The authors acknowledge the Swiss Light Source (Villigen, Switzerland) for beamtime allocation to the project on beamlines X10SA (PXII) and X06DA (PXIII), Alexandra Bluhm for her contribution to structure refinement, Clarissa Worsdale for the recording of the voiceover and Fran&#231;ois Schnell (Universit&#233; de Strasbourg) for his assistance in video editing and SFX. This work was supported by the French Centre National de la Recherche Scientifique (CNRS), the University of Strasbourg, the LabEx consortium &#34;NetRNA&#34; (ANR-10-LABX-0036_NETRNA), a PhD funding to R.dW from the Excellence initiative (IdEx) of the University of Strasbourg in the frame of the French National Program &#34;Investissements d&#39;Avenir&#34;, a PhD funding to K.R. from the French-German University (UFA-DFH, grant no. CT-30-19), the Deutsche Forschungsgemeinschaft (grant no. Mo 634/10-1). The authors benefitted from the PROCOPE Hubert Curien cooperation program (French Ministry of Foreign Affair and Deutscher Akademischer Austauschdienst).

1. Setting up crystallization assays in ChipX
NOTE: The ChipX microfluidic device can be obtained from the authors. A description of the chip is given in Figure 1. Solutions containing the crystallant (or crystallizing agent) used to trigger crystallization may be of commercial origin or prepared by the experimenter.
<ol>
	<li>Loading the biomolecule sample 
	&#8203;NOTE: The sample volume actually required to perform an individual counter-diffusion assay in the straight section of each channel of ChipX is 300 nL. However for convenience we suggest to load 5 &#181;L to completely fill the eight channels taking into account the variable length of their curved section and inlet dead volumes.
	<ol>
		<li>Pipet 5 &#181;L of enzyme solutions using standard 10 &#181;L pipet and tip.</li>
		<li>Introduce the tip vertically in the sample inlet and inject the solution until the eight channels are filled up to their opposite end (entry of the crystallant reservoir).</li>
		<li>Inject 1 &#181;L of paraffin oil in the sample inlet in order to disconnect the channels from each other.</li>
		<li>Recover the extra solution in the crystallant reservoir at the extremity of each channel using a standard 10 &#181;L pipet.</li>
		<li>Seal the sample inlet with a 1 cm x 1 cm piece of tape.</li>
	</ol>
	</li>
	<li>Loading the crystallization solutions
	<ol>
		<li>Pipet 5 &#181;L of crystallization solution using standard 10 &#181;L pipet and tip. The reservoir volume is 10 &#181;L, but loading only half of it avoids overflow when sealing with tape and facilitates further addition of ligand for soaking experiments. If initial crystallization conditions were obtained by vapor diffusion, increase the crystallant concentration by a factor of 1.5 - 2. Solutions can be different in every reservoir (in the presented case, 1 M diammonium hydrogen phosphate, 100 mM sodium acetate, pH 4.5 was used throughout).</li>
		<li>Orient the pipet tip towards the entry of the channel in the funnel shaped part of the reservoir to avoid the formation of an air bubble upon solution deposition. It would prevent the contact between the two solutions and crystallant diffusion into the channel.</li>
		<li>Inject the crystallant solution into the reservoir.</li>
		<li>Seal the reservoirs with a 2.5 cm x 1 cm piece of tape.</li>
		<li>Incubate the chip at 20 &#176;C (temperature can be adjusted depending on the target, typically between 4 and 37 &#176;C 4).</li>
	</ol>
	</li>
</ol>
2. Protein labeling with carboxyrhodamine for fluorescence detection
NOTE: This step is optional. It must be performed prior to sample loading to facilitate the detection of crystals in the chip using fluorescence. The detailed method of trace fluorescent labeling was described by Pusey and coworkers7. All steps are carried out at room temperature.
<ol>
	<li>Dissolve 5 mg of carboxyrhodamine ester powder in 1 mL anhydrous dimethyl-formamide, split the solution in 0.6 &#181;L aliquots to be stored at -20 &#176;C.</li>
	<li>Prepare a 1 M Na-borate pH 8.75 stock solution.</li>
	<li>Dilute the stock to prepare the reaction buffer at 0.05 M Na-borate pH 8.75.</li>
	<li>Rinse a desalting column (7 kDa MWCO, 0.5 mL) with 800 &#181;L of reaction buffer.</li>
	<li>Centrifuge the column for 1 min at 1400 x g, remove the filtrate.</li>
	<li>Repeat this operation twice (steps 2.4-2.5) to wash the column.</li>
	<li>Deposit 80 &#181;L of protein in its storage buffer on the column (the protein can be diluted down to 1 mg/mL to increase the volume if needed).</li>
	<li>Centrifuge the column for 1 min at 1400 x g. This step is intended to transfer the protein from its storage buffer to the reaction buffer.</li>
	<li>Recover the flow-through (containing the protein in the reaction buffer) and mix it with 0.6 &#181;L of carboxyrhodamine solution.</li>
	<li>Incubate 5 min at room temperature.</li>
	<li>Meanwhile, rinse the column 3 x with the storage buffer, centrifuge the column for 1 min at 1400 x g and discard the filtrate.</li>
	<li>Deposit the reaction solution on the column.</li>
	<li>Centrifuge the column for 1 min at 1400 x g and recover the flow-through (i.e., solution of labeled protein in its storage buffer).</li>
	<li>Supplement the stock protein solution with 0.1-1 % (w/w) of labeled protein.</li>
