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>
	...

<ol>
	<li>Br&#228;nd&#233;n, G., Neutze, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+and+challenges+in+time-resolved+macromolecular+crystallography.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+room+temperature+crystallography.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+X-rays:+an+overview+of+emerging+structural+biology+methods.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-EM:+A+unique+tool+for+the+visualization+of+macromolecular+complexity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffraction+before+destruction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-CHIP:+an+integrated+platform+for+high-throughput+protein+crystallization+and+on-the-chip+X-ray+diffraction+data+collection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+combined+crystallization+plate+for+high-throughput+crystal+screening+and+in+situ+data+collection+at+a+crystallography+beamline.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-plate+protein+crystallization,+in+situ+ligand+soaking+and+X-ray+diffraction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+serial+Laue+diffraction+on+a+microfluidic+crystallization+device.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal-on-crystal+chips+for+in+situ+serial+diffraction+at+room+temperature.">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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+crystal+quartz+chips+for+protein+crystallization+and+X-ray+diffraction+data+collection+and+related+methods.">Single crystal quartz chips for protein crystallization and X-ray diffraction data collection and related methods.</a> US patent. , (2017).</li><li>Ren, Z., 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=An+automated+platform+for+in+situ+serial+crystallography+at+room+temperature.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+solving+traveling+salesman+problems.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+far-red-sensing+cyanobacteriochrome+reveals+an+atypical+bilin+conformation+and+spectral+tuning+mechanism.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+molecular+logic+OR+in+a+dual-sensor+histidine+kinase.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-scan+cryocrystallography+reveals+reaction+intermediates+in+bacteriophytochrome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+prokaryotic+(6-4)+photolyase+with+an+Fe-S+cluster+and+a+6,7-dimethyl-8-ribityllumazine+antenna+chromophore.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+crystallography+reveals+early+signaling+events+in+ultraviolet+photoreceptor+UVR8.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+base+selectivity+from+the+1.8+&#197;+resolution+structure+of+an+RB69+DNA+polymerase+ternary+complex.">Insights into base selectivity from the 1.8 &#197; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryocrystallography.">Cryocrystallography.</a> Structure. 2, 1135-1140 (1994).</li><li>Zhao, F. -. Z., 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+guide+to+sample+delivery+systems+for+serial+crystallography.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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, &#216;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 &#216;1&#34; 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

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...

Watch this Scientific Journal Video about On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature at JoVE.com

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

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 ...

This protocol demonstrate protein crystallization on a chip-like device in x-ray diffraction data recollection. This device is called a crystal-on-crystal because protein crystals grow on a single quartz crystal. Upon successful growth of protein crystals on such devices, thousands of diffraction images are collected from each device at room temperature without even touching the protein crystals.X-ray diffraction at room temperature is very essential for studying protein function involving many conformational states. This informative protein changes or protein actions can be frozen, thus not detectable in cryo-crystallography. This devices can be used to study light-induced signaling processes and redox changes.Only a handful of devices gives complete and redundant datasets. To begin device pre-assembly, label the outer ring for the sample identification. Include the project name, device number, crystallization condition, and date as desired.Then place the labeled ring upside down on a clean surface and place one quartz wafer inside the outer ring. Next, pour a small amount of microscope immersion oil into a Petri dish and dip a shim in the oil ensuring that both sides of the shim are well coated. Remove any excess oil by dabbing the shim on a clean surface.Then place the oiled shim on top of the first quartz wafer. Mix the protein solution and crystallization buffer on the first quartz wafer using a pipette. Try to avoid air bubbles when mixing.The total volume of the crystallization solution should not exceed the maximum capacity of the crystallization chamber determined by the shim size and thickness. Place the second quartz wafer over the mixed solution. The solution will spread out spontaneously.Then 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. If necessary, use a tightening tool.Avoid over-tightening as it may cause delicate quartz wafers to deform or crack. Store the assembled devices in a box at room temperature or inside a temperature-controlled incubator. After a few hours or days, place the crystallization device under a microscope and monitor the crystal growth.If necessary, optimize crystallization conditions as described in the manuscript. For calibration, install a thin YAG crystal on the chip holder, then install the beam stop. Next, open the Insitux software and run the indicated program to take x-ray fluorescence images of the direct beam where the device is a user-selected name for the crystallization device and device.param is the filename that contains device-specific control parameters. Then find the precise position of the direct x-ray beam by running the beam profile fitting program where the burn image is the filename of the x-ray fluorescence image. For optical scanning, place a crystallization device in the chip holder and secure using a thumbscrew.Then mount the chip holder onto the translation stage of the diffractometer through a kinematic mechanism. Depending on the light sensitivity of the protein sample and the purpose of the experiment, install a white or infrared light source to take micrographs from the optical window of the device. Once the setup is ready, run the scan program by entering the indicated command for scanning in motion at the beam line.Then run the tiling program on a user computer where x is the initial value for the column and y is the initial value for rogue displacements of micrographs. The program stitches all micrographs into a montage of one to three micrometer pixel resolution. After stitching the micrographs, enter the indicated command to run the crystal finding program.This program performs crystal recognition and shot planning and the key parameters in this program enables specific crystal selection and target planning. Remove the light source and position the beam stop. Then run the data collection program for serial diffraction.The suggested command triggers data collection by visiting the planned shots in a pre-programmed sequence. Each targeted crystal is translocated to the beam position. At each stop, x-ray exposure is taken either with or without a laser illumination at a scheduled time delay.In the study, the crystallization conditions between vapor diffusion and an on-chip batch were compared. Four case studies of on-chip crystallization and representative datasets collected directly from quartz devices are demonstrated here. The dynamic crystallography experiments revealed light-induced changes in far red photoreceptor protein by comparing data from 4, 352 crystals in the dark and 8, 287 crystals after light illumination.The dark dataset from in situ serial Laue diffraction resulted in better resolved electron densities, enabling confident model building of a bilin chromophore in an all Z sign conformation. The light-induced difference maps have revealed concerted motions in the central beta sheet suggesting the importance of the pi-pi stacking between the pyrrole rings of the chromophore and several aromatic residues. This platform is called Insitux.The unique advantage of this platform is that one doesn't have to do any crystal manipulation once the crystallization is done. It is necessary to collect many datasets under changing conditions to capture the protein motions. And this becomes possible with Insitux because it enables large-scale data collection from thousands of protein crystals at room temperature.With this new capability including crystallography, the light sensitive systems can be triggered inside the device and the light inert system can be studied if the crystal-on-crystal device is converted into outflows.

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

Research

JoVE Journal

Biochemistry

1.7K visualizações.  University of Illinois at Chicago. Este protocolo demonstra a cristalização de proteínas em um dispositivo semelhante a um chip na coleta de dados de difração de raios X. Este dispositivo é chamado de cristal sobre cristal porque os cristais de proteína crescem em um único cristal de quartzo. Após o crescimento bem-sucedido de cristais de proteína em tais dispositivos, milhares de imagens de difração são coletadas de cada dispositivo à temperatura ambiente sem sequer tocar nos cristais de proteína.A difra...