scholarly journals Gentle, fast and effective crystal soaking by acoustic dispensing

2016 ◽  
Author(s):  
Patrick M Collins ◽  
Jia Tsing Ng ◽  
Romain Talon ◽  
Karolina Nekrosiute ◽  
Tobias Krojer ◽  
...  
Keyword(s):  
High Throughput ◽  
Bright Light ◽  
Protein Crystals ◽  
Solvent Tolerance ◽  
Light Sources ◽  
X Ray Diffraction ◽  
Ligand Complex ◽  
Structure Based Drug Design ◽  
X Ray ◽  
Droplet Ejection

SynopsisA high-throughput method is described for crystal soaking using acoustic droplet ejection, and its effectiveness demonstrated. AbstractBright light sources, agile robotics, and fast detectors are continually reducing the time it takes to perform an X-ray diffraction experiment, making high throughput experiments more feasible than ever. But this is also pushing the upstream bottleneck towards sample preparation, even for robust and well characterised crystal systems. Crystal soaking is routinely used to generate protein-ligand complex structures, yet protein crystals are often sensitive to changes in solvent composition, and frequently require gentle or careful stepwise soaking techniques, limiting overall throughput. Here, we describe the use of acoustic droplet ejection for soaking of protein crystals with small molecules, and show that it is both gentle on crystals and allows very high throughput, with 1000 unique soaks easily performed in under 10 minutes. In addition to having very low compound consumption (tens of nanolitres per sample), the positional precision of acoustic droplet ejection enables targeted placement of the compound/solvent away from crystals and towards drop edges, allowing for gradual diffusion of solvent across the drop. This ensures both an improvement in reproducibility of X-ray diffraction and an increased solvent tolerance of the crystals, thus enabling higher effective compound soaking concentrations. We detail the technique here with examples from the protein target JMJD2D, a histone lysine demethylase, having roles in cancer and the focus of active structure based drug design efforts.

scholarly journals Gentle, fast and effective crystal soaking by acoustic dispensing

2017 ◽  
Vol 73 (3) ◽  
pp. 246-255 ◽  
Author(s):  
Patrick M. Collins ◽  
Jia Tsing Ng ◽  
Romain Talon ◽  
Karolina Nekrosiute ◽  
Tobias Krojer ◽  
...  
Keyword(s):  
High Throughput ◽  
Solvent Tolerance ◽  
X Ray Diffraction ◽  
Binding Studies ◽  
Structure Based Drug Design ◽  
X Ray ◽  
Active Structure ◽  
Droplet Ejection ◽  
Lysine Demethylase ◽  
Positional Precision

The steady expansion in the capacity of modern beamlines for high-throughput data collection, enabled by increasing X-ray brightness, capacity of robotics and detector speeds, has pushed the bottleneck upstream towards sample preparation. Even in ligand-binding studies using crystal soaking, the experiment best able to exploit beamline capacity, a primary limitation is the need for gentle and nontrivial soaking regimens such as stepwise concentration increases, even for robust and well characterized crystals. Here, the use of acoustic droplet ejection for the soaking of protein crystals with small molecules is described, and it is shown that it is both gentle on crystals and allows very high throughput, with 1000 unique soaks easily performed in under 10 min. In addition to having very low compound consumption (tens of nanolitres per sample), the positional precision of acoustic droplet ejection enables the targeted placement of the compound/solvent away from crystals and towards drop edges, allowing gradual diffusion of solvent across the drop. This ensures both an improvement in the reproducibility of X-ray diffraction and increased solvent tolerance of the crystals, thus enabling higher effective compound-soaking concentrations. The technique is detailed here with examples from the protein target JMJD2D, a histone lysine demethylase with roles in cancer and the focus of active structure-based drug-design efforts.

