scholarly journals Hitting the target: fragment screening with acousticin situco-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes

2014 ◽  
Vol 70 (5) ◽  
pp. 1177-1189 ◽  
Author(s):  
Xingyu Yin ◽  
Alexander Scalia ◽  
Ludmila Leroy ◽  
Christina M. Cuttitta ◽  
Gina M. Polizzo ◽  
...  
Keyword(s):  
Data Collection ◽  
High Speed ◽  
Screening Strategy ◽  
Specimen Preparation ◽  
Fragment Screening ◽  
Liquid Handling ◽  
Droplet Ejection ◽  
Crystallization Experiments ◽  
Single Data ◽  
Fragment Libraries

Acoustic droplet ejection (ADE) is a powerful technology that supports crystallographic applications such as growing, improving and manipulating protein crystals. A fragment-screening strategy is described that uses ADE to co-crystallize proteins with fragment libraries directly on MiTeGen MicroMeshes. Co-crystallization trials can be prepared rapidly and economically. The high speed of specimen preparation and the low consumption of fragment and protein allow the use of individual rather than pooled fragments. The Echo 550 liquid-handling instrument (Labcyte Inc., Sunnyvale, California, USA) generates droplets with accurate trajectories, which allows multiple co-crystallization experiments to be discretely positioned on a single data-collection micromesh. This accuracy also allows all components to be transferred through small apertures. Consequently, the crystallization tray is in equilibrium with the reservoir before, during and after the transfer of protein, precipitant and fragment to the micromesh on which crystallization will occur. This strict control of the specimen environment means that the crystallography experiments remain identical as the working volumes are decreased from the few microlitres level to the few nanolitres level. Using this system, lysozyme, thermolysin, trypsin and stachydrine demethylase crystals were co-crystallized with a small 33-compound mini-library to search for fragment hits. This technology pushes towards a much faster, more automated and more flexible strategy for structure-based drug discovery using as little as 2.5 nl of each major component.

scholarly journals High-density grids for efficient data collection from multiple crystals

2016 ◽  
Vol 72 (1) ◽  
pp. 2-11 ◽  
Author(s):  
Elizabeth L. Baxter ◽  
Laura Aguila ◽  
Roberto Alonso-Mori ◽  
Christopher O. Barnes ◽  
Christopher A. Bonagura ◽  
...  
Keyword(s):  
Data Collection ◽  
Room Temperature ◽  
High Density ◽  
Cubic Phase ◽  
Structural Investigations ◽  
Storage Container ◽  
Liquid Handling ◽  
Crystallization Experiments ◽  
Linac Coherent Light Source ◽  
Efficient Data

Higher throughput methods to mount and collect data from multiple small and radiation-sensitive crystals are important to support challenging structural investigations using microfocus synchrotron beamlines. Furthermore, efficient sample-delivery methods are essential to carry out productive femtosecond crystallography experiments at X-ray free-electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS). To address these needs, a high-density sample grid useful as a scaffold for both crystal growth and diffraction data collection has been developed and utilized for efficient goniometer-based sample delivery at synchrotron and XFEL sources. A single grid contains 75 mounting ports and fits inside an SSRL cassette or uni-puck storage container. The use of grids with an SSRL cassette expands the cassette capacity up to 7200 samples. Grids may also be covered with a polymer film or sleeve for efficient room-temperature data collection from multiple samples. New automated routines have been incorporated into theBlu-Ice/DCSSexperimental control system to support grids, including semi-automated grid alignment, fully automated positioning of grid ports, rastering and automated data collection. Specialized tools have been developed to support crystallization experiments on grids, including a universal adaptor, which allows grids to be filled by commercial liquid-handling robots, as well as incubation chambers, which support vapor-diffusion and lipidic cubic phase crystallization experiments. Experiments in which crystals were loaded into grids or grown on grids using liquid-handling robots and incubation chambers are described. Crystals were screened at LCLS-XPP and SSRL BL12-2 at room temperature and cryogenic temperatures.