	<li>Setup the ChipX crystallization assays as described in section 1.</li>
	<li>Check for the presence of protein crystals in the assays by exciting the fluorescent probe with a 520 nm wavelength light source.</li>
</ol>
3. Crystal observation
NOTE: The ChipX device may be handled without special care, even with crystals inside, except if the temperature needs to be controlled.
<ol>
	<li>Use any stereomicroscope to check the outcome of crystallization assays in ChipX. Its footprint has the standard dimensions of microscope slides and is compatible with any system and slide holder.</li>
	<li>Check the content of microfluidic channels starting from the reservoir where the crystallant concentration is the highest to the sample inlet where the crystallant concentration is the lowest. The ChipX material is transparent to visible light, compatible with the use of polarizers, as well as with UV illumination for protein crystal identification by intrinsic tryptophane fluorescence8.</li>
	<li>Record crystal positions using the labels embossed along the channels or mark crystal locations with a permanent marker by drawing color dots next to them on the chip surface.</li>
</ol>
4. Crystal soaking with ligands
NOTE: This procedure is optional. It is used to introduce ligands, enzyme substrates or heavy atoms into the crystals and should be carried out at least 24-48 h before X-ray analysis to allow the compound diffusion along the channels and into the crystals.
<ol>
	<li>Gently remove the sealing tape from the reservoirs.</li>
	<li>Add up to 5 &#181;L of ligand solution in one or several reservoirs using a 10 &#181;L micropipet (in the example, 3 &#181;L of 10 mM cytidine-5&#39;-[(&#945;,&#946;)-methyleno] triphosphate (CMPcPP) solution was added to achieve a final concentration of 3.75 mM). CMPcPP is a non-hydrolizable analog of CTP, a natural substrate of the enzyme.</li>
	<li>Seal the reservoirs with a 2.5 cm x 1 cm piece of tape.</li>
	<li>Incubate the chip under controlled temperature for 24-48 h to allow ligand diffusion along the channels of the chip.</li>
</ol>
5. Crystal analysis by serial crystallography
NOTE: This part of the protocol needs to be adapted depending on the beamline setup and the diffraction properties of the crystals. Only general indications are given for the crystallographic analysis based on experiments performed at X06DA beamline (SLS, Villigen, Switzerland).
<ol>
	<li>ChipX mounting on the beamline goniometer 
	&#8203;NOTE: The file for 3D printing the ChipX holder is provided in ref4.
	<ol>
		<li>Turn off the cryo-jet of the beamline. The analysis here is carried out at room temperature.</li>
		<li>Mount the ChipX on a dedicated holder with the channel containing the crystals to be analyzed positioned at the center of the holder. The ChipX holder4&#160;does not require any screw or additional part, as it was designed to provide a perfect fit for ChipX.</li>
		<li>Attach the holder to the goniometer.</li>
	</ol>
	</li>
	<li>Data collection
	<ol>
		<li>Orient the thickest layer (top layer, Figure 1) of ChipX (in this orientation labels along the channels are directly readable using the centering camera of the beamline), towards the direct beam and the thinnest face behind the crystal to minimize the attenuation of the diffracted signal as described in ref3.</li>
		<li>To avoid collision of ChipX with the surrounding material (beamstop, collimator), restrict goniometer&#160;movements in the range &#177;30&#176; (0&#176; corresponding to the channels being perpendicular to the X-ray beam).</li>
		<li>Find crystals position with the help of the labels embossed along the channels.</li>
		<li>Select a crystal position.</li>
		<li>Center the crystal either by standard low dose grid/raster screening or 1-click procedure (the video shows an example of grid screening).</li>
		<li>Collect diffraction data within the range -30&#176; / +30&#176;.</li>
		<li>Restart the procedure at steps 5.2.4-5.2.6 on another crystal in the same channel after translation of the chip.</li>
		<li>Manually realign another ChipX channel at the center of the holder and carry on data collection on crystals present in this channel.</li>
		<li>Use standard crystallographic packages and procedures to process and merge the data, then to solve and refine the structure.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Gieg&#233;, R., Sauter, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocrystallography:+past,+present,+future.">Biocrystallography: past, present, future.</a> HFSP Journal. 4 (3-4), 109-121 (2010).</li><li>Dhouib, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+chips+for+the+crystallization+of+biomacromolecules+by+counter-diffusion+and+on-chip+crystal+X-ray+analysis.">Microfluidic chips for the crystallization of biomacromolecules by counter-diffusion and on-chip crystal X-ray analysis.</a> Lab on a Chip. 9 (10), 1412-1421 (2009).</li><li>Pinker, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChipX:+A+Novel+Microfluidic+Chip+for+Counter-Diffusion+Crystallization+of+Biomolecules+and+in+Situ+Crystal+Analysis+at+Room+Temperature.">ChipX: A Novel Microfluidic Chip for Counter-Diffusion Crystallization of Biomolecules and in Situ Crystal Analysis at Room Temperature.</a> Crystal Growth &amp; Design. 13 (8), 3333-3340 (2013).