High-throughput counter-diffusion capillary crystallization andin situdiffraction using high-pressure freezing in protein crystallography

Post-crystallization treatments such as manual fishing of crystals and soaking in cryoprotectant solutions, especially of large macromolecular complexes and membrane proteins, are cumbersome and often lead to crystal damage and reduced diffraction data quality. Here, a capillary crystallization plate is presented that simultaneously allows counter-diffusion crystallization at the nanolitre scale in a high-throughput screening mode, low-temperaturein situdiffraction data collection from crystals after cryoprotection and low-temperaturein situdata collection of crystals without the addition of any cryoprotectant after high-pressure (HP) freezing. The development of this plate and plunge cooling of crystals in the capillaries is a major step towards implementing automatedin situhigh-throughput crystal diffraction data collection at a synchrotron beamline. In combination with HP freezing this offers a new opportunity to obtain structural information from fragile crystals of supramolecular complexes that might otherwise not be feasible.

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Chapter 1. Practical Approaches for In Situ X-ray Crystallography: from High-throughput Screening to Serial Data Collection

Protein Crystallography - Chemical Biology ◽

10.1039/9781788010504-00001 ◽

2018 ◽

pp. 1-27 ◽

Cited By ~ 1

Author(s):

Isabelle Martiel ◽

Vincent Olieric ◽

Martin Caffrey ◽

Meitian Wang

Keyword(s):

Data Collection ◽

High Throughput ◽

High Throughput Screening ◽

X Ray ◽

X Ray Crystallography ◽

Serial Data

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Novel combined crystallization plate for high-throughput crystal screening and in situ data collection at a crystallography beamline

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x21008104 ◽

2021 ◽

Vol 77 (9) ◽

pp. 319-327

Author(s):

Miao Liang ◽

Li Yu ◽

Zhijun Wang ◽

Huan Zhou ◽

Yi Zhang ◽

...

Keyword(s):

Data Collection ◽

High Throughput ◽

Diffraction Data ◽

Large Scale ◽

Liquid Drop ◽

In Situ Data ◽

Combined Crystallization ◽

Manual Manipulation ◽

Large Scale Screening

In situ microplates are small in size, crystal cultivation and operation are difficult, and the efficiency of crystal screening is relatively low. To solve this problem, a novel combined crystallization plate was designed for high-throughput crystal cultivation and in situ data collection. A frame was used to hold 48 in situ microplates, and the in situ microplates were sealed on one side with an ultralow background-scattering Kapton film. An automatic liquid handler (Mosquito) was used to add a liquid drop to the in situ microplates in the frame, and CrystalClear HD tape was used to seal the frame. A sealed frame holding 48 microplates was developed as a novel combined crystallization plate and was used for crystal cultivation under different conditions and in situ data collection at the synchrotron beamline. Moreover, individual microplates can be separated from the combined crystal plate and then fixed on a magnetic base or loaded onto a UniPuck for in situ data collection. Automatic grid scanning was used to locate crystals. The efficiency of the combined crystallization plate for crystal screening was verified. This method avoids the manual manipulation of crystals during crystal screening and diffraction data collection; therefore, the combined crystallization plate is suitable for large-scale screening of microcrystals.

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Attaining atomic resolution from in situ data collection at room temperature using counter-diffusion-based low-cost microchips

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798320008475 ◽

2020 ◽

Vol 76 (8) ◽

pp. 751-758

Author(s):

Jose A. Gavira ◽

Isaac Rodriguez-Ruiz ◽

Sergio Martinez-Rodriguez ◽

Shibom Basu ◽

Sébastien Teychené ◽

...

Keyword(s):

Data Collection ◽

Diffraction Data ◽

Room Temperature ◽

Low Cost ◽

Atomic Resolution ◽

Microfluidic Channels ◽

High Symmetry ◽

X Ray ◽

Counter Diffusion

Sample handling and manipulation for cryoprotection currently remain critical factors in X-ray structural determination. While several microchips for macromolecular crystallization have been proposed during the last two decades to partially overcome crystal-manipulation issues, increased background noise originating from the scattering of chip-fabrication materials has so far limited the attainable resolution of diffraction data. Here, the conception and use of low-cost, X-ray-transparent microchips for in situ crystallization and direct data collection, and structure determination at atomic resolution close to 1.0 Å, is presented. The chips are fabricated by a combination of either OSTEMER and Kapton or OSTEMER and Mylar materials for the implementation of counter-diffusion crystallization experiments. Both materials produce a sufficiently low scattering background to permit atomic resolution diffraction data collection at room temperature and the generation of 3D structural models of the tested model proteins lysozyme, thaumatin and glucose isomerase. Although the high symmetry of the three model protein crystals produced almost complete data sets at high resolution, the potential of in-line data merging and scaling of the multiple crystals grown along the microfluidic channels is also presented and discussed.

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Integration of an In Situ MALDI-Based High-Throughput Screening Process: A Case Study with Receptor Tyrosine Kinase c-MET

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555217727701 ◽

2017 ◽

Vol 22 (10) ◽

pp. 1203-1210 ◽

Cited By ~ 5

Author(s):

Katrin Beeman ◽

Jens Baumgärtner ◽

Manuel Laubenheimer ◽

Karlheinz Hergesell ◽

Martin Hoffmann ◽

...

