Name: On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature (Video) | JoVE Journal
Uploaded: 2022-03-11T12:30:00.000+00:00
Duration: 7 min 42 s
Description: Learn protein crystallization on a chip-like device for X-ray diffraction data collection at room temperature. This method enables automated serial data collection from thousands of crystals without physical contact.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

Use of Advanced Photon Source, an Office of Science User Facility operated for US Department of Energy by Argonne National Laboratory, was supported by contract DE-AC02-06CH11357. Use of BioCARS was supported by the National Institute of General Medical Sciences of the National Institutes of Health under grant number R24GM111072. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Use of LS-CAT Sector 21 was supported by Michigan Economic Development Corporation and Michigan Technology Tri-Corridor grant 085P1000817. This work is supported by grants from the University of Illinois at Chicago, National Institutes of Health (R01EY024363), and National Science Foundation (MCB 2017274) to XY.

1. Device pre-assembly
<ol>
	<li>Label the outer ring (30 mm diameter) for sample identification. If necessary, include the project name, device number, crystallization condition, and date (Figure 1A). Place the outer ring upside down on a clean surface (Figure 1B), and carefully place one quartz wafer inside the ring (Figure 1C). This first quartz wafer serves as an entrance window for the incident X-rays.</li>
	<li>Pour a small amount of microscope immersion oil (viscosity of 150 cSt) into a Petri dish. Dip a shim in the oil, and make sure that both sides of the shim are properly oiled (Figure 1D). Remove the excess oil by dabbing the shim on a clean surface.</li>
	<li>Place the oiled shim on top of the first quartz wafer (Figure 1E). 
	&#8203;NOTE: Immersion oil is an excellent sealant that protects the crystallization chamber from potential vapor loss. Properly assembled chips usually last for weeks without visible drying. This pre-assembly step is performed under room light. For light-sensitive samples, all subsequent steps, including sample loading, device storage, and observation, must be carried out under safety light.</li>
</ol>
2. Sample loading and device assembly
<ol>
	<li>Use a pipette to thoroughly mix the protein solution and crystallization buffer on the first quartz wafer. The volume ratio between the protein sample and buffer typically ranges from 2:1 to 1:2 (Figure 1F). Make sure that the total volume of the crystallization solution does not exceed the maximum capacity of the crystallization chamber determined by the shim size and thickness. Avoid air bubbles during mixing. 
	NOTE: The composition of a crystallization buffer varies from one experiment to another. Refer to Table 1 for crystallization conditions.</li>
	<li>Place the second quartz wafer over the mixed solution as the solution starts to spread out (Figure 1G). This second quartz wafer serves as the&#160;exit window of the diffracted X-rays.</li>
	<li>Tap the second quartz wafer lightly on the edge to help spread the oil while pushing air out. Secure the device by screwing a retaining ring into the outer ring (Figure 1H). Use a tightening tool if necessary (Figure 1I). Be mindful that over-tightening may cause delicate quartz wafers to deform or even crack.</li>
</ol>
3. Device storage and crystallization optimization
<ol>
	<li>Store the assembled devices (Figure 1J) in a box at room temperature or inside an incubator with temperature control. 
	NOTE: Protein crystals may appear in a few hours to days after a crystallization device is assembled. Typical results from on-chip crystallization are shown for several representative protein samples (Figure 2).</li>
	<li>Monitor crystal growth by observing the crystallization device under a microscope. If necessary, optimize crystallization conditions by iterations of sections 1-3.</li>
</ol>
4. Calibration
NOTE: The programs and commands mentioned in the sections below are run in inSituX software.
<ol>