Use of a crystallization robot to set up sitting-drop vapor-diffusion crystallization andin situcrystallization screens

2000 ◽  
Vol 33 (2) ◽  
pp. 344-349 ◽  
Author(s):  
Christopher F. Snook ◽  
Michael D. Purdy ◽  
Michael C. Wiener
Keyword(s):  
Membrane Proteins ◽  
Protein Crystallization ◽  
Integral Membrane Proteins ◽  
Vapor Diffusion ◽  
Liquid Handling ◽  
General Utility ◽  
Crystallization Experiments ◽  
Automated Screening ◽  
Set Up

A commercial crystallization robot has been modified for use in setting up sitting-drop vapor-diffusion crystallization experiments, and for setting up protein crystallization screensin situ. The primary aim of this effort is the automated screening of crystallization of integral membrane proteins in detergent-containing solutions. However, the results of this work are of general utility to robotic liquid-handling systems. Sources of error that can prevent the accurate dispensing and mixing of solutions have been identified, and include local environmental, machine-specific and solution conditions. Solutions to each of these problems have been developed and implemented.

scholarly journals Through-grid wicking enables high-speed cryoEM specimen preparation

2020 ◽  
Vol 76 (11) ◽  
pp. 1092-1103
Author(s):  
Yong Zi Tan ◽  
John L. Rubinstein
Keyword(s):  
Glass Fiber ◽  
High Speed ◽  
Protein Complexes ◽  
Absorption Capacity ◽  
Specimen Preparation ◽  
Water Interface ◽  
Time Resolved ◽  
Glass Fiber Filter ◽  
Liquid Absorption ◽  
Air Water Interface

Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air–water interface. Here, it is shown that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass-fiber filter held against the opposite side, often called the `back', of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. Ultrasonic specimen application and through-grid wicking were combined in a high-speed specimen-preparation device that was named `Back-it-up' or BIU. The high liquid-absorption capacity of the glass fiber compared with self-wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The speed of the device increases the number of views for a specimen that suffers from preferred orientations.

Airplane and Drop Experiments on Crystallization of InxGa1−xSb Semiconductor under Different Gravity Conditions

2001 ◽  
Vol 692 ◽  
Author(s):  
Krishnan Balakrishnan ◽  
Yasuhiro Hayakawa ◽  
Hideki Komatsu ◽  
Noriaki Murakami ◽  
Tetsuo Nakamura ◽  
...  
Keyword(s):  
Crystal Growth ◽  
High Speed ◽  
Normal Gravity ◽  
Ccd Camera ◽  
Reduced Gravity ◽  
Gravity Condition ◽  
Crystallization Experiments ◽  
Melting And Crystallization ◽  
Normal Gravity Condition

AbstractMelting and crystallization experiments of InGaSb were done under the reduced gravity condition (10−2G) in an airplane and at the normal gravity condition (1G) in the laboratory. Crystallized InGaSb was found to contain many needle crystals in both the cases. Reduced gravity condition was found to be more conducive for crystal growth than the normal gravity condition. Formation of spherical projections on the surface of InGaSb during its crystallization was in-situ observed using a high speed CCD camera in the drop experiment. Spherical projections showed dependence of gravity during its growth. Indium compositions in the spherical projections were found to vary depending on the temperature.

High-Throughput DNA Plasmid Transfection Using Acoustic Droplet Ejection Technology

2018 ◽  
Vol 24 (4) ◽  
pp. 492-500
Author(s):  
Béatrice Colin ◽  
Benoit Deprez ◽  
Cyril Couturier
Keyword(s):  
High Throughput ◽  
Plasmid Dna ◽  
High Speed ◽  
Protein Crystallization ◽  
Dna Transfection ◽  
Dna Amount ◽  
Biological Assays ◽  
Droplet Ejection ◽  
Plasmid Transfection ◽  
Automated Protocol