</li><li>de Wijn, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+versatile+microfluidic+device+for+efficient+biomacromolecule+crystallization+and+structural+analysis+by+serial+crystallography.">A simple and versatile microfluidic device for efficient biomacromolecule crystallization and structural analysis by serial crystallography.</a> IUCrJ. 6 (3), 454-464 (2019).</li><li>Garc&#237;a-Ruiz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+supersaturation+wave+of+protein+crystallization.">A supersaturation wave of protein crystallization.</a> Journal of Crystal Growth. 232 (1-4), 149-155 (2001).</li><li>Ot&#225;lora, F., Gavira, J. A., Ng, J. D., Garc&#237;a-Ruiz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counterdiffusion+methods+applied+to+protein+crystallization.">Counterdiffusion methods applied to protein crystallization.</a> Progress in Biophysics and Molecular Biology. 101 (1-3), 26-37 (2009).</li><li>Pusey, M., Barcena, J., Morris, M., Singhal, A., Yuan, Q., Ng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+fluorescent+labeling+for+protein+crystallization.">Trace fluorescent labeling for protein crystallization.</a> Acta Crystallographica Section F Structural Biology Communications. 71 (7), 806-814 (2015).</li><li>Meyer, A., Betzel, C., Pusey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Latest+methods+of+fluorescence-based+protein+crystal+identification.">Latest methods of fluorescence-based protein crystal identification.</a> Acta Crystallographica Section F Structural Biology Communications. 71 (2), 121-131 (2015).</li><li>Betat, H., Rammelt, C., M&#246;rl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=tRNA+nucleotidyltransferases:+ancient+catalysts+with+an+unusual+mechanism+of+polymerization.">tRNA nucleotidyltransferases: ancient catalysts with an unusual mechanism of polymerization.</a> Cellular and Molecular Life Sciences. 67 (9), 1447-1463 (2010).</li><li>Ernst, F. G. M., Erber, L., Sammler, J., J&#252;hling, F., Betat, H., M&#246;rl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+adaptation+of+tRNA+nucleotidyltransferases:+A+tradeoff+in+activity,+stability+and+fidelity.">Cold adaptation of tRNA nucleotidyltransferases: A tradeoff in activity, stability and fidelity.</a> RNA Biology. 15 (1), 144-155 (2018).</li><li>Kabsch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XDS.">XDS.</a> Acta Crystallographica. Section D, Biological Crystallography. 66 (2), 125-132 (2010).</li><li>Adams, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PHENIX:+a+comprehensive+Python-based+system+for+macromolecular+structure+solution.">PHENIX: a comprehensive Python-based system for macromolecular structure solution.</a> Acta Crystallographica. Section D, Biological Crystallography. 66 (2), 213-221 (2010).</li><li>Dessau, M. A., Modis, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Crystallization+for+X-ray+Crystallography.">Protein Crystallization for X-ray Crystallography.</a> Journal of Visualized Experiments. (47), e2285 (2011).</li><li>Sauter, C., Lorber, B., McPherson, A., Gieg&#233;, R., Arnold, E., Himmel, D. M., Rossmann, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+-+General+Methods.">Crystallization - General Methods.</a> International Tables of Crystallography, Vol. F, Crystallography of Biological Macromolecules. , 99-120 (2012).</li><li>Garman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Cool"+crystals:+macromolecular+cryocrystallography+and+radiation+damage.">"Cool" crystals: macromolecular cryocrystallography and radiation damage.</a> Current Opinion in Structural Biology. 13 (5), 545-551 (2003).</li><li>Li, D., Boland, C., Aragao, D., Walsh, K., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvesting+and+Cryo-cooling+Crystals+of+Membrane+Proteins+Grown+in+Lipidic+Mesophases+for+Structure+Determination+by+Macromolecular+Crystallography.">Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography.</a> Journal of Visualized Experiments. (67), e4001 (2012).</li><li>Diederichs, K., Karplus, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+R-factors+for+diffraction+data+analysis+in+macromolecular+crystallography.">Improved R-factors for diffraction data analysis in macromolecular crystallography.</a> Nature Structural Biology. 4 (4), 269-275 (1997).</li><li>Karplus, P. A., Diederichs, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+Crystallographic+Model+and+Data+Quality.">Linking Crystallographic Model and Data Quality.</a> Science. 336 (6084), 1030-1033 (2012).</li><li>de la Mora, E., Coquelle, N., Bury, C. S., Rosenthal, M., Holton, J. M., Carmichael, I., Garman, E. F., Burghammer, M., Colletier, J. -. P., Weik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+damage+and+dose+limits+in+serial+synchrotron+crystallography+at+cryo-+and+room+temperatures.">Radiation damage and dose limits in serial synchrotron crystallography at cryo- and room temperatures.</a> Proceedings of the National Academy of Sciences of the United States of America. 117 (8), 4142-4151 (2020).</li><li>Nave, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Description+of+Imperfections+in+Protein+Crystals.">Description of Imperfections in Protein Crystals.</a> Acta Crystallographica. Section D, Biological Crystallography. 54 (5), 848-853 (1998).</li></ol>