	<li>Install a thin crystal of doped yttrium aluminum garnet on the chip holder (Figure 3). Install the beam stop. Take X-ray fluorescence images of the direct beam by running the program: 
	burnmark.py &#60;device&#62;.param 
	where &#60;device&#62; is a user-selected name for the crystallization device. &#60;device&#62;.param is a filename that contains device-specific control parameters. The default values will be gradually replaced by specific values along the protocol. A sample &#60;device&#62;.param file is shown in Supplementary File 1.</li>
	<li>Find the precise position of the direct X-ray beam by running the beam profile fitting program: 
	beam.py &#60;burn image&#62; -d &#60;device&#62; 
	where &#60;burn image&#62; is the filename of the X-ray fluorescence image (Figure 4). 
	NOTE: This program calculates the precise direct beam positions as well as the beam size. The beam position marks the translocation destination for all crystals from the same device. The beam size is also used for target planning.</li>
</ol>
5. Optical scanning
<ol>
	<li>Place a crystallization device in the chip holder and secure the device using a thumbscrew (Figure 3A).</li>
	<li>Mount the chip holder onto the translation stage of the diffractometer through a kinematic mechanism (Figure 3B).</li>
	<li>Install a proper light source for taking micrographs from the optical window of the device. White light, IR light, or other light of choice can be used depending on the light sensitivity of the protein sample as well as the purpose of the experiment.</li>
	<li>Run the scan program: 
	scan.py &#60;device&#62;.param 
	This program captures a set of micrographs that are automatically transferred to specified user computers.</li>
	<li>Run the tiling program on a user computer: 
	tile.py &#60;device&#62; -x &#60;x&#62; -y &#60;y&#62; 
	where &#60;x&#62; and &#60;y&#62; are the initial values for column and row displacements of micrographs, respectively. This program stitches all micrographs into a montage of 1-3 &#956;m/pixel resolution (Figure 5). 
	NOTE: Steps 5.4 and 5.5 usually take a few minutes. The total number of micrographs ranges from several tens to hundreds depending on the scan area and magnification.</li>
	<li>Run the crystal-finding program: 
	findX.py &#60;montage&#62; -c &#60;length&#62; &#60;width&#62; -w &#60;wedge&#62; -x &#60;beam size&#62; 
	where &#60;montage&#62; is the tiled image. This program performs crystal recognition and shot planning. &#60;length&#62; and &#60;width&#62; indicate the crystal size to be found. If the user wishes to avoid smaller crystals, &#60;width&#62; can be used as a cutoff by setting a number larger than the sizes of unwanted small crystals. &#60;wedge&#62; is an angular value that sets the tolerance for crystals of irregular shape. &#60;beam size&#62; refers to the direct beam size obtained from the profile fitting above (step 4.2; Figure 4). Also, a nominal value can be set by users to further space out targeted shots. These key parameters enable specific crystal selection and target planning (Figure 6).</li>
</ol>
6. X-ray diffraction
<ol>
	<li>Remove the light source and install the beam stop. Set an appropriate detector distance. Follow the beamline safety protocol to search the X-ray hutch. Open the X-ray shutter and laser shutter if applicable.</li>
	<li>Run the data collection program for serial diffraction: 
	collect.py &#60;device&#62;.param -l &#60;light duration&#62; 
	This command triggers data collection in which all planned shots are visited one after another according to a preprogrammed sequence. Each targeted crystal is translocated to the beam position (step 4.2). At each stop, X-ray exposure is taken with or without a laser illumination at a scheduled time delay. Movie 1 shows an automated data collection sequence operated at a frequency of 1 Hz. Routinely tens to hundreds of diffraction images are collected from a single crystallization device (Movie 2). 
	NOTE: Section 4 calibration and section 5 optical scan are self-contained in the inSituX platform, therefore, completely transferable to another beamline. Section 6 X-ray diffraction has to involve some detail in beamline operation.</li>
</ol>