The Labcyte Echo acoustic liquid handler allows accurate droplet ejection at high speed from a source well plate to a destination plate. It has already been used in various miniaturized biological assays, such as quantitative PCR (q-PCR), quantitative real-time PCR (q-RT-PCR), protein crystallization, drug screening, cell dispensing, and siRNA transfection. However, no plasmid DNA transfection assay has been published so far using this dispensing technology. In this study, we evaluated the ability of the Echo 550 device to perform plasmid DNA transfection in 384-well plates. Due to the high throughput of this device, we simultaneously optimized the three main parameters of a transfection process: dilution of the transfection reagent, DNA amount, and starting DNA concentration. We defined a four-step protocol whose optimal settings allowed us to transfect HeLa cells with up to 90% efficiency and reach a co-expression of nearly 100% within transfected cells in co-transfection experiments. This fast, reliable, and automated protocol opens new ways to easily and rapidly identify optimal transfection settings for a given cell type. Furthermore, it permits easy software-based transfection control and multiplexing of plasmids distributed on wells of a source plate. This new development could lead to new array applications, such as human ORFeome protein expression or CRISPR-Cas9-based gene function validation in nonpooled screening strategies.

scholarly journals A Calculation Method for Vehicle Movement Reconstruction from Videos

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Hao Feng ◽  
Weiguo Shi ◽  
Feng Chen ◽  
Young-Ji Byon ◽  
Weiwei Heng ◽  
...  
Keyword(s):  
Data Collection ◽  
High Speed ◽  
Video Sequences ◽  
One Dimensional ◽  
Spatial Consistency ◽  
Active Calibration ◽  
Actual Movement ◽  
Camera Perspective ◽  
Vehicle Movement ◽  
Data Collection Procedure

This paper proposes a new enhanced method based on one-dimensional direct linear transformation for estimating vehicle movement states in video sequences. The proposed method utilizes a contoured structure of target vehicles, and the data collection procedure is found to be relatively stable and effective, providing a better applicability. The movements of vehicles in the video are captured by active calibration regions while the spatial consistency between the vehicle’s driving track and the calibration information are in sync. The vehicle movement states in the verification phase are estimated using the proposed method first, and then the estimated states are compared with the actual movement states recorded in the experimental test. The results show that, in the case of camera perspective of 90 degrees, in all driving states of low speed, high speed, or deceleration, the error between estimated speed and recorded speed is less than 1.5%, the error of accelerations is less than 7%, and the error of distances is less than 2%; similarly, in the case of camera perspective of 30 degrees, the errors of speeds, distances, and accelerations are less than 4%, 5%, and 10%, respectively. It is found that the proposed method is superior to other existing methods.

Mechanism of Parameters Variation about Electronic Components Subjected to High Impact Loads

2010 ◽  
Vol 44-47 ◽  
pp. 256-259 ◽  
Author(s):  
Bo Li ◽  
Ya Zhang ◽  
Hong Xiang Zhang
Keyword(s):  
Data Collection ◽  
Stress Level ◽  
Main Parameter ◽  
High Speed ◽  
Dynamic Testing ◽  
High Impact ◽  
Impact Loads ◽  
Electronic Components ◽  
Collection System ◽  
High Speed Data

Large quantity dynamic testing experiments are carried out to testing the parameter of electronic components in high over loading impact condition with a standard hammer machine and a high speed data collection system. And the experiments were performed for about 5000 times to more than 30 types of resistors, capacitors, transistors to test their main parameter in different stress level and fixing mode. The parameter of some electronic components appears more or less unstable, namely temporary failure, and it is sensitive to different stress level and impact direct in experiments, which can offer important value of references for reliability lifespan.

scholarly journals Label-Free Primary Screening and Affinity Ranking of Fragment Libraries Using Parallel Analysis of Protein Panels

2008 ◽  
Vol 13 (3) ◽  
pp. 202-209 ◽  
Author(s):  
Markku D. Hämäläinen ◽  
Andrei Zhukov ◽  
Maria Ivarsson ◽  
Tomas Fex ◽  
Johan Gottfries ◽  
...  
Keyword(s):  
Binding Site ◽  
Parallel Analysis ◽  
Label Free ◽  
Glutathione S Transferase ◽  
Chip Surface ◽  
Fragment Screening ◽  
Fragment Library ◽  
Direct Binding ◽  
Resonance Detection ◽  
Fragment Libraries

