We acknowledge funding from the United Kingdom&#39;s Department of Business, Energy, and Industrial Strategy (BEIS). We thank Alex R. Jones and Mike Shaw from the National Physical Laboratory for their feedback on the manuscript.

1. Protein sample preparation
<ol>
	<li>Prepare (through recombinant methods or otherwise) and purify the protein(s)&#160;of interest in a suitable buffer (i.e., one that is amenable for protein stability and crystallization) that has been filtered with a 0.22 &#181;m filter (see Table of Materials). Evaluate the protein quality (in terms of purity, polydispersity, and stability) prior to crystallization31. 
	NOTE: Please see the representative results section for details on the proteins and buffers used in the present study.</li>
	<li>Concentrate the sample up to 10-50 mg.mL-1 or higher (depending on the protein target) using the correct MWCO concentrator (see Table of Materials). 
	NOTE: The concentrated sample can be used immediately for crystallization or stored frozen at -80 &#176;C.</li>
	<li>If the protein has been frozen, centrifuge the sample for 10 min in a bench-top centrifuge (&#8805;16,000 &#215; g, at room temperature or 4 &#176;C) to pellet and remove any unwanted material (e.g., precipitates).</li>
	<li>Aliquot the protein into clean 200 &#181;L PCR tubes. Keep it on ice or at 4 &#176;C if the protein is unstable at room temperature.</li>
</ol>
2. Setting up the microdialysis plate
NOTE: The commercially available microdialysis plate (see Table of Materials) consists of two sides (&#39;protein side&#39; and &#39;buffer side&#39;) with a regenerated cellulose membrane (available in different MWCOs), as shown in Figure 2.
<ol>
	<li>With the plate orientated protein side up, peel back and remove the adhesive cover tape (Figure 2A) from the 200 &#181;m pressure adhesive spacer. Take note of the position of well A1.</li>
	<li>Using a multichannel pipette, load a maximum of 3.2 &#181;L of protein (step 1.4) into each well (Figure 2B). 
	NOTE: The protein can also be loaded using a repeat dispensing pipette. Using a single-channel pipette is possible, but it will increase the likelihood of partial dehydration of the drops occurring due to the extended loading time.</li>
	<li>Position the 200 &#181;m UV cover film (see Table of Materials) over the 96-well plate and check its integrity (Figure 2C). Ensure that the protective film is facing up.</li>
	<li>Use a sealing paddle (see Table of Materials), press down the UV cover film to activate and seal the pressure adhesive.</li>
	<li>Invert the plate and take note of the position of well A1. Ensure that the plate is oriented buffer side up and that the well positions are mirrored (Figure 2D).</li>
	<li>Using a multichannel pipette, load a maximum of 350 &#181;L of the dialysis solution into each of the wells (Figure 2D). 
	NOTE: In this step, either commercially available crystallization screens (sparse matrix screens) or laboratory-made customized screens (grid screens) can be used (see Table of Materials).</li>
	<li>Carefully seal the wells with the &#39;reservoir cover film&#39; (see Table of Materials) (Figure 2E).</li>
	<li>Place the plate (buffer side up) in a suitable temperature-controlled incubator (20 &#176;C) for crystal growth. 
	NOTE: For the proteins selected for the present study, the crystals started to appear between 1-8 h at 20 &#176;C.</li>
	<li>To inspect for crystals under the microscope, invert the plate to bring the protein side up and remove the protective film from the 200 &#181;m UV cover film (step 2.3), as shown in Figure 2F,G.</li>
</ol>
3. Large-scale crystallization using dialyzer tubes
NOTE: Once crystallization conditions have been explored using the microdialysis plate, large-scale crystallization of the protein (for serial crystallography or other purposes) can be performed using the dialyzer tube, as shown in Figure 3. Similar to the microdialysis plate, these devices are available with 3, 5, 10, and 30 kDa MWCO dialysis membranes.
<ol>
	<li>Prepare the optimized crystallization condition for the growth of microcrystals in large containers (adopting the conditions optimized using the microdialysis plate). Ensure the buffer has been previously filtered with a 0.2 &#181;m filter.</li>
	<li>Pipette the protein into the dialyzer tube (maximum volume of 0.5 mL) and seal the end using the included red plastic cap (Figure 3A).</li>
	<li>Place the 500 &#181;L dialyzer tube into a suitably sized container (depending on the total volume of dialysis solution) containing the floating rack (see Table of Materials), as shown in Figure 3B. 
	NOTE: The floating rack provided with the dialyzer kit holds up to 18 dialyzer tubes simultaneously.</li>
	<li>Place the container stationary in a suitable temperature-controlled incubator (20 &#176;C) for crystal growth.</li>
	<li>To inspect for crystals, pipette 1-2 &#181;L of the solution onto a glass cover slide. Cover with a second glass cover slide on top (to prevent excess dehydration) and view under a microscope (see Table of Materials). 
	NOTE: Crystals may be damaged when sandwiched between two glass cover slides during the inspection. Crystals may also be viewed directly by placing the dialyzer tube under a stereo high-magnification microscope.</li>
</ol>