<ol><li>Brändén, G., Neutze, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+and+challenges+in+time-resolved+macromolecular+crystallography%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Advances and challenges in time-resolved macromolecular crystallography.</a> Science. 373, (2021).</li>
<li>Fischer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macromolecular+room+temperature+crystallography%5BTitle%5D)+AND+%22Quarterly+Reviews+of+Biophysics%22%5BJournal%5D)">Macromolecular room temperature crystallography.</a> Quarterly Reviews of Biophysics. 54, (2021).</li>
<li>Schaffer, J. E., Kukshal, V., Miller, J. J., Kitainda, V., Jez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beyond+X-rays%3A+an+overview+of+emerging+structural+biology+methods%5BTitle%5D)+AND+%22Emerging+Topics+in+Life+Sciences%22%5BJournal%5D)">Beyond X-rays: an overview of emerging structural biology methods.</a> Emerging Topics in Life Sciences. 5 (2), 221-230 (2021).</li>
<li>Nogales, E., Scheres, S. H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryo-EM%3A+A+unique+tool+for+the+visualization+of+macromolecular+complexity%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Cryo-EM: A unique tool for the visualization of macromolecular complexity.</a> Molecular Cell. 58, 677-689 (2015).</li>
<li>Chapman, H. N., Caleman, C., Timneanu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diffraction+before+destruction%5BTitle%5D)+AND+%22Philosophical+Transactions+of+the+Royal+Society+B+Biological+Sciences%22%5BJournal%5D)">Diffraction before destruction.</a> Philosophical Transactions of the Royal Society B Biological Sciences. 369, 20130313(2014).</li>
<li>Kisselman, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((X-CHIP%3A+an+integrated+platform+for+high-throughput+protein+crystallization+and+on-the-chip+X-ray+diffraction+data+collection%5BTitle%5D)+AND+%22Acta+Crystallographica+Section+D+Biological+Crystallography%22%5BJournal%5D)">X-CHIP: an integrated platform for high-throughput protein crystallization and on-the-chip X-ray diffraction data collection.</a> Acta Crystallographica Section D Biological Crystallography. 67 (6), 533-539 (2011).</li>
<li>Liang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+combined+crystallization+plate+for+high-throughput+crystal+screening+and+in+situ+data+collection+at+a+crystallography+beamline%5BTitle%5D)+AND+%22Acta+Crystallographica+Section+F+Structural+Biology+Communications%22%5BJournal%5D)">Novel combined crystallization plate for high-throughput crystal screening and in situ data collection at a crystallography beamline.</a> Acta Crystallographica Section F Structural Biology Communications. 77, 319-327 (2021).</li>
<li>le Maire, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In-plate+protein+crystallization%2C+in+situ+ligand+soaking+and+X-ray+diffraction%5BTitle%5D)+AND+%22Acta+Crystallographica+Section+D+Biological+Crystallography%22%5BJournal%5D)">In-plate protein crystallization, in situ ligand soaking and X-ray diffraction.</a> Acta Crystallographica Section D Biological Crystallography. 67, 747-755 (2011).</li>
<li>Perry, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+serial+Laue+diffraction+on+a+microfluidic+crystallization+device%5BTitle%5D)+AND+%22Journal+of+Applied+Crystallography%22%5BJournal%5D)">In situ serial Laue diffraction on a microfluidic crystallization device.</a> Journal of Applied Crystallography. 47, 1975-1982 (2014).</li>
<li>Ren, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crystal-on-crystal+chips+for+in+situ+serial+diffraction+at+room+temperature%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">Crystal-on-crystal chips for in situ serial diffraction at room temperature.</a> Lab on a Chip. 18, 2246-2256 (2018).</li>
