MBS acknowledges the support of the MI / CNRS under the contract Instrumentation at the limits 2014-2015. NJ acknowledges CEA&#39;s International Doctoral Research Program (Irtelis) for the PhD Fellowship. MBS and SJ acknowledge funding from the European Union&#39;s Horizon 2020 Research and Innovation Programme under Marie Sk&#322;odowska-Curie grant agreement number 722687. MBS, SJ, and NJ thank LIPhy (UGA) for the clean room establishment for microfabrication experiments. IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA).

The authors have nothing to disclose.

A microfluidic device has been developed for on-chip protein crystallization with the microdialysis method and in situ X-ray diffraction experiments at room temperature. NOA 81 chips integrating RC dialysis membranes of any MWCO in order to use microdialysis for on-chip protein crystallization can be fabricated. Fabrication materials with a relatively high X-ray transparency were used, rendering the chips compatible for in situ protein crystallography. The fabrication materials that compose the compartment for protein crystallization of the device (PMMA, Kapton, RC dialysis membrane) were evaluated to generate low background noise. Specifically, the background noise generated by the dialysis chip is mainly observed at low resolution (&#62; 6 &#197;) and does not affect the treatment of high-resolution diffraction data of the large lysozyme crystals required for protein structure determination. The automation of the data collection is amplified using a 3D printed support that can be mounted directly in macromolecular crystallography beamlines and carry up to three microchips simultaneously. This way, manual harvesting and manipulation of the fragile protein crystals is avoided. Moreover, the data collection takes place at room temperature, avoiding the need for cryoprotection which can be related to conformational changes from the native protein structure2,3.
The use of microdialysis as a method to grow crystals on-chip allows to accurately monitor and control the crystallization process. As discussed in the introduction, most of the conventional protein crystallization methods have been implemented using microfluidic devices11,14. However, the advantages of dialysis for protein crystallization had not yet been fully exploited at the microscale. On-chip microdialysis provides the possibility to study phase diagrams and perform screening and optimization of crystallization conditions with the same protein sample19. For the prototypes presented in this work, the protein consumption per chip is limited down to 0.1 or 0.3 &#181;L. Based on the experimental work so far, the most critical steps of the protocol do not derive from the chips&#39; fabrication procedure but from the crystallization process. The fabrication protocol includes many steps but it is straightforward and enables the fabrication of numerous devices (20 to 30 chips) in a single day in the clean room, with relatively inexpensive materials. However, the on-chip crystallization of proteins can be a delicate procedure due to the intrinsic stochastic nature of nucleation and crystal growth, especially in the microscale. A case study has been described, where well-established conditions were used for the crystallization of lysozyme that yielded robust, well-defined crystals suitable for in situ X-ray diffraction data collection. Nevertheless, difficulties may arise by the use of more challenging protein targets, such as membrane proteins, where the crystallization medium is much more complicated, phase diagrams are not known and well-working crystallization conditions are not yet well established. The dialysis chip offers the possibility to surpass these difficulties and study phase diagrams on-chip, without disposing the valuable and frequently costly protein sample, by merely exchanging the crystallization solution within the microfluidic channel.
The versatility of the microfluidic devices stems from exploiting microdialysis for on-chip protein crystallization in order to reversibly control crystallization conditions and map concentration and temperature varied phase diagrams using low protein volume. Moreover, the device is compatible with in situ X-ray diffraction experiments and the prototyping of the devices is inexpensive and rapid. Numerous, isomorphous crystals of soluble and membrane proteins (in preparation) can be grown on-chip and it is expected that all these features can be utilized for serial X-ray crystallography studies of challenging protein targets at synchrotron and XFEL facilities. Finally, performing on-chip and in situ time-resolved studies is a future possibility that could be of significant interest to the crystallographic community. Therefore, by growing crystals on the dialysis chip and introducing the reagents into the microfluidic channel, either manually (using a syringe) or automatically (with a pressure-control fluid system or a syringe-pump), future efforts will focus on proving that the microfluidic chips can be successfully used to trigger time-resolved experiments on synchrotron beamlines.