Keyword(s):

Drug Discovery ◽

High Throughput ◽

High Throughput Screening ◽

Maldi Ms ◽

Kinase Assay ◽

Label Free ◽

Screening Process ◽

Label Free Detection ◽

Ic50 Values

Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated “in-line reader” for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid format. We use the MALDI-MS to directly measure the ratio of c-MET substrate and phosphorylated product to acquire IC50 curves and demonstrate that the pharmacology is unaffected. The resulting IC50 values correlate well between the common label-based capillary electrophoresis and the label-free MALDI-MS detection method. We predict that label-free MALDI-MS-based high-throughput screening will become increasingly important and more widely used for drug discovery.

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X-CHIP: an integrated platform for high-throughput protein crystallization and on-the-chip X-ray diffraction data collection

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444911011589 ◽

2011 ◽

Vol 67 (6) ◽

pp. 533-539 ◽

Cited By ~ 38

Author(s):

Gera Kisselman ◽

Wei Qiu ◽

Vladimir Romanov ◽

Christine M. Thompson ◽

Robert Lam ◽

...

Keyword(s):

Data Collection ◽

High Throughput ◽

Diffraction Data ◽

Protein Crystallization ◽

X Ray Diffraction ◽

X Ray ◽

Integrated Platform

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A multicrystal diffraction data-collection approach for studying structural dynamics with millisecond temporal resolution

IUCrJ ◽

10.1107/s2052252516016304 ◽

2016 ◽

Vol 3 (6) ◽

pp. 393-401 ◽

Cited By ~ 11

Author(s):

Robin Schubert ◽

Svetlana Kapis ◽

Yannig Gicquel ◽

Gleb Bourenkov ◽

Thomas R. Schneider ◽

...

Keyword(s):

Data Collection ◽

Radiation Damage ◽

Temporal Resolution ◽

Diffraction Data ◽

Structural Changes ◽

Structural Information ◽

Data Sets ◽

Enzymatic Reactions ◽

Time Resolved ◽

Site Specific

Many biochemical processes take place on timescales ranging from femtoseconds to seconds. Accordingly, any time-resolved experiment must be matched to the speed of the structural changes of interest. Therefore, the timescale of interest defines the requirements of the X-ray source, instrumentation and data-collection strategy. In this study, a minimalistic approach forin situcrystallization is presented that requires only a few microlitres of sample solution containing a few hundred crystals. It is demonstrated that complete diffraction data sets, merged from multiple crystals, can be recorded within only a few minutes of beamtime and allow high-resolution structural information of high quality to be obtained with a temporal resolution of 40 ms. Global and site-specific radiation damage can be avoided by limiting the maximal dose per crystal to 400 kGy. Moreover, analysis of the data collected at higher doses allows the time-resolved observation of site-specific radiation damage. Therefore, our approach is well suited to observe structural changes and possibly enzymatic reactions in the low-millisecond regime.

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High-pressure developments for resonant X-ray scattering experiments at I16

Journal of Synchrotron Radiation ◽

10.1107/s1600577519016308 ◽

2020 ◽

Vol 27 (2) ◽

pp. 351-359

Author(s):

I. Povedano ◽

A. Bombardi ◽

D. G. Porter ◽

M. Burt ◽

S. Green ◽

...

Keyword(s):

High Pressure ◽

Low Temperature ◽

The Body ◽

X Ray ◽

X Ray Scattering ◽

Scattering Geometry ◽

Diamond Anvil ◽

Membrane Cell ◽

Ray Scattering

An experimental setup to perform high-pressure resonant X-ray scattering (RXS) experiments at low temperature on I16 at Diamond Light Source is presented. The setup consists of a membrane-driven diamond anvil cell, a panoramic dome and an optical system that allows pressure to be measured in situ using the ruby fluorescence method. The membrane cell, inspired by the Merrill–Bassett design, presents an asymmetric layout in order to operate in a back-scattering geometry, with a panoramic aperture of 100° in the top and a bottom half dedicated to the regulation and measurement of pressure. It is specially designed to be mounted on the cold finger of a 4 K closed-cycle cryostat and actuated at low-temperature by pumping helium into the gas membrane. The main parts of the body are machined from a CuBe alloy (BERYLCO 25) and, when assembled, it presents an approximate height of 20–21 mm and fits into a 57 mm diameter. This system allows different materials to be probed using RXS in a range of temperatures between 30 and 300 K and has been tested up to 20 GPa using anvils with a culet diameter of 500 µm under quasi-cryogenic conditions. Detailed descriptions of different parts of the setup, operation and the developed methodology are provided here, along with some preliminary experimental results.

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Fluorescence Detection-Based Functional Assay for High-Throughput Screening for MraY

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.3.897-902.2004 ◽

2004 ◽

Vol 48 (3) ◽

pp. 897-902 ◽

Cited By ~ 51

Author(s):

Thérèse Stachyra ◽

Christophe Dini ◽

Paul Ferrari ◽

Ahmed Bouhss ◽

Jean van Heijenoort ◽

...

Keyword(s):

Escherichia Coli ◽

High Pressure ◽

Cell Wall ◽

High Throughput ◽

High Throughput Screening ◽

Reaction Medium ◽

The Other ◽

Functional Assay ◽

Liquid Chromatography Separation ◽

Fluorescent Derivative

ABSTRACT We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan. This was accomplished by using UDP-MurNAc-Nε -dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an Escherichia coli overproducing strain. Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium. The latter assay was validated with a set of natural and synthetic MraY inhibitors.

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