<li>Ren, Z. Single crystal quartz chips for protein crystallization and X-ray diffraction data collection and related methods. US patent. , 9632042 (2017).</li>
<li>Ren, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+automated+platform+for+in+situ+serial+crystallography+at+room+temperature%5BTitle%5D)+AND+%22IUCrJ%22%5BJournal%5D)">An automated platform for in situ serial crystallography at room temperature.</a> IUCrJ. 7, 1009-1018 (2020).</li>
<li>Croes, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+for+solving+traveling+salesman+problems%5BTitle%5D)+AND+%22Operations+Research%22%5BJournal%5D)">A method for solving traveling salesman problems.</a> Operations Research. 6, 791-812 (1958).</li>
<li>Bandara, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crystal+structure+of+a+far-red-sensing+cyanobacteriochrome+reveals+an+atypical+bilin+conformation+and+spectral+tuning+mechanism%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Crystal structure of a far-red-sensing cyanobacteriochrome reveals an atypical bilin conformation and spectral tuning mechanism.</a> Proceedings of the National Academy of Sciences of the United States of America. 118, 2025094118(2021).</li>
<li>Shin, H., Ren, Z., Zeng, X., Bandara, S., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+basis+of+molecular+logic+OR+in+a+dual-sensor+histidine+kinase%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Structural basis of molecular logic OR in a dual-sensor histidine kinase.</a> Proceedings of the National Academy of Sciences of the United States of America. 116, 19973-19982 (2019).</li>
<li>Yang, X., Ren, Z., Kuk, J., Moffat, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temperature-scan+cryocrystallography+reveals+reaction+intermediates+in+bacteriophytochrome%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Temperature-scan cryocrystallography reveals reaction intermediates in bacteriophytochrome.</a> Nature. 479, 428-432 (2011).</li>
<li>Zhang, F., Scheerer, P., Oberpichler, I., Lamparter, T., Krauss, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crystal+structure+of+a+prokaryotic+%286-4%29+photolyase+with+an+Fe-S+cluster+and+a+6%2C7-dimethyl-8-ribityllumazine+antenna+chromophore%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Crystal structure of a prokaryotic (6-4) photolyase with an Fe-S cluster and a 6,7-dimethyl-8-ribityllumazine antenna chromophore.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 7217-7222 (2013).</li>
<li>Zeng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+crystallography+reveals+early+signaling+events+in+ultraviolet+photoreceptor+UVR8%5BTitle%5D)+AND+%22Nature+Plants%22%5BJournal%5D)">Dynamic crystallography reveals early signaling events in ultraviolet photoreceptor UVR8.</a> Nature Plants. 1, 14006(2015).</li>
<li>Wang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insights+into+base+selectivity+from+the+1.8+%C3%85+resolution+structure+of+an+RB69+DNA+polymerase+ternary+complex%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Insights into base selectivity from the 1.8 Å resolution structure of an RB69 DNA polymerase ternary complex.</a> Biochemistry. 50, 581-590 (2011).</li>
<li>Rodgrs, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryocrystallography%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">Cryocrystallography.</a> Structure. 2, 1135-1140 (1994).</li>
<li>Zhao, F. -Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+guide+to+sample+delivery+systems+for+serial+crystallography%5BTitle%5D)+AND+%22TheFEBS+Journal%22%5BJournal%5D)">A guide to sample delivery systems for serial crystallography.</a> TheFEBS Journal. 286, 4402-4417 (2019).</li>
</ol>