The authors present fragment screening data obtained using a label-free parallel analysis approach where the binding of fragment library compounds to 4 different target proteins can be screened simultaneously using surface plasmon resonance detection. They suggest this method as a first step in fragment screening to identify and select binders, reducing the demanding requirements on subsequent X-ray or nuclear magnetic resonance studies, and as a valuable “clean-up” tool to eliminate unwanted promiscuous binders from libraries. A small directed fragment library of known thrombin binders and a general 500-compound fragment library were used in this study. Thrombin, blocked thrombin, carbonic anhydrase, and glutathione-S-transferase were immobilized on the sensor chip surface, and the direct binding of the fragments was studied in real time. Only 12 µg of each protein is needed for screening of a 3000-compound fragment library. For screening, a binding site-blocked target as reference facilitates the identification of binding site-selective hits and the signals from other reference proteins for the elimination of false positives. The scope and limitations of this screening approach are discussed for both target-directed and general fragment libraries. ( Journal of Biomolecular Screening 2008:202-209)

Absorption and Scatter Corrections for Transflectance Measurements in Nonhomogeneous Fiber Samples

1998 ◽  
Vol 6 (A) ◽  
pp. A35-A44 ◽  
Author(s):  
R.A. Taylor
Keyword(s):  
High Speed ◽  
Near Infrared ◽  
Light Transmission ◽  
Accurate Determination ◽  
Fiber Optic Sensor ◽  
Infrared Light ◽  
Specimen Preparation ◽  
Absorption Bands ◽  
Speed Sensor ◽  
Wide Range

A new fiber optic sensor was developed to measure the mass of optically thin cotton samples by integrating light transmission and reflectance signals. High speed measurements of cotton strength requires an accurate determination of the specimen mass without use of laborious cut-and-weigh methods. A previous high speed sensor measured changes in visible light transmission which required secondary measurements of fiber fineness to adjust the data for light scattering. Fiber orientation also affected scattering which required a precise control on specimen preparation. The new sensor measures fiber specimen transflectance using near infrared light. Because cellulose (the basic compound in cotton) exhibits strong absorption bands, its concentration can be accurately measured using near infrared absorbance. In this report we show that an integratinq sensor gave the best measurement of cotton fiber mass. Additionally, we demonstrated its accuracy over a wide range of fiber orientations using a novel fiber specimen tension experiment.

scholarly journals Parallel Screening of Low Molecular Weight Fragment Libraries

2012 ◽  
Vol 18 (2) ◽  
pp. 147-159 ◽  
Author(s):  
Jerome Wielens ◽  
Stephen J. Headey ◽  
David I. Rhodes ◽  
Roger J. Mulder ◽  
Olan Dolezal ◽  
...  
Keyword(s):  
Screening Methods ◽  
Resonance Spectroscopy ◽  
Lead Discovery ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fragment Screening ◽  
Lead Compounds ◽  
Preliminary Identification ◽  
Std Nmr ◽  
Fragment Libraries

Fragment screening is becoming widely accepted as a technique to identify hit compounds for the development of novel lead compounds. In neighboring laboratories, we have recently, and independently, performed a fragment screening campaign on the HIV-1 integrase core domain (IN) using similar commercially purchased fragment libraries. The two campaigns used different screening methods for the preliminary identification of fragment hits; one used saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR), and the other used surface plasmon resonance (SPR) spectroscopy. Both initial screens were followed by X-ray crystallography. Using the STD-NMR/X-ray approach, 15 IN/fragment complexes were identified, whereas the SPR/X-ray approach found 6 complexes. In this article, we compare the approaches that were taken by each group and the results obtained, and we look at what factors could potentially influence the final results. We find that despite using different approaches with little overlap of initial hits, both approaches identified binding sites on IN that provided a basis for fragment-based lead discovery and further lead development. Comparison of hits identified in the two studies highlights a key role for both the conditions under which fragment binding is measured and the criteria selected to classify hits.

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