Elucidating the three-dimensional (3D) structure of biological macromolecules is an unceasing pursuit in structural biology where X-ray crystallography remains the principal investigation technique. Applied for unraveling the structural details of complex macromolecules, such as proteins, it aims at facilitating the understanding of their mechanisms of actions and their involvement in various biological functions. Powerful X-ray sources at synchrotrons and X-ray free-electron lasers (XFELs) provide all the tools required for a deeper insight into the proteins&#39; structure at near atomic resolution. Despite the advantages that come along with the use of X-rays for structural studies, there are intrinsic limitations to X-ray radiation and the crystallization process itself. Radiation damage provoked by high X-ray flux and long exposure times of the protein crystal in front of the X-ray beam are restrictive parameters that crystallographers have to surpass using cryogenic cooling1. However, finding the optimal cryocooling conditions can be laborious since conformational changes from the native protein structure or artifacts can be concealed2,3. Moreover, recent studies indicate that performing diffraction experiments at room temperature leads to lower specific radiation damage4. Another bottleneck in structural biology is the acquisition of well-diffracting crystals with a sufficient size5. Small crystals are easier to produce, especially in the case of membrane proteins, but are more susceptible to radiation damage even under cryocooling conditions because a high radiation dose must be directed in a smaller volume compared to the case of larger protein crystals6. The novel approach of serial crystallography7,8 at synchrotrons and XFELs can circumvent the restrains of radiation damage and at the same time exploit smaller crystals (200 nm to 2 &#181;m)7 by merging data sets from multiple, isomorphous and randomly oriented protein crystals and profiting from the associated technological advances such as femtosecond pulses, shorter exposure times and micro-focused X-ray beams5,7,9,10.
Microfluidic technology is valuable to X-ray crystallography, exhibiting manifold advantages for the crystallization of biological macromolecules and their structural investigation. Conducting crystallization experiments in microfluidic devices requires small volumes of protein sample, therefore constraining the production cost of these high-valued bio macromolecules and facilitating high-throughput screening and optimization of numerous crystallization conditions. Moreover, the inherent large surface area-to-volume ratio at the microfluidic scale and diffusion-limited transport phenomena enable fine control over flows and temperature or concentration gradients11,12,13,14, rendering microfluidic devices suitable for growing uniformly sized crystals and exploring phase diagrams15,16,17,18,19. Moreover, microfluidic tools display a distinctive potential to address another hurdle in protein crystallography, which is the sample delivery, and the necessity to handle and harvest protein crystals prior to their use for X-ray diffraction experiments. The method of on-chip and in situ X-ray crystallography eliminates the crystal manipulation and the potential deterioration of crystal quality prior to data collection. A wide range of microfluidic chips compatible for in situ X-ray protein crystallography have been designed, developed, and tested by many research groups confronting the related restrictions arising from the nature of the microfabrication materials and their interactions with X-rays14,19,20,21,22,23. The fabrication materials must be optically transparent, biologically inert and demonstrate high transparency to X-ray radiation and an optimal signal-to-noise ratio during data collection.
Most of the crystallization methods applied in conventional protein crystallography24,25 have also been implemented at the microfluidic scale11,14 for on chip crystallization and in situ X-ray diffraction analysis. Simple, hybrid, or multi-layered microfluidic apparatus incorporating vapor diffusion26, evaporation27, free interface diffusion (FID)28, microbatch26, or even seeding29 have been used to crystallize soluble and membrane proteins. High throughput screening and optimization of crystallization conditions can be achieved30,31 in well-based32, droplet-based33, or valve-actuated34 devices. In situ X-ray diffraction experiments of challenging protein targets at room temperature have been conducted in microchips fabricated from various materials such as PDMS (polydimethylsiloxane), COC (cyclic olefin copolymer), PMMA (poly(methyl methacrylate))21,22,26,28,29, graphene films23, Kapton35, epoxy glue6, or NOA (Norland Optical Adhesive)19 and the materials&#39; transparency to X-ray radiation and their contribution to background noise have been evaluated. Moreover, microchips have been designed to couple the in situ and the serial data collection strategies in a single tool for X-ray protein crystallography experiments at synchrotron sources23,35,36 and XFELs7.