The authors have nothing to disclose.

Current protocols in biocrystallography involve the preparation of crystals using methods such as vapor diffusion or batch13, 14, and their transfer into a microloop for cryo-cooling15, 16&#160;before performing the diffraction analysis in a nitrogen jet at cryogenic conditions. In contrast, direct crystal cryo-cooling is not possible in ChipX3 and crystals cannot be extracted from their microfluidic channel, which can be seen as limitations of this setup. However, the protocol described in the article provides a fully integrated pipeline for the determination of crystal structures at room temperature (i.e., in more physiological conditions). Even though data collection at room-temperature causes an increase of radiation damage19, this effect is counterbalanced by fast data acquisition time (a maximum of 60&#176; rotation is collected on each crystal) and by merging of several partial datasets. Both ChipX design and material were optimized to reduce background scattering and diffraction signal attenuation3, and data collection can be performed on crystals with dimensions equivalent to half the size of the channels (40 &#181;m)4.
To summarize, the main advantages of the protocol are the following. The crystals are produced in a convection-free environment (microfluidic channels), which is very favorable to the growth of high-quality crystals. The counter-diffusion method implemented in ChipX is very efficient at screening the supersaturation landscape; the diffusion of crystallants into the chip channel creates a concentration and supersaturation wave that helps determine appropriate nucleation and growth conditions5. Crystals are never directly handled, but are analyzed in situ, inside the chip, which preserves their genuine diffraction properties (i.e., does not alter crystal mosaicity by physical interaction or cryocooling)20. The diffraction analysis is performed on a series of crystals distributed along the chip channels with low dose exposure to minimize radiation damage, and a full dataset is assembled by merging partial data from the series. The standard footprint and simple design of ChipX will allow in the future a complete automation of in situ data collection using synchrotron or XFEL facilities. All steps of the protocol are carried out in ChipX. From the experimenter point-of-view, chip setup is simple and easy to perform with standard pipets and does not require any extra equipment. The tree-like channel connection at the sample inlet minimizes dead volumes in the system, which is important when working with samples that are difficult to purify or that are only available in limited quantity.
In conclusion, the lab-on-a-chip approach implemented in ChipX simplifies and efficiently miniaturizes the process of crystallization by counter-diffusion and crystal structure determination, allowing to go from the sample to its 3D structure in a single device. It is widely applicable and offers a user-friendly, cost-effective solution for routine serial biocrystallography investigations at room temperature.