ZR is the inventor of the crystal-on-crystal chips on the US patent 9632042 granted to Renz Research, Inc.

Protein crystallography in the early years conducted at room temperature experienced tremendous difficulty in battling X-ray radiation damage. Thus, it has been superseded by the more robust cryocrystallography method as synchrotron X-ray sources became readily available20. With the advent of X-ray free-electron lasers, room-temperature protein crystallography has been revived in recent years, with many new developments driven by a desire to observe protein structural dynamics at a physiologically...

Due to the ionizing effects of X-ray radiation, protein crystallography, to a great extent, has been limited to cryogenic conditions in the last three decades. Therefore, the current knowledge of protein motions during its function largely arises from comparisons between static structures observed in different states under cryogenic conditions. However, cryogenic temperatures inevitably hinder the progression of a biochemical reaction or interconversion between different conformational states while protein molecules are at work. To directly observe protein structural dynamics at atomic resolution by crystallography, robust and routine methods are needed for conducting diffraction experiments at room temperature, which calls for technical innovations in sample delivery, data collection, and posterior data analysis. To this end, recent advances in serial crystallography have offered new avenues to capture the molecular images of intermediates and short-lived structural species at room temperature1,2,3. In contrast to the &#34;one-crystal-one-dataset&#34; strategy widely used in conventional cryocrystallography, serial crystallography adopts a data collection strategy similar to that of single-particle cryo-electron microscopy. Specifically, the experimental data in serial crystallography are collected in small fractions from a large number of individual samples, followed by intensive data processing in which data fractions are evaluated and combined into a complete dataset for 3D structure determination4. This &#34;one-crystal-one-shot&#34; strategy effectively eases the X-ray radiation damage to protein crystals at room temperature via a diffraction before destruction strategy5.
Since serial crystallography requires a large number of protein crystals to complete a dataset, it poses major technical challenges for many biological systems where protein samples are limited and/or delicate crystal handling is involved. Another important consideration is how to best preserve crystal integrity in serial diffraction experiments. The in situ diffraction methods address these concerns by allowing protein crystals to diffract directly from where they grow without breaking the seal of the crystallization chamber6,7,8,9. These handling-free methods are naturally compatible with large-scale serial diffraction. We have recently reported the design and implementation of a crystallization device for in situ diffraction based on a crystal-on-crystal concept - protein crystals grown directly on monocrystalline quartz11. This &#34;crystal-on-crystal&#34; device offers several advantages. First, it features an X-ray and light transparent window made of a monocrystalline quartz substrate, which produces little background scattering, hence resulting in excellent signal-to-noise ratios in diffraction images from protein crystals. Second, the single-crystal quartz is an excellent vapor barrier equivalent to glass, thereby providing a stable environment for protein crystallization. In contrast, other crystallization devices using polymer-based substrates are prone to drying due to vapor permeability unless the polymer material has a substantial thickness, which consequently contributes to high background scattering10. Third, this device enables the delivery of a large number of protein crystals to the X-ray beam without any form of crystal manipulation or harvesting, which is critical for preserving crystal integrity11.
To streamline serial X-ray diffraction experiments using the crystal-on-crystal devices, we have developed a diffractometer prototype to facilitate easy switching between the optical scanning and X-ray diffraction modes12. This diffractometer has a small footprint and has been used for serial data collection at two beamlines of the Advanced Photon Source (APS) at Argonne National Laboratory. Specifically, we used BioCARS 14-ID-B for Laue diffraction and LS-CAT 21-ID-D for monochromatic oscillation. This diffractometer hardware is not required if a synchrotron or X-ray free-electron laser beamline is equipped with two key capabilities: (1) motorized sample positioning with a travel range of &#177;12 mm around the X-ray beam in all directions; and (2) an on-axis digital camera for crystal viewing under light illumination that is safe to protein crystals under study. The monocrystalline quartz device together with a portable diffractometer and the control software for optical scanning, crystal recognition, and automated in situ data collection collectively constitute the inSituX platform for serial crystallography. Although this development is primarily motivated by its dynamic crystallography applications using a polychromatic X-ray source, we have demonstrated the potential of this technology to support monochromatic oscillation methods10,12. With automation, this platform offers a high-throughput serial data collection method at room temperature with affordable protein consumption.
In this contribution, we describe in detail how to set up on-chip crystallization in a wet lab and how to perform serial X-ray data collection at a synchrotron beamline using the inSituX platform.
The batch method is used to set up on-chip crystallization under a condition similar to that of the vapor diffusion method obtained for the same protein sample (Table 1). As a starting point, we recommend using precipitant at 1.2-1.5x concentration of that for the vapor diffusion method. If necessary, the batch crystallization condition can be further optimized via fine grid screening. Quartz wafers are not necessary for optimization trials; glass coverslips can be used instead (see below). Partially loaded crystallization devices are recommended to keep optimization trials on a smaller scale. A number of protein samples have been successfully crystallized on such devices using the batch method10 (Table 1).
The device itself consists of the following parts: 1) an outer ring; 2) two quartz wafers; 3) one washer-like shim of plastic or stainless steel; 4) a retaining ring; 5) microscope immersion oil as sealant (Figure 1). The total volume of the crystallization solution loaded on one chip depends on the purpose of the experiment. The capacity of the crystallization chamber can be adjusted by choosing a shim of different thicknesses and/or inner diameter. We routinely set up crystallization devices of 10-20 &#181;L in capacity using shims of 50-100 &#956;m in thickness. A typical device can produce tens to thousands of protein crystals adequate for serial data collection (Figure 2).
When successful, on-chip crystallization will produce tens to hundreds or even thousands of protein crystals on each quartz device ready for X-ray diffraction. At a synchrotron beamline, such a device is mounted on a three-axis translation stage of the diffractometer using a kinematic mechanism. The crystallization window of a mounted device is optically scanned and imaged in tens to hundreds of micrographs. These micrographs are then stitched into a high-resolution montage. For photosensitive crystals, optical scanning can be carried out under infrared (IR) light to avoid unintended photoactivation. A computer vision software has been developed to identify and locate protein crystals randomly distributed on the device. These crystals are then ranked according to their size, shape, and position to inform or guide the data collection strategy in serial crystallography. For example, single or multiple shots can be located on each targeted crystal. Users could plan a single pass or multiple routes through targeted crystals. We have implemented software to compute various traveling routes. For example, the shortest route is computed using algorithms that address the traveling salesman problem13. For pump-probe dynamic crystallographic applications, the timing and duration of laser (pump) and X-ray (probe) shots can be chosen. An automated serial data collection is programmed to translocate each targeted crystal into the X-ray beam one after another.
The key components of the insituX diffractometer include: 1) a device holder; 2) a three-axis translation stage; 3) a light source for optical scanning; 4) an X-ray beam stop; 5) pump lasers if light-sensitive proteins are studied; 6) Raspberry Pi microcomputer equipped with an IR-sensitive camera; 7) control software to synchronize motors, camera, light sources, pump laser, and to interface with beamline controls.