Room temperature&#160;in situ data collection has also been implemented in various delivery methods and devices. For example, Nogly et al.54 used a lipidic cubic phase (LCP) injector in order to study the structure of the light-driven photon pump bacteriorhodopsin (bR) by serial femtosecond crystallography (SFX) using an XFEL source. The crystal structure of bR was solved to 2.3 &#197; resolution, demonstrating the compatibility of an LCP injector with time-resolved serial femtosecond crystallography (TR-SFX). Baxter et al.55 designed a high-density multi-crystal grid, fabricated by a 100 or 200 &#181;m thick polycarbonate plastic with laser-cut holes of various sizes. An additional 5 &#181;m thick polycarbonate film can be fixed to one side of the grid when using the device for sitting- or hanging-drop crystallization experiments. This high-density grid can be used in multiple ways as crystals can be loaded directly onto the ports of the device or crystals can be grown on the device by vapor diffusion or the LCP method. Moreover, the grid can be adjusted in a standard magnetic base and used for in situ X-ray data collection at cryogenic or room temperature conditions. More recently, Feiler et al.56 developed a sample holder for macromolecular in situ&#160;X-ray crystallography at cryogenic and ambient temperature with minimal background noise contribution. Specifically, the holder comprises of a plastic support, a transparent COC foil and a microporous structured polyimide foil. It was designed to replace the commonly used cover slides for setting up crystallization drops, while allowing in-place manipulation such as ligand soaking, complex formation, and cryogenic protection without opening the crystallization drop or manually handling the crystals. Moreover, the sample holder can be removed from the crystallization plate and placed onto a magnetic base for in situ data collection at standard goniometer-based beamlines. For ambient temperature data collection, the COC foil is removed prior to the experiment and only the 21 &#181;m-thick polyimide foil contributes to background scattering, which in this case is minimal. These examples compose only a small fraction of the ongoing research and the multitude of versatile microchips developed for X-ray protein crystallography.
However, the dialysis protein crystallization method has not been widely incorporated within microfluidics. Dialysis is a diffusion-based method aiming for the equilibration of precipitant concentration through a semi-permeable membrane in order to approach the nominal concentration for protein crystallization and enables precise and reversible control over the crystallization conditions24. The Molecular Weight Cut-Off (MWCO) of the semi-permeable dialysis membrane can be chosen depending on the molecular weight of the macromolecule and the precipitants to allow the diffusion of small precipitant molecules while retaining the macromolecule of interest. Due to the reversibility of the dialysis process, it can be used in combination with temperature control to decouple and optimize nucleation and crystal growth independently37 for investigating phase diagrams by altering the precipitant concentration while using the same protein sample. The integration of membranes in microfluidics is reviewed by de Jong et al.38 and the case studies in biology implanting dialysis into microchips can be principally listed in sample preparation, concentration or filtration applications39,40,41,42 or cell-related studies43,44. Pervaporation through PDMS was used by Shim et al.37 to study the nucleation and growth of xylanase in various conditions. Water permeated through the 15 &#181;m thick PDMS membrane into the protein reservoir of the microfluidic device, subsequently altering the protein and precipitant concentration.
The protocol developed by Junius et al.19,45 for the fabrication of a microfluidic chip compatible for both on-chip protein crystallization via microdialysis and in situ X-ray diffraction experiments at room temperature is presented. The protocol for the device fabrication is directly inspired by the pioneering work accomplished by Studer and coworkers12,46 for micro-patterned stickers of photo-curable thiolene-based resin NOA 81 embedding commercially available membranes, using soft imprint lithography. An innovative modification of the method resulted in microchips enabling the use of microdialysis to accurately monitor and control the experimental parameters for the on-chip growth of protein crystals and simultaneously exploit&#160;the advantages of microfluidics, such as reduced consumption of protein samples per experiment (&#60;1 &#181;L). In a previous work, the principles of dialysis applied to a macro-scale system (typical volume &#62;20 &#181;L) for screening and optimizing crystallization conditions by mapping temperature-precipitant concentration phase diagrams were demonstrated47. In this work, a protocol is described for producing dialysis microchips incorporating regenerated cellulose (RC) dialysis membranes of different MWCO in order to perform crystallization assays on-chip and in situ X-ray diffraction data collection. The materials comprising the microchips have been evaluated for their transparency to X-rays19 and the devices can be set directly in front of the X-ray beam for room temperature in situ diffraction experiments, excluding the manual handling and minimizing the degradation of fragile protein crystals. In a case study, hen egg-white lysozyme crystals were grown on-chip via microdialysis generating a uniformly sized population. The microchip was then mounted in front of the X-ray beam with a 3D-printed support19 and complete in situ diffraction data sets were collected at room temperature from multiple, isomorphous crystals, demonstrating the high potential and relevance of the chips for synchrotron serial crystallography studies of challenging macromolecular targets.