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The co-authors Pascelle Draper and Paul Reardon are employed by SWISSCI.

Currently, crystallization by dialysis is the most underutilized crystallization method, namely due to the low-throughput and tedious nature of the existing approaches, such as microdialysis with buttons. Here, a simple but robust protocol follows an HTP workflow for protein crystal growth by dialysis through a commercially available microdialysis plate and dialyzer tube. Depending on the target protein, the microdialysis plate and dialyzer tube used in the procedure come with a choice of a dialysis membrane with a 3, 5, 10, or 30 kDa MWCO. The protocol can be easily set up in any standard crystallization facility and has the great advantage of being applicable to both soluble and membrane proteins. However, protein-protein and protein-nucleic acid complexes were not tested during this protocol.
As in any crystallization approach by dialysis, the volume ratio between the protein sample and dialysis solution is critical. In this protocol, a ratio of 1:100 between the sample and dialysis solution is recommended, but since the microdialysis plate allows a maximum capacity of 350 &#181;L of dialysis solution, these ratios can be explored to obtain crystal hits. A volume of 1-2 &#181;L of protein is used in the presented protocol when setting up crystallization plates. This is to ensure that drops set accurately with a manual multichannel pipette. By using electronic pipettes (multichannel or repeat dispensing pipettes) or HTP liquid dispensing robots, drops of lower volumes can be achieved accurately, thus lowering the amount of protein required. Furthermore, due to the relatively low volumes of dialysis buffer required by the microdialysis plate (contrary to other conventional dialysis methods), it is possible (without the extensive use of resources) to explore large chemical spaces, not only using commercially available crystallization screens but also with optimization screens (designed around the initial crystallization hit condition).
A critical step in the presented HTP procedure is the timely application of small protein volumes (0.50-3.2 &#181;L per condition) to the dialysis plate to limit dehydration and sample loss. This can be easily mitigated by using a multichannel pipette, a repeat dispensing pipette, or a robotic crystallization system. The long incubation time, such as more than 2 weeks, of the plates at 20 &#176;C might lead to dehydration of the protein droplets or damage to the recently formed crystals. Keeping the dialysis plates inside a humidification chamber or a sealable bag can alleviate this effect. In addition, using sterile materials and techniques are recommended to avoid bacterial growth.
As mentioned in the introduction, recently, with the increased need to understand protein structural dynamics for disease mechanisms, protein-ligand binding interactions, and protein-protein interactions, the field of protein X-ray crystallography has been revolutionized through the development of new and existing crystallization techniques, modern approaches to crystal sample delivery, new generations of X-ray sources, and new sophisticated methods for data acquisition and processing36,37,38. Therefore, the advent of room temperature serial micro-crystallography, either performed using XFELs or synchrotron light sources, has emerged as a remarkable tool in structural biology, specifically in the field of membrane proteins39. However, thousands of microcrystals are required to generate enough data for a robust structure solution, which is not an easy task (at least by conventional crystallization methods). The dialysis crystallization method described here enables the production of a large number of microcrystals. Once the crystallization condition for the production of microcrystals (1-10 &#181;m) has been determined through the use of a microdialysis plate, large quantities of high-density microcrystals can be produced using the 0.5 mL dialyzer device (Figure 5).&#160;These crystals are ideally suited for data collection using fixed targets or liquid jet sample delivery systems27,40. Crystals obtained through this method can also be appropriate for MicroED applications. However, these may need to be milled down to a suitable size and thickness for this specific application, as electrons interact much more strongly with crystals than X-ray photons41.
In conclusion, the crystallization by dialysis approach described here adds to the evolving strategies in protein crystallization for structure determination and expands the range of efforts than can be employed to determine novel protein targets that have previously been unsuccessful with other conventional methods.

