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>
	...

<ol>
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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 ...

Dieses Protokoll demonstriert die Proteinkristallisation auf einem chipähnlichen Gerät in der Röntgenbeugungsdatensammlung. Dieses Gerät wird als Kristall-auf-Kristall bezeichnet, da Proteinkristalle auf einem einzigen Quarzkristall wachsen. Nach erfolgreichem Wachstum von Proteinkristallen auf solchen Geräten werden Tausende von Beugungsbildern von jedem Gerät bei Raumtemperatur gesammelt, ohne die Proteinkristalle zu berühren.Die Röntgenbeugung bei Raumtemperatur ist sehr wichtig für die Untersuchung der Proteinfunktion mit vielen Konformationszuständen. Diese informativen Proteinveränderungen oder Proteinaktionen können eingefroren werden und sind daher in der Kryokristallographie nicht nachweisbar. Mit diesen Geräten können lichtinduzierte Signalprozesse und Redoxänderungen untersucht werden.Nur eine Handvoll Geräte liefert vollständige und redundante Datensätze. Um mit der Gerätevormontage zu beginnen, beschriften Sie den äußeren Ring für die Probenidentifikation. Geben Sie den Projektnamen, die Gerätenummer, die Kristallisationsbedingung und das gewünschte Datum an.Legen Sie dann den beschrifteten Ring kopfüber auf eine saubere Oberfläche und legen Sie einen Quarzwafer in den äußeren Ring. Als nächstes gießen Sie eine kleine Menge Mikroskop-Tauchöl in eine Petrischale und tauchen Sie eine Unterlegscheibe in das Öl, um sicherzustellen, dass beide Seiten der Unterlegscheibe gut beschichtet sind. Entfernen Sie überschüssiges Öl, indem Sie die Unterlegscheibe auf eine saubere Oberfläche tupfen.Legen Sie dann die geölte Unterlegscheibe auf den ersten Quarzwafer. Mischen Sie die Proteinlösung und den Kristallisationspuffer auf dem ersten Quarzwafer mit einer Pipette. Versuchen Sie, Luftblasen beim Mischen zu vermeiden.Das Gesamtvolumen der Kristallisationslösung sollte die maximale Kapazität der Kristallisationskammer, die durch die Größe und Dicke der Unterlegscheibe bestimmt wird, nicht überschreiten. Legen Sie den zweiten Quarzwafer über die Mischlösung. Die Lösung wird sich spontan ausbreiten.Klopfen Sie dann den zweiten Quarzwafer leicht auf den Rand, um das Öl zu verteilen und gleichzeitig Luