Name: on-chip crystallization and large-scale serial diffraction at room temperature
Uploaded: 2022-03-11T12:30:00
Description: Watch this Scientific Journal Video about On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature at JoVE.com

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>
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<ol>
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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>

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.

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Research

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Biochemistry

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 Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

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

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

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

Microcrystallography of Protein Crystals and In Cellulo Diffraction

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

Microcrystallography of Protein Crystals and In Cellulo Diffraction

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

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

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

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

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

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.

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

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

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments.
In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD,&#160;allows for the differentiation of extracellular from cytosolic superoxide. The mitochondrial 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido] piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide).
Spin probes and EPR spectroscopy can also be applied to in vivo models. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 &#176;C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 &#176;C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.

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.

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of ...

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

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

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

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.

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

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

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

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

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

Fixed Target Serial Data Collection at Diamond Light Source

Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is presented with detailed step-by-step instructions, figures, and videos for smooth data collection.

We present a comprehensive guide to fixed target sample preparation, data collection, and data processing for serial synchrotron crystallography at Diamond beamline I24.

Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is ...

Microcrystal Electron Diffraction of Small Molecules

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.

Here, we describe the procedures developed in our laboratory for preparing powders of small molecule crystals for microcrystal electron diffraction (MicroED) experiments.

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been ...