Crystallography is a method to decipher the 3D architecture of biological macromolecules. The latter is important to understand how an enzyme selects and processes its substrates. The determination of a crystal structure requires the crystallization of the target macromolecule and the conditioning of the crystals for their analysis by X-ray diffraction1. Both crystal preparation and handling are crucial but delicate steps that can affect crystal quality and diffraction properties, and, thus, the resolution (i.e., the accuracy) of the resulting 3D structure. To facilitate the preparation of high-quality crystals and eliminate unnecessary handling to preserve their diffraction properties, we designed a user-friendly and versatile microfluidic device called ChipX2,3,4.
In this article, we will demonstrate how to load the protein solution into ChipX channels using conventional laboratory material to prepare crystals by counter-diffusion. This crystallization method provides an efficient screening of supersaturation and of potential nucleation conditions along the microfluidic channels containing the enzyme solution due to the concentration gradient generated by the diffusion of the crystallizing agent5,6.
The chip setup is simple, it uses only standard laboratory pipets and does not require any costly equipment. When crystals have grown in ChipX, ligands of the enzyme can be introduced by diffusion. Diffraction data are then collected at room temperature on a series of crystals contained in the channels of the chip using a synchrotron X-ray source. The structural study described here led to the determination of structures of a tRNA maturation enzyme in its apo form and in complex with an analog&#160;of its CTP substrate introduced by soaking. This protein called CCA-adding enzyme polymerizes the CCA trinucleotide tail at the 3&#39; end of tRNAs. The comparison of the two 3D images obtained by serial crystallography reveals the local conformational changes related to the binding of the ligand in conditions that are more physiological than those used in cryo-crystallography. The protocol described in this video is generally applicable to any biomolecule, be it a protein, a nucleic acid or a multi-component complex.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Axioscope A1 stereomicroscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Crystal observation&#160; (step 3)</td>
		</tr>
		<tr>
			<td>Carboxyrhodamine succinimidyl ester</td>
			<td>Invitrogen</td>
			<td>C-6157</td>
			<td>Protein labeling (step 2)</td>
		</tr>
		<tr>
			<td>CMPcPP</td>
			<td>Jena Bioscience</td>
			<td>NU-438</td>
			<td>Crystal soaking (step 4)</td>
		</tr>
		<tr>
			<td>Crystal clear sealing tape</td>
			<td>Hampton research</td>
			<td>HR3-511</td>
			<td>ChipX sealing&#160; (step 1)</td>
		</tr>
		<tr>
			<td>Parafin oil</td>
			<td>Hampton research</td>
			<td>HR3-411</td>
			<td>ChipX loading&#160; (step 1)</td>
		</tr>
		<tr>
			<td>Ultimaker 2 extended+</td>
			<td>Ultimaker</td>
			<td></td>
			<td>3D printer - Representative results</td>
		</tr>
		<tr>
			<td>UV light source</td>
			<td>Xtal Concepts Gmbh</td>
			<td>XtalLight100c</td>
			<td>Crystal observation&#160; (step 3)</td>
		</tr>
		<tr>
			<td>Zeba spin desalting column 7K MWCO</td>
			<td>ThermoFisher Scientific</td>
			<td>89882</td>
			<td>Protein labeling&#160; (step 2)</td>
		</tr>
	</tbody>
</table>

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.