<table><tbody><tr><td>3 in wafer</td><td>Silicon Materials Inc.</td><td></td><td>Silicon wafer</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>Minispin</td><td>Bench-top centrifuge</td></tr><tr><td>CleWin 3.0</td><td>WieWeb software</td><td></td><td>Designing software</td></tr><tr><td>Epoxy glue</td><td>Devcon</td><td></td><td>5 minutes epoxy glue</td></tr><tr><td>Fluidic connectors</td><td>Cluzeau Info Lab</td><td>N-333</td><td>NanoPort kit for 1/16&quot; OD tubing</td></tr><tr><td>Hen egg-white lysozyme</td><td>Roche</td><td>10 837 059 001</td><td>Lyophilized protein powder</td></tr><tr><td>High-vacuum silicone grease</td><td>Sigma-Aldrich</td><td>Z273554</td><td>Dow Corning&amp;nbsp;high-vacuum silicone grease</td></tr><tr><td>HMDS</td><td>Sigma-Aldrich</td><td>440191</td><td>Silane, chemical</td></tr><tr><td>Hot plate</td><td>Sawatec</td><td>HP-200-Z-HMDS BM</td><td>Hot plate</td></tr><tr><td>Isopropyl alcohol</td><td>Sigma-Aldrich</td><td></td><td>Solvent</td></tr><tr><td>Kapton tape</td><td>DuPont</td><td></td><td>Polyimide tape</td></tr><tr><td>Mask aligner</td><td>SUSS MicroTec</td><td>MJB4</td><td>Mask aligner, UV source</td></tr><tr><td>Membrane filter</td><td>Millipore</td><td>GSWP04700</td><td>0.22 &amp;mu;m pore size filter</td></tr><tr><td>Microscope glass slide</td><td>Fisher Scientific</td><td>12164682</td><td>3 x 1 in glass slides</td></tr><tr><td>NOA81</td><td>Norland Products Inc.&amp;nbsp;</td><td>NOA81</td><td>Photocurable resin</td></tr><tr><td>Oven</td><td>Memmert</td><td></td><td>Oven</td></tr><tr><td>Parafilm</td><td>Sigma-Aldrich</td><td>P6543</td><td>Parafilm M roll size 20&amp;nbsp;in. &amp;times; 50&amp;nbsp;ft</td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>Sylgard 184</td><td>Silicone</td></tr><tr><td>PEG 400</td><td>Hampton Research</td><td>HR2-603</td><td>Chemical</td></tr><tr><td>Petri dish</td><td>Sigma-Aldrich</td><td>P5731</td><td>100 x 15 mm</td></tr><tr><td>PGMEA</td><td>Sigma-Aldrich</td><td>484431</td><td>Developer</td></tr><tr><td>Plasma equipment</td><td>Diener Electronic</td><td>ZEPTO</td><td>Plasma treatment</td></tr><tr><td>PMMA</td><td>Goodfellow</td><td>137-745-63</td><td>PMMA sheets 150x150 mm, 0.175 mm thickness</td></tr><tr><td>Pressure driven system</td><td>Elveflow</td><td>OB1 MK3+</td><td>Pressure/vacuum controller</td></tr><tr><td>PTFE tubing</td><td>Elveflow/Darwin microfluidics</td><td>LVF-KTU-15</td><td>PTFE tubing roll 1/16&quot; OD X 1/32&quot; ID</td></tr><tr><td>RC dialysis membrane</td><td>Spectra/Por</td><td></td><td>Various MWCOs</td></tr><tr><td>Scalpel</td><td>Swann-Morton</td><td></td><td>Carbon steel surgical blades</td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td>Chemical</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>746398</td><td>Chemical</td></tr><tr><td>Solidworks</td><td>Dassault Systemes</td><td></td><td>3D-CAD designing software</td></tr><tr><td>Spin coater</td><td>SPS</td><td>Spin150</td><td>Wafer spinner</td></tr><tr><td>SU-8 3000 series</td><td>MicroChem Corp.</td><td>SU-8 3050</td><td>Photoresist</td></tr><tr><td>Syringe</td><td>BD</td><td>309628</td><td>1 mL Luer-Lok syringe</td></tr><tr><td>UV crosslinker</td><td>Uvitec</td><td>CL-508</td><td>UV crosslinker</td></tr></tbody></table>