Technology of high quality protein crystals’ obtaining aboard the ISS at execution of joint Japanese–Russian experiments

2020 ◽  
pp. 5-25
Author(s):  
Koji INAKA ◽  
Saori ICHIMIZU ◽  
Izumi YOSHIZAKI ◽  
Kiyohito KIHIRA ◽  
Elena G. LAVRENKO ◽  
...  
Keyword(s):  
Drug Design ◽  
International Space Station ◽  
Space Station ◽  
Protein Crystals ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
High Quality ◽  
International Space ◽  
X Ray ◽  
High Quality Protein

A series of space experiments aboard the International Space Station (ISS) associated with high-quality Protein Crystal Growth (PCG) in microgravity conditions can be considered as a unique and one of the best examples of fruitful collaboration between Japanese and Russian scientists and engineers in space, which includes also other ISS International Partners. X-ray diffraction is still the most powerful tool to determine the protein three dimensional structure necessary for Structure based drug design (SBDD). The major purpose of the experiment is to grow high quality protein crystals in microgravity for X-ray diffraction on Earth. Within one and a half decade, Japan and Russia have established an efficient process over PCG in space to support latest developments over drug design and structural biology. One of the keys for success of the experiment lies in how precisely pre-launch preparations are made. Japanese party provides flight equipment for crystallization and ensures the required environment to support the experiment aboard of the ISS’s Kibo module, and also mainly takes part of the experiment ground support such as protein sample characterization, purification, crystallization screening, and solution optimization for microgravity experiment. Russian party is responsible for integration of the flight items equipped with proteins and precipitants on board Russian transportation space vehicles (Soyuz or Progress), for delivery them at the ISS, transfer to Kibo module, and returning the experiments’ results back on Earth aboard Soyuz manned capsule. Due to close cooperation of the parties and solid organizational structure, samples can be launched at the ISS every half a year if the ground preparation goes smoothly. The samples are crystallized using counter diffusion method at 20 degree C for 1–2.5 months. After samples return, the crystals are carefully taken out from the capillary, and frozen for X-ray diffraction at SPring8 facility in Japan. Extensive support of researchers from both countries is also a part of this process. The paper analyses details of the PCG experiment scheme, unique and reliable technology of its execution, and contains examples of the application. Key words: International Space Station, Protein crystals, Microgravity, International collaboration.

Microwave-Assisted Hydrothermal Synthesis of ZnFe2O4/TiO2 Composite and Photocatalytic Properties

2018 ◽  
Vol 788 ◽  
pp. 102-107
Author(s):  
Pavels Rodionovs ◽  
Jānis Grabis ◽  
Aija Krūmiņa
Keyword(s):  
Photocatalytic Activity ◽  
Photon Absorption ◽  
Light Sources ◽  
Mass Loading ◽  
Spherical Nanoparticles ◽  
X Ray Diffraction ◽  
X Ray ◽  
Microwave Assisted ◽  
Average Size ◽  
Prepared Nanostructures

In order to improve TiO2 photocatalytic activity ZnFe2O4/TiO2 nanocomposites with different ZnFe2O4 mass loading were produced. Obtained ZnFe2O4 nanoparticles were coupled with TiO2 via microwave-assisted hydrothermal method in order to improve photon absorption in a range of visible light. Prepared nanostructures were characterized with scanning electron microscopy and X-ray diffraction. Photocatalytic activity of prepared samples was investigated by degradation of methylene blue under different light sources – LED, Hg and Osram Vitalux lamps. ZnFe2O4 consists of spherical nanoparticles with average size of 15 nm. TiO2 spherical nanoparticles size is in a range of 30÷45 nm. The results show that doping TiO2 with ZnFe2O4 nanoparticles increases photocatalytic activity. Photocatalytic activity increases as mass loading of ZnFe2O4 decreases.

scholarly journals Advances in the Use of Microfluidics to Study Crystallization Fundamentals