<table><tbody><tr><td>Analysis software</td><td>In-house developed</td><td></td><td></td></tr><tr><td>Cerium doped yttrium aluminum garnet</td><td>MSE Supplies</td><td></td><td>Ce:Y3Al5O12, YAG single crystal substrates</td></tr><tr><td>Chip holder</td><td>In-house developed</td><td></td><td></td></tr><tr><td>Control software</td><td>In-house developed</td><td></td><td></td></tr><tr><td>Immersion oil</td><td>Cargille Laboratories</td><td>16482</td><td>Type A low viscosity 150 cSt</td></tr><tr><td>inSituX platform</td><td>In-house developed</td><td></td><td></td></tr><tr><td>IR light source</td><td>Thorlabs Incorporated</td><td>LED1085L</td><td>LED with a Glass Lens, 1085 nm, 5 mW, TO-18</td></tr><tr><td>Microscope</td><td>Zeiss</td><td></td><td>SteREO Discovery V8</td></tr><tr><td>Outer ring</td><td>In-house developed</td><td></td><td></td></tr><tr><td>Petri dish</td><td>Fisher Scietific</td><td>FB0875713</td><td></td></tr><tr><td>Pipette</td><td>Pipetman</td><td>F167380</td><td>P10</td></tr><tr><td>Pump lasers</td><td>Thorlabs Incorporated</td><td>LD785-SE400</td><td>785 nm, 400 mW, &amp;Oslash;9 mm, E Pin Code, Laser Diode</td></tr><tr><td>Raspberry Pi</td><td>Raspberry Pi Fundation</td><td></td><td></td></tr><tr><td>Retaining ring</td><td>Thorlabs Incorporated</td><td>SM1RR</td><td>SM1 retaining ring for &amp;Oslash;1&quot; lens tubes and mounts</td></tr><tr><td>Seedless quartz crystal</td><td>University Wafers, Inc.</td><td>U01-W2-L-190514</td><td>25.4 mm diameter Z-cut 0.05 mm thickness double side polish 8 mm on -X</td></tr><tr><td>Shim</td><td>In-house developed</td><td></td><td></td></tr><tr><td>X-ray beam stop</td><td>In-house developed</td><td></td><td></td></tr></tbody></table>

on-chip crystallization and large-scale serial diffraction at room temperature

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

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

Several representative datasets have been published In the past few years10,12 along with the crystallographic results and scientific findings from a diverse range of protein samples, including photoreceptor proteins and enzymes, for example, a plant UV-B photoreceptor UVR8, a light-driven DNA repair photolyase PhrB10, a novel far-red-light sensing protein from a multi-domain sensory histidine kinase14, ligand/light...

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

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

on-chip-crystallization-large-scale-serial-diffraction-at-room

Research

JoVE Journal

Biochemistry

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

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

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

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

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.

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

We present a protocol for the crystallization of proteins using the crystallization facility in the Research Complex at Harwell and subsequent in situ X-ray crystallographic data collection from crystals within the plates at Diamond's Versatile Macromolecular Crystallography in situ (VMXi) beamline. We describe sample requirements, crystallization protocols, and data collection guidelines.

Crystallization and In Situ Room Temperature Data Collection Using the Crystallization Facility at Harwell and Beamline VMXi, Diamond Light Source

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.

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

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.

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

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

Microcrystallography of Protein Crystals and In Cellulo Diffraction

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Electron paramagnetic resonance (EPR) spectroscopy is an unambiguous method to measure free radicals. The use of selective spin probes allows for detection of free radicals in different cellular compartments. We present a practical, efficient method to collect biological samples that facilitate treating, storing, and transferring samples for EPR measurements.

Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Learn protein crystallization on a chip-like device for X-ray diffraction data collection at room temperature. This method enables automated serial data collection from thousands of crystals without physical contact.

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

Explore More Videos