Over the last century, X-ray crystallography has been critical in elucidating and understanding the structure-function paradigm of biological macromolecules. To date, it continues to be one of the most successful methods in elucidating atomic resolution structures of many uniquely different proteins that are crucial to the fundamental understanding of cell biochemistry, medicine, and early drug discovery1,2. However, protein crystallization remains a bottleneck in studying many protein targets, particularly membrane proteins and large protein complexes3. Consequently, protein crystallization is almost always considered an art due to the labor-intensive trial-and-error approaches employed4,5,6.
A precipitating agent is usually added to a protein solution at high concentration to form a well-ordered, regular, and repeating lattice arrangement of protein molecules, known as crystals. Under favorable conditions, such as temperature, pH, concentration, and precipitant agent, a supersaturated solution eventually forms, followed by crystal nucleation and growth7,8. Although there have been many advances in crystallization trial setups, predominantly with the development of high-throughput robotic systems and the availability of ready-made &#34;sparse matrix&#34; screens, the general approaches to protein crystallization have largely remained unchanged over the years. Common experimental protein crystallization techniques include vapor diffusion (hanging drop and sitting drop)9, microbatch (under oil)10,11, free-interface diffusion (microfluidic devices)12, and dialysis (using buttons and other techniques)13,14,15. However, other more specialized setups also exist, such as mesophase approaches for crystallizing membrane proteins16,17. While the majority of X-ray protein structures deposited in the Protein Data Bank have so far been solved through crystallization by vapor diffusion methods6,18,other approaches, such as crystallization by dialysis, seem to be underutilized, likely due to the practical aspects related to their experimental setup.
Crystallization by dialysis simply relies on the slow diffusion of solutes (precipitants, ions, additives, and buffers) through a semi-permeable membrane that simultaneously prevents protein molecules from circulating. This way, the protein solution is slowly brought into equilibrium, with the precipitant reaching the necessary concentration to crystallize. The system&#39;s kinetics depends on temperature, precipitant concentration, and the cellulose membrane molecular weight cut-off (MWCO)19. To date, the most popular crystallization setup by dialysis has been using microdialysis buttons made of transparent acrylic sheets. These are usually immersed in reservoirs (mostly using vapor diffusion hanging drop plates) containing the crystallization precipitant solutions. However, this lower-throughput method also requires specific assembly to seal the protein solution within the dialysis membrane placed over the button chamber, as illustrated in Figure 1. Moreover, air bubbles trapped between the dialysis membrane and the protein solution are a frequent problem that impair crystal growth. Another constraint of the method is the sample requirements, whereby much higher concentrations and volumes are necessary compared to vapor diffusion methods, to accommodate the dialysis buttons. Therefore, crystallization using microdialysis buttons has been perceived as an unappealing method, especially for difficult targets such as membrane proteins, whose purification yields are frustratingly low. Recently, microfluidic devices have been developed to facilitate protein crystallization by dialysis15. These chips have also been designed to have high X-ray transparency with low background, allowing the chips to be used for in situ data collection at room temperature, thus eliminating the inconvenience of harvesting and cryocooling crystals. Despite these advances, the approach is still very low-throughput and expensive.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64744/64744fig01.jpg" /> 
Figure 1: Schematic representation of crystallization by dialysis using dialysis buttons. (A)&#160;Schematic representation of a crystallization dialysis button. (B) The protein solution is added to the microdialysis button chamber. (C) The dialysis membrane is held to the microdialysis button with the help of a rubber ring (O-ring) applied via an applicator. (D) The dialysis button is ready to be immersed in the reservoir containing the crystallization solution (dialysis solution), as shown in (E). The vial containing the immersed dialysis button must be sealed to avoid evaporation. <a href="https://www.jove.com/files/ftp_upload/64744/64744fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Here, a straightforward protocol is presented for screening protein crystallization conditions and crystal growth using the 96-well high-throughput dialysis plate. These disposable plates are designed to be used similarly to the vapor diffusion crystallization plates (pipette then seal), as shown in Figure 2. The plates can accommodate up to 3.2 &#181;L of protein and 350 &#181;L of dialysis solution. Each well features a separate regenerated cellulose membrane to prevent cross-contamination between the wells. The setup takes around 10 min to complete and does not require any specialized equipment besides what can be found in all standard crystallization laboratories. Four different proteins, including two membrane proteins, are used to demonstrate and validate this approach as an effective method for high-throughput (HTP) protein crystallography.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64744/64744fig02.jpg" /> 