crystallization of proteins on chip by microdialysis for in situ x-ray diffraction studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (&#60;1 &#181;L) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Watch this Scientific Journal Video about Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies at JoVE.com

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

crystallization-proteins-on-chip-microdialysis-for-situ-x-ray

Research

JoVE Journal

Chemistry

6.4K Views.  Université Grenoble Alpes, CEA, CNRS, IBS. This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

Video: Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

Microcrystallography of Protein Crystals and In Cellulo Diffraction

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in their largest dimension, which used to represent a challenge. We present two alternative workflows for the structure determination of protein microcrystals by X-ray crystallography with a particular focus on crystals grown in vivo. The microcrystals are either extracted from cells by sonication and purified by differential centrifugation, or analyzed in cellulo after cell sorting by flow cytometry of crystal-containing cells. Optionally, purified crystals or crystal-containing cells are soaked in heavy atom solutions for experimental phasing. These samples are then prepared for diffraction experiments in a similar way by application onto a micromesh support and flash cooling in liquid nitrogen. We briefly describe and compare serial diffraction experiments of isolated microcrystals and crystal-containing cells using a microfocus synchrotron beamline to produce datasets suitable for phasing, model building and refinement.
These workflows are exemplified with crystals of the Bombyx mori cypovirus 1 (BmCPV1) polyhedrin produced by infection of insect cells with a recombinant baculovirus. In this case study, in cellulo analysis is more efficient than analysis of purified crystals and yields a structure in ~8 days from expression to refinement.

Microcrystallography of Protein Crystals and In Cellulo Diffraction

A protocol is presented for X-ray crystallography using protein microcrystals. Two examples analyzing in vivo-grown microcrystals after purification or in cellulo are compared.

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

Biochemistry

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since cryogenic temperatures are non-physiological and may prevent, deter, or even alter protein structural dynamics, a robust method for routine X-ray diffraction experiments at room temperature is highly desirable. The crystal-on-crystal device and its accompanying hardware and software used in this protocol are designed to enable in situ X-ray diffraction at room temperature for protein crystals of different sizes without any sample manipulation. Here we present the protocols for the key steps from device assembly, on-chip crystallization, optical scanning, crystal recognition to X-ray shot planning and automated data collection. Since this platform requires no crystal harvesting nor any other sample manipulation, hundreds to thousands of protein crystals grown on chip can be introduced into an X-ray beam in a programmable and high-throughput manner.

