitored using time-resolved X-ray diffraction with synchrotron radiation as the X-ray source. A time resolution of 250 ms was achieved. The combustion synthesis of BaTiO3 was followed by observing the intensities of reactant and product Bragg diffraction peaks in order to qualitatively identify the phases present. Because BaTiO3 forms initially as a cubic phase, X-ray diffraction of the product was monitored for a period of 20 min after the reaction to observe the phase transformation to the tetragonal form. This transformation is evident in these post-reaction scans as the cubic 110 and 220 peaks are split to the tetragonal 101/110 and 202/220 ones, respectively.

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