Figure 2: Crystallization workflow using the microdialysis plate. (A) Removal of the red adhesive &#34;cover film&#34;. (B) Dispensing the protein droplets into each of the drop wells. (C) The wells are covered with the UV &#34;cover film&#34;. (D) The plate is inverted to add the dialysis solutions (or crystallization screen). (E) The plate is sealed and incubated. (F,G) Microscope inspection of the drops. <a href="https://www.jove.com/files/ftp_upload/64744/64744fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The use of this crystallization by dialysis protocol was demonstrated using the 0.5 mL dialyzer tube (Figure 3) for the large-scale (hundreds to thousands) production of microcrystals, suitable for state-of-the-art data collection methods such as serial crystallography at both XFEL facilities20,21,22,23,24 and synchrotrons25,26,27, as well as for MicroED28,29,30 approaches.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64744/64744fig03.jpg" /> 
Figure 3: Large-scale microdialysis crystallization using the dialyzer tube. (A) Schematic representation of the 0.5 mL dialyzer tube. (B) Side view of a beaker containing the crystallization solution and the floating tube rack holding a dialyzer tube. <a href="https://www.jove.com/files/ftp_upload/64744/64744fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>0.2 mL tubes</td><td>Thermo Scientific</td><td>AB0620</td><td>For aliquoting protein solutions.</td></tr><tr><td>0.2 &amp;micro;m syringe filter</td><td>Sartorius</td><td>17823----------K</td><td>Surfactant-free cellulose acetate filters. For filtering dialysis solutions.</td></tr><tr><td>0.22 &amp;micro;m membrane filters</td><td>Millipore</td><td>GSTF04700</td><td>Membrane filters for filtering large volumes of buffers</td></tr><tr><td>12-channel, variable 0.5 &amp;ndash; 10 &amp;micro;L Research plus pipette</td><td>Eppendorf</td><td>3125000028</td><td>For dispensing protein drops onto the Diaplate.</td></tr><tr><td>12-channel, variable 30 &amp;ndash; 300 &amp;micro;L Eppendorf Research plus pipette&amp;nbsp;</td><td>Eppendorf</td><td>3125000060</td><td>For dispensing dialysis solutions on the Diaplate reservoirs.</td></tr><tr><td>20 mL syringe</td><td>Fisherbrand</td><td>15889152</td><td>For use with syringe filters.</td></tr><tr><td>96 well 2.2 mL deep-well plates</td><td>Thermo Scientific</td><td>AB0788</td><td>Polypropylene deep-well storage plates; for preparing screens using the Hamilton Microlab STARlet.</td></tr><tr><td>Centrifuge 5425</td><td>Eppendorf</td><td>5405000565</td><td>With rotor FA-24x2 with a maximum g-force of 21,300 x g.</td></tr><tr><td>Diacon dialyser</td><td>SWISSCI</td><td>W72010</td><td>Dialyzer tubes with a regenerated cellulose membrane with a molecular weight cut-off of 10 kDa. Ideal for protein solutions of up to 0.5 mL.</td></tr><tr><td>Diaplate 96-well plate</td><td>SWISSCI</td><td>W82010</td><td>Microdialysis plate. The Diaplate consists of two sides with a regenerated cellulose membrane in-between with a molecular weight cut-off of 10 kDa.</td></tr><tr><td>Falcon 50 mL High Clarity PP Centrifuge Tube</td><td>Corning</td><td>352070</td><td>For holding dialysis solutions.</td></tr><tr><td>Floating rack</td><td>SWISSCI</td><td>n/a</td><td>Included in the Diacon kit</td></tr><tr><td>Floor-standing vibration-free incubator</td><td>Molecular Dimensions</td><td>MD5-01</td><td>400 L temperature-controlled incubator set to 20 &amp;deg;C.</td></tr><tr><td>Leica M205 C stereo microscope</td><td>Leica</td><td></td><td>Planapo 1.0x objective, 7.8x &amp;ndash; 160x zoom range with DMC 4500 camera</td></tr><tr><td>Lysozyme from chicken egg white</td><td>Sigma Aldrich</td><td>62971</td><td>Lyophilized protein</td></tr><tr><td>Memgold2</td><td>Molecular Dimensions</td><td>MD1-64</td><td>Sparse-matrix screen</td></tr><tr><td>Microlab STARlet</td><td>Hamilton</td><td>n/a</td><td>Liquid handler system.</td></tr><tr><td>Reservoir cover film</td><td>SWISSCI</td><td>n/a</td><td>Included in the Diaplate kit</td></tr><tr><td>Reusable bottle top filter</td><td>Thermo Scientific</td><td>DS0320-5045</td><td>For fitering large volumes of buffers, for use with 0.22 &amp;micro;m membrane filters</td></tr><tr><td>Sealing paddle</td><td>SWISSCI</td><td>n/a</td><td>Included in the Diaplate kit</td></tr><tr><td>Thaumatin from &lt;em&gt;Thaumatococcus daniellii&lt;/em&gt;</td><td>Sigma Aldrich</td><td>T7638</td><td>Lyophilized protein</td></tr><tr><td>UV cover film</td><td>SWISSCI</td><td>n/a</td><td>Included in the Diaplate kit</td></tr></tbody></table>