The microfluidic chip described here was designed to enable easy setup of crystallization assays and crystal analysis at room temperature. The procedure described above and in the video was applied in the frame of the structural characterization of the CCA-adding enzyme from the cold-adapted bacterium Planococcus halocryophilus. This enzyme belongs to an essential polymerase family that catalyses the sequential addition of the 3&#39; CCA sequence on tRNAs using CTP and ATP9,10.
The chip was first used to prepare crystals of the enzyme for structural analysis by the method of counter-diffusion. To this end, the enzyme solution was loaded in the eight microfluidic channels (crystallization chambers) by a single injection in the sample inlet of the chip (see Figure 1). The enzyme was used at 5.5 mg/mL in its storage buffer containing 20 mM Tris/HCl pH 7.5, 200 mM NaCl and 5 mM MgCl2. This step was performed manually with a standard 10 &#181;L micropipet. Crystallization solutions (100 mM sodium acetate pH 4.5,1 M diammonium hydrogen phosphate) were then deposited in the reservoirs at the other extremity of the channels.
The loading procedure is straightforward and does not take longer than five minutes (Figure 2). The crystallant then diffuses into the channels, creates a gradient of concentration that triggers crystal nucleation and growth. This gradient evolves dynamically and explores a continuum of supersaturation states5,6&#160;until reaching an equilibrium of crystallant concentration between the channels and the reservoir. Crystallization assays are typically checked under the micoscope over a period of 2 - 4 weeks to track the growth of crystals. Bipyramidal crystals of CCA-adding enzyme appeared throughout the channels after a few days of incubation at 20 &#176;C (Figure 3). The optional fluorescent labeling7&#160;of the protein greatly facilitates the identification of protein crystals and their discrimination from salt crystals (Figure 4).
We exploited the diffusive environment in chip channels to deliver a substrate to the enzyme that builds up the crystals. In the present case, CMPcPP, a CTP analog, was added to the reservoir solutions at a final concentration of 3.75 mM (Figure 5). This addition was performed two days before the crystallographic analysis to allow CMPcPP to reach and occupy the catalytic site of the enzyme, as later confirmed by the crystal structure (see below).
We manufactured a chip holder (Figure 6) in polylactic acid using a 3D printer. The holder enables chip mounting on goniometers using standard magnetic heads. Hence the chip can be easily positioned and translated in the X-ray beam to bring the crystals in diffraction position. The data collection strategy needs to be adapted depending on beamline characteristics and on crystal properties. In the case of the CCA-adding enzyme, data were collected at X06DA and X10SA beamlines, Swiss Light Source (SLS), with an X-ray wavelength of 1.0 &#197; and Pilatus 2M-F and 6M pixel detectors, respectively. 30-60&#176; of rotation were collected on each crystal at room temperature with images of 0.1&#176; or 0.2&#176; and 0.1 s exposure (see Table 1). Partial datasets were processed individually and cut when the resolution of diffraction patterns started to decay due to radiation damage (detected by the decrease of signal-to-noise ratio <img alt="Equation 1" src="/files/ftp_upload/61972/61972eq1.png" style="margin: 0" />&#160;and CC1/2, and an increase of Rmeas in the high resolution shell). Full datasets were reconstituted by merging data from 5 crystals (Table 1). Crystal structures were derived by molecular replacement using standard crystallographic packages and procedures for data processing11&#160;and refinement12. The comparison of the structures of the enzyme and of its complex with CMPcPP reveals the local conformational adaptation that accompanies substrate binding in the active site of the CCA-adding enzyme (Figure 7).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61972/61972fig01.jpg" /> 
Figure 1: ChipX design. The chip consists of a top layer made of COC (thickness: 1 mm) in which eight microfluidic channels and reservoirs are imprinted. The entire chip is sealed with a layer of COC (thickness: 0.1 mm). All channels are connected to a single inlet on the left hand side for simultaneous sample injection and to individual reservoirs on the right hand side in which crystallization solutions are deposited. The channels, which constitute the actual crystallization chambers of the chip, are 4 cm long and have a cross section of 80 &#181;m x 80 &#181;m. Labels (A1, A2, A3, etc.) embossed along the channels facilitate crystal positioning under the microscope and the preparation of a sample list for data collection. ChipX has the size of a standard microscope slide (7.5 cm x 2.5 cm). <a href="https://www.jove.com/files/ftp_upload/61972/61972fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61972/61972fig02.jpg" /> 
Figure 2: Setting up crystallization assays in ChipX.&#160;1) Deposit 5-6 &#181;L of enzyme solutions using standard 10 &#181;L pipet and tip. 2) Introduce the tip vertically in the sample inlet and inject the solution in the eight channels. 3) Pipet 1 &#181;L of paraffin oil. 4) Introduce the tip vertically in the sample inlet and inject the oil in order to disconnect the channels from each other. 5) Seal the inlet with a piece of tape. 6) Pipet 5 &#181;L of crystallization solution using standard 10 &#181;L pipet and tip. Solutions can be different in every reservoir (e.g., from a screening kit). 7) Orient the pipet tip towards the entry of the channel in the funnel shaped part of the reservoir (to avoid the formation of an air bubble upon solution deposition) and inject the crystallant solution in the reservoir. 8) Seal the reservoirs with a piece of tape and incubate the chip at controlled temperature. <a href="https://www.jove.com/files/ftp_upload/61972/61972fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61972/61972fig03.jpg" /> 