2019 ◽  
Vol 10 (1) ◽  
pp. 59-83 ◽  
Author(s):  
Nadine Candoni ◽  
Romain Grossier ◽  
Mehdi Lagaize ◽  
Stéphane Veesler
Keyword(s):  
Phase Diagram ◽  
Diffraction Analysis ◽  
High Throughput ◽  
Concentration Measurement ◽  
Ultraviolet Spectroscopy ◽  
X Ray Diffraction ◽  
Chemical Library ◽  
Microfluidic Platform ◽  
X Ray ◽  
Phase Diagram Determination

This review compares droplet-based microfluidic systems used to study crystallization fundamentals in chemistry and biology. An original high-throughput droplet-based microfluidic platform is presented. It uses nanoliter droplets, generates a chemical library, and directly solubilizes powder, thus economizing both material and time. It is compatible with all solvents without the need for surfactant. Its flexibility permits phase diagram determination and crystallization studies (screening and optimizing experiments) and makes it easy to use for nonspecialists in microfluidics. Moreover, it allows concentration measurement via ultraviolet spectroscopy and solid characterization via X-ray diffraction analysis.

scholarly journals High-Throughput Crystallization Pipeline at the Crystallography Core Facility of the Institut Pasteur

Molecules ◽  
2019 ◽  
Vol 24 (24) ◽  
pp. 4451 ◽  
Author(s):  
Patrick Weber ◽  
Cédric Pissis ◽  
Rafael Navaza ◽  
Ariel E. Mechaly ◽  
Frederick Saul ◽  
...  
Keyword(s):  
High Throughput ◽  
Sequence Data ◽  
Data Bank ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
X Ray Diffraction ◽  
Sequencing Technology ◽  
X Ray ◽  
Set Up ◽  
General User

The availability of whole-genome sequence data, made possible by significant advances in DNA sequencing technology, led to the emergence of structural genomics projects in the late 1990s. These projects not only significantly increased the number of 3D structures deposited in the Protein Data Bank in the last two decades, but also influenced present crystallographic strategies by introducing automation and high-throughput approaches in the structure-determination pipeline. Today, dedicated crystallization facilities, many of which are open to the general user community, routinely set up and track thousands of crystallization screening trials per day. Here, we review the current methods for high-throughput crystallization and procedures to obtain crystals suitable for X-ray diffraction studies, and we describe the crystallization pipeline implemented in the medium-scale crystallography platform at the Institut Pasteur (Paris) as an example.

Preparation of N-doped TiO2 by oxidizing TiN and its application on phenol degradation

2013 ◽  
Vol 68 (4) ◽  
pp. 934-939 ◽  
Author(s):  
Ji-Guo Huang ◽  
Xiao-Guang Zhao ◽  
Meng-Yang Zheng ◽  
Sen Li ◽  
Yu Wang ◽  
...  
Keyword(s):  
Photoelectron Spectroscopy ◽  
Diffuse Reflectance ◽  
Phenol Degradation ◽  
Light Sources ◽  
X Ray Diffraction ◽  
Simple Method ◽  
Doped Tio2 ◽  
X Ray ◽  
Nitrogen Doped ◽  
Different Temperatures

Incomplete oxidation of titanium nitride (TiN) to prepare nitrogen-doped TiO2 was verified by calcining TiN at different temperatures in air for 30 min. The as-prepared samples were characterized by X-ray diffraction, UV-Vis diffuse reflectance spectra and X-ray photoelectron spectroscopy. The results confirmed that oxidizing TiN incompletely is an effective and simple method to prepare nitrogen-doped TiO2. Photocatalytic degradation of phenol was conducted to evaluate the photocatalytic activity of as-prepared samples. The results showed that phenol can be degraded efficiently by the as-prepared samples under visible light; low phenol concentration was conducive to degradation; the optimum calcination temperature and photocatalyst dosage are 650 °C and 0.5 g/L, respectively. The effects of different light sources on phenol degradation were compared. The reusability of nitrogen-doped TiO2 was tested and the results indicated a relatively good reusability under laboratory conditions.

Sign in / Sign up

Export Citation Format

Share Document