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.

Four proteins were crystallized using the microdialysis plate (with 10 kDa MWCO membranes), including two membrane proteins. The chicken egg-white lysozyme from the lyophilized powder (see Table of Materials) was prepared at 50 mg.mL-1 in 20 mM NaOAc (pH 4.5), and the lyophilized thaumatin (see Table of Materials) was dissolved in water to a final concentration of 25 mg.mL-1. The two membrane proteins used in this study were the E. coli multidrug efflux pump AcrB and the E. coli lactose transporter&#160;LacY. AcrB was expressed using C43 (DE3) cells and purified by Ni-NTA affinity chromatography and size-exclusion chromatography in 10 mM Tris (pH 7.5), 300 mM NaCl, 0.03% (w/v) n-dodecyl-&#946;-D-maltoside (DDM), and 5% (v/v) glycerol32. Subsequently, the protein was concentrated using a 100 kDa MWCO centrifugal concentrator to a final concentration of 6 mg.mL-1. LacY was also expressed in C43 (DE3) cells, purified by Ni-sepharose affinity chromatography followed by size-exclusion chromatography in 20 mM Tris (pH 7.5), 150 mM NaCl, 0.03% (w/v) DDM, and concentrated using a 100 kDa MWCO concentrator to 10 mg.mL-1 4,33.
For the lysozyme and thaumatin proteins, the 96-well microdialysis plate was filled with a crystallization grid screen prepared in-house as the dialysis solution, while for the membrane protein AcrB, a grid screen was prepared in-house from a previously published crystallization condition34. For LacY, initial hit conditions were found from a commercial screen (see Table of Materials) and further optimized with a grid screen made in-house. The crystallization drop ratio for lysozyme, thaumatin, and AcrB was 1:100, with 2 &#181;L of protein to 200 &#181;L of precipitant (dialysis solution). LacY crystallization drops were also at a 1:100 ratio, with 1 &#181;L of protein to 100 &#181;L of dialysis solution, due to a lower amount of available protein.
Crystals from all four proteins, including the membrane proteins, started to appear between 1-8 h at 20 &#176;C following setup with the microdialysis plate. In this case, it was not necessary to supplement the dialysis solution with detergent for the membrane protein crystallization, typically done during the standard dialysis process to prevent membrane protein aggregation, as the detergent micelles were expected to be larger than the MWCO of the dialysis membranes35.
Following the successful crystallization of the proteins on the microdialysis plate (Figure 4), crystallization conditions were noted, and large-scale crystallization by dialysis was performed using the dialyzer tube with the same 10 kDa MWCO dialysis membranes. Thousands of microcrystals for each of the individual proteins grew inside the dialyzer, as shown in Figure 5. For thaumatin crystallization, 250 &#181;L of protein was loaded onto the dialyzer and dialyzed against 50 mL (1:200 ratio). For AcrB, 250 &#181;L of protein was dialyzed against 25 mL of dialysis solution (1:100). LacY crystallization was also set up at the same ratio with 100 &#181;L of protein to 10 mL of dialysis solution.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64744/64744fig04.jpg" /> 
Figure 4: Crystals are grown by dialysis using the microdialysis plate. The protocol demonstrates the applicability of the setup with soluble proteins: (A) Lysozyme was dialyzed against 0.1 M NaOAc (pH 4.0), 0.5 M NaCl, and 25% (v/v) glycerol. (B) Thaumatin was dialyzed against 0.1 M Bis-tris propane (pH 6.6), 1 M K/Na tartrate, and 18% (v/v) ethylene glycol. Crystals were also obtained for membrane proteins: (C) AcrB was dialysed against 0.1 M MES (pH 5.5), 0.3 M NaCl, and 20% (v/v) PEG-400. (D) LacY was dialysed against 0.1 M MES (pH 6.5), 0.1 M NaCl, and 32% (v/v) PEG-300. Images were captured using a stereo high-magnification microscope with a cross polariser. Scale bars: (A,B,D) = 1 mm; (C) = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64744/64744fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64744/64744fig05.jpg" /> 
Figure 5: Representative images of membrane protein crystals grown by dialysis using the dialyzer tube. (A) Image showing the tube full of microcrystals (indicated by the black arrow). (B) Microscopic image of 2 &#181;L from a slurry of thaumatin crystals grown by dialysis using the 0.5 mL dialyzer tube, dialyzed against 0.1 M Bis-Tris propane (pH 6.6), 1.4 M K/Na tartrate, and 18% (v/v) ethylene glycol. (C) Dark-field (left) and bright-field (right) images of AcrB microcrystals grown by dialysis against 0.1 M MES (pH 5.5), 0.3 M NaCl, and 20% (v/v) PEG-400. (D) Dark-field (left) and bright-field (right) images of LacY microcrystals grown by dialysis against 0.1 M MES (pH 6.5), 0.1 M NaCl, and 32% (v/v) PEG-300. Some precipitates are observed. Scale bars: (B,D) = 200 &#181;m; (C) = 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64744/64744fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about High-Throughput Protein Crystallization via Microdialysis at JoVE.com