Figure 3:&#160;Crystals of CCA-adding enzyme grown by counter-diffusion in the microfluidic channels of ChipX. Scale bar is 0.1 mm. <a href="https://www.jove.com/files/ftp_upload/61972/61972fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61972/61972fig04.jpg" /> 
Figure 4: Crystal soaking procedure.&#160;1) Gently remove the tape from the reservoirs. 2) Deposit up to 5 &#181;L of ligand solution using a 10 &#181;L micropipet. 3) Add the ligand to one or several reservoirs. 4) Seal again the reservoirs with a piece of tape and incubate the chip under controlled temperature for 24-48 h before data collection. <a href="https://www.jove.com/files/ftp_upload/61972/61972fig04large.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/61972/61972fig05.jpg" /> 
Figure 5: Trace fluorescent labeling discriminates protein (left) from salt (right) crystals. The CCA-adding enzyme solution contained 0.4 % (w/w) of protein labeled with carboxyrhodamine. On the right, crystals are illuminated with a 520 nm wavelength light source and the image is taken with a low pass filter at 550 nm (LP550); (inset) structure of carboxyrhodamine-succinimidyl ester. <a href="https://www.jove.com/files/ftp_upload/61972/61972fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61972/61972fig06.jpg" /> 
Figure 6: (Left) Drawing of the ChipX holder and (Right) ChipX mounted on the goniometer of beamline X06DA at SLS (Villigen, Switzerland) for serial crystal analysis. <a href="https://www.jove.com/files/ftp_upload/61972/61972fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61972/61972fig07.jpg" /> 
Figure 7: Comparison of CCA-adding enzyme active site in the apo form (in pink) and in the complex with a CTP analog (in green). Although the overall conformation of the enzyme is not affected, the binding of the CMPcPP ligand is accompanied by a slight reorganization of side chains in the active site. The 2Fo-Fc electron density map (in blue) is contoured at 1.2 sigma. The difference electron density map contoured at 4 sigma (in green) confirms the presence of the ligand in the active site. <a href="https://www.jove.com/files/ftp_upload/61972/61972fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Crystallized sample</td>
			<td>CCA-adding enzyme</td>
			<td>CCA-adding enzyme + CMPcPP</td>
		</tr>
		<tr>
			<td>Crystal analysis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray beamline</td>
			<td>SLS &#8211; X06DA</td>
			<td>SLS &#8211; X10SA&#160;</td>
		</tr>
		<tr>
			<td>Wavelength (&#197;)</td>
			<td>1.000</td>
			<td>1.000</td>
		</tr>
		<tr>
			<td>Temperature (K)</td>
			<td>293</td>
			<td>293</td>
		</tr>
		<tr>
			<td>Detector</td>
			<td>Pilatus 2M-F</td>
			<td>Pilatus 6M</td>
		</tr>
		<tr>
			<td>Crystal-detector distance (mm)</td>
			<td>300</td>
			<td>400</td>
		</tr>
		<tr>
			<td>Crystals collected</td>
			<td>6</td>
			<td>14</td>
		</tr>
		<tr>
			<td>Crystals selected</td>
			<td>5</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Rotation range per image (&#176;)</td>
			<td>0.1</td>
			<td>0.2</td>
		</tr>
		<tr>
			<td>Exposure time per image (s)</td>
			<td>0.1</td>
			<td>0.1</td>
		</tr>
		<tr>
			<td>No. of images selected</td>
			<td>1000</td>
			<td>540</td>
		</tr>
		<tr>
			<td>Total rotation range (&#176;)</td>
			<td>100</td>
			<td>108</td>
		</tr>
		<tr>
			<td>Space group</td>
			<td>P43212</td>
			<td>P43212</td>
		</tr>
		<tr>
			<td>a, c (&#197;)</td>
			<td>71.5, 293.8</td>
			<td>71.4, 293.6</td>
		</tr>
		<tr>
			<td>Mean mosaicity (&#176;)</td>
			<td>0.04</td>
			<td>0.04</td>
		</tr>
		<tr>
			<td>Resolution range (&#197;)</td>
			<td>46 &#8211; 2.54 (2.6 &#8211; 2.54)</td>
			<td>48 &#8211; 2.3 (2.4 &#8211; 2.3)</td>
		</tr>
		<tr>
			<td>Total No. of reflections</td>
			<td>176105 (9374)</td>
			<td>232642 (32937)</td>
		</tr>
		<tr>
			<td>No. of unique reflections</td>
			<td>23922 (1598)</td>
			<td>34862 (4066)</td>
		</tr>
		<tr>
			<td>Completeness (%)</td>
			<td>90.6 (84.6)</td>
			<td>99.5 (100.0)</td>
		</tr>
		<tr>
			<td>Redundancy</td>
			<td>7.5 (6.0)</td>
			<td>6.7 (8.1)</td>
		</tr>
		<tr>
			<td><img alt="Equation 1" src="/files/ftp_upload/61972/61972eq1.png" /></td>
			<td>8.1 (1.3)</td>
			<td>6.9 (0.7)</td>
		</tr>
		<tr>
			<td>Rmeas (%) &#167;</td>
			<td>18.6 (126.0)</td>
			<td>18.0 (231.2)</td>
		</tr>
		<tr>
			<td>CC1/2 (%) &#163;</td>
			<td>98.7 (55.0)</td>
			<td>98.7 (46.9)</td>
		</tr>
		<tr>
			<td>Overall B factor from Wilson plot (&#197;2)</td>
			<td>57.4</td>
			<td>60.6</td>
		</tr>
		<tr>
			<td>Crystallographic refinement</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>No. of reflections, working set / test set</td>
			<td>23583 / 1180</td>
			<td>34840 / 3405</td>
		</tr>
		<tr>
			<td>Final Rcryst (%) / Rfree (%)</td>
			<td>18.8 / 21.4</td>
			<td>20.0 / 22.9</td>
		</tr>
		<tr>
			<td>No. of non-H atoms: overall / protein / ligand / solvent</td>
			<td>2998 / 2989 / 0 / 9</td>
			<td>3057 / 2989 / 29 / 10</td>
		</tr>
		<tr>
			<td>R.m.s. deviations for bonds (&#197;) / angles (&#176;)</td>
			<td>0.009 / 1.23</td>
			<td>0.010 / 1.22</td>
		</tr>
		<tr>
			<td>Average B factors (&#197;2):&#160; overall / protein / ligand / solvent</td>
			<td>60.1 / 60.1 / 0 / 52.7</td>
			<td>62.5 / 62.6 / 60.1 / 55.5&#160;</td>
		</tr>
		<tr>
			<td>Ramachandran plot: most favored (%) / allowed (%)</td>
			<td>98.1 / 1.9</td>
			<td>97.2 /&#160; 2.8</td>
		</tr>
		<tr>
			<td>PDB id</td>
			<td>6IBP</td>
			<td>6Q52</td>
		</tr>
	</tbody>
</table>
Table 1: Data collection and refinement statistics
&#167; Redundancy-independent Rmeas = &#931;hkl(N/N-1)1/2&#931;i&#160;| Ii(hkl)- &#60;I(hkl)&#62;| / &#931;hkl&#931;i Ii(hkl), where N is the data multiplicity 17.
&#163; Data with low &#60;I/&#963;(I)&#62; in outer shell (&#60;2.0) were included based on CC1/2 criterion (correlation between two random halves of the dataset &#62; 50%) as proposed by Karplus &#38; Diederichs 18.