This contribution describes how to set up protein crystallization on crystal-on-crystal devices and how to perform automated serial data collection at room temperature using the on-chip crystallization platform.

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since ...

High-Throughput Protein Crystallization via Microdialysis

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug discovery. To date, X-ray crystallography remains the most successful method for solving three-dimensional protein structures at atomic resolution. With recent advances in serial crystallography, either using X-ray free electron lasers (XFELs) or synchrotron light sources, protein crystallography has progressed to the next frontier, where the ability to acquire time-resolved data provides important mechanistic insights into the behavior of biological molecules at room temperature. This protocol describes a straightforward high-throughput (HTP) workflow for screening crystallization conditions through the use of a 96-well dialysis plate. These plates follow the Society for Biomolecular Screening (SBS) standard and can be easily set up using any standard crystallization laboratory. Once optimal conditions are identified, large quantities of crystals (hundreds of microcrystals) can be produced using the dialyzer. To validate the robustness and versatility of this approach, four different proteins were crystallized, including two membrane proteins.

High-Throughput Protein Crystallization via Microdialysis

The presented protocol describes a straightforward approach for screening protein crystallization conditions and crystal growth using a 96-well high-throughput dialysis plate. The use of dialyzer tubes for the large-scale growth of microcrystals is also demonstrated for serial crystallography and MicroED applications.

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug ...

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Biology

Protein Crystallization

Protein crystallization, obtaining a solid lattice of biomolecules, elucidates protein structure and enables the study of protein function. Crystallization involves drying purified protein under a combination of many factors, including pH, temperature, ionic strength, and protein concentration. Once crystals are obtained, the protein structure can be elucidated by x-ray diffraction and computation of an electron density model.
This video introduces protein crystallization and shows a general procedure. Protein expression and purification, crystallization, and x-ray diffraction are covered in the procedure. Applications of protein crystallization include in silico drug design, binding site determination, and membrane protein structure analysis.
<div class="chapter_text" id="script_0">
Protein crystallization is the process of obtaining a latticed solid form of a protein. These crystals are especially valuable to structural biologists, assisting in the study of protein function. Other techniques, such as mass spec or SDS-PAGE, can only provide information on the one-dimensional structure of proteins. Protein crystallization is complemented by the techniques of recombinant protein expression and x-ray diffraction. This video will show the principles of protein crystallization, a general laboratory procedure, and several of its applications in the biochemical field.
</div>
<div class="chapter_text" id="script_1">
The first step required in the process is to obtain milligram quantities of very pure protein, typically using recombinant protein expression. The gene corresponding to the protein of interest is cloned into an expression vector, and the expressed protein is fused to an affinity tag, such as poly-histidine, to assist in the purification by affinity chromatography. To learn more, see this collection&#39;s video on affinity chromatography.
Formation of the purified protein into crystals is dependent on the proper combination of many factors, including pH, ionic strength, concentrations of precipitant and protein, temperature, and rate of equilibration. The most common method used is vapor diffusion, of which there are two categories: hanging drop and sitting drop. A droplet containing pure protein, buffer, and precipitant, which is an ionic solid that binds water molecules, reducing water availability for the protein and mimicking higher protein concentration, is in an enclosed microwell with a reservoir with a more highly concentrated mixture of the same buffer and precipitant. At the beginning, the concentrations of protein and precipitant are too low to cause crystallization. During the course of the experiment, water vaporizes from the droplet and collects in the reservoir; a decrease in the amount of water in the droplet causes the system to become supersaturated, and nucleation, followed by crystallization, can occur. The net transfer of water from the droplet is in equilibrium, and the system is maintained until the process is complete.
To visualize the 3D structure, x-ray diffraction is used. To obtain x-ray data from a crystal, it is placed in a monochromatic x-ray beam, where it is exposed to the beam at all angles. Each exposure provides an image, where each spot is a diffracted x-ray, which emerges from the crystal and is registered by a detector. The data are combined to produce a model of the arrangement of atoms within the crystal. The resulting crystal structure demonstrates the 3-dimensional placement of the atoms, with a typical resolution of 2 angstroms.