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.

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 ...

high-throughput-protein-crystallization-via-microdialysis

Research

JoVE Journal

Biochemistry

2.2K Views.  National Physical Laboratory. 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.

Video: High-Throughput Protein Crystallization via Microdialysis

Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize proteins, DNAs, RNAs, and their complexes can however not be predicted. Employing a broad variety of conditions is a way to increase the yield of quality diffraction crystals. Two fully automated systems have been developed at the MRC Laboratory of Molecular Biology (Cambridge, England, MRC-LMB) that facilitate crystallization screening against 1,920 initial conditions by vapor diffusion in nanoliter droplets. Semi-automated protocols have also been developed to optimize conditions by changing the concentrations of reagents, the pH, or by introducing additives that potentially enhance properties of the resulting crystals. All the corresponding protocols will be described in detail and briefly discussed. Taken together, they enable convenient and highly efficient macromolecular crystallization in a multi-user facility, while giving the users control over key parameters of their experiments.

Automated systems and protocols for the routine preparation of a large number of screens and nanoliter crystallization droplets for vapor diffusion experiments are described and discussed.

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize ...

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 ...

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

This article presents a method to generate protein crystals derivatized with I3C (5-amino-2,4,6-triiodoisophthalic acid) using microseeding to generate new crystallization conditions in sparse matrix screens. The trays can be set up using liquid dispensing robots or by hand.

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...

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.

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.

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

Chemistry

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization.

Author Spotlight: High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography  

This protocol details high-throughput crystallization screening, ranging from the 1,536 microassay plate preparation to the end of a 6 week experimental time window. Details are included about the sample setup, the imaging obtained, and how users can perform analyses using an artificial intelligence-enabled graphical user interface to quickly and efficiently identify macromolecular crystallization conditions.

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...

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

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand.
In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams.

Structural studies of biomacromolecules by crystallography require high-quality crystals. Here we demonstrate a protocol that can be used by OptiCrys (a fully automated instrument developed in our lab) and/or microdialysis buttons for growing large high-quality crystals based on knowledge of the crystallization phase diagram.

High-Throughput Protein Crystallization via Microdialysis

Summary

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