Watch this Scientific Journal Video about Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip at JoVE.com

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

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 ...

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Biochemistry

8.1K Views.  Institut de Biologie Moléculaire et Cellulaire. 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.

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

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 &#181;m crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural ...

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

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Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

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A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its precursor p100. p52 functions as a homodimer or heterodimer with another NF-&#954;B subunit, RelB. These dimers in turn regulate the expression of hundreds of genes involved in inflammation, cell survival, and cell cycle. IKK1/&#945; primarily remains associated with IKK2/&#946; and NEMO as a ternary complex. However, a small pool of it is also observed as a low molecular weight complex(es). It is unknown if the p100 processing activity is triggered by activation of IKK1/&#945; within the larger or the smaller complex pool. Constitutive activity of IKK1/&#945; has been detected in several cancers and inflammatory diseases. To understand the mechanism of activation of IKK1/&#945;, and enable its use as a drug target, we expressed recombinant IKK1/&#945; in different host systems, such as E. coli, insect, and mammalian cells. We succeeded in expressing soluble IKK1/&#945; in baculovirus infected insect cells, obtaining mg quantities of highly pure protein, crystallizing it in the presence of inhibitors, and determining its X-ray crystal structure. Here, we describe the detailed steps to produce the recombinant protein, its crystallization, and its X-ray crystal structure determination.

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&amp;#945;

I&#954;B Kinase 1/&#945; (IKK1/&#945; CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-&#954;B transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein.

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Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

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Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and IKK&#946;. Upon activation, the IKK complex phosphorylates the I&#954;B molecules leading to NF-&#954;B nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-&#954;B pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.