</div>
<div class="chapter_text" id="script_2">
Now that we have covered the principles of protein crystallization, let us look at a generalized protocol.
To begin the procedure an expression vector containing the gene of interest is transformed into cells. The cells are incubated and at mid-log phase, expression is initiated by adding an inducer, such as IPTG, which triggers transcription of the gene&#39;s mRNA. After protein expression, the crude material is suspended in lysis buffer, and then clarified by centrifugation.
The clarified lysate is then loaded onto a nickel column, and the polyhistidine-tagged protein binds to the column while all other biomolecules are washed away.
Once several milligrams of pure protein have been obtained, it is ready for crystallization by vapor diffusion. A 24-well hanging/sitting drop tray is filled with varying concentrations of sodium chloride and sodium acetate buffer solutions. For the sitting drop method, equal volumes of protein and reservoir solution are pipetted onto the shelf above each well, and then the tray is covered with transparent tape. The tray is then placed in an incubation chamber, and the wells are monitored for growth the following day, then every few days.
Once a proper crystal has been obtained it is ready for x-ray diffraction analysis. The crystal is mounted on a goniometer to position the crystal at selected orientations. The crystal is illuminated with a monochromatic beam of x-rays at all angles, producing a diffraction pattern. The software converts the two-dimensional images, taken at different orientations, to a three-dimensional model of the density of electrons within the crystal by determining the positions of the atoms in the crystal.
</div>
<div class="chapter_text" id="script_3">
Now that we have reviewed a procedure, let&#39;s review some useful applications of protein crystallization, and another crystallization technique.
Protein crystallization may be used for in silico drug design. The three-dimensional structure of Influenza virus&#39;s polymerase basic protein 2, which has been linked to viral infection in mammals, was determined by crystallization and x-ray diffraction. Potential binding sites in the protein are visualized, and with the use of a docking program, a three-dimensional molecule was designed that would insert into a cleft in the protein.
Co-crystallization of protein-DNA complexes is also a useful technique. DNA-binding proteins modulate a wide variety of biological functions such as transcription and DNA polymerization and DNA repair; and crystal structures of these complexes can provide insight into protein function, mechanism, and the nature of the specific interaction. The E. coli protein SeqA, a negative regulator of DNA replication, was co-crystallized with hemimethylated DNA.
Integral membrane proteins such as G-protein coupled receptors, or GCPRs, are difficult to crystallize due to their limited amount of polar surface area available for forming crystal lattice contacts, which has led to the development of fusion-protein-assisted protein crystallization. Genes encoding &#946;2 adrenergic receptor, a GCPR, and a lysozyme were inserted into an expression vector. The crystallization of the &#946;2AR- lysozyme fusion protein was achieved due to the increased extracellular hydrophilic surface over the naturally hydrophobic &#946;2AR, provided by the lysozyme, necessary for forming packing interactions in the crystal lattice.
</div>
<div class="chapter_text" id="script_4">
You&#39;ve just watched JoVE&#39;s video on protein crystallization. This video described its principles, a generalized protocol, and some its uses in the biomedical field. Thanks for watching!
</div>

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

Summary

Explore More Videos

Chapters in this video

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

Microcrystallography of Protein Crystals and In Cellulo Diffraction

Dynamic Electrochemical Measurement of Chloride Ions

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

High-Throughput Protein Crystallization via Microdialysis

Protein Crystallization for X-ray Crystallography

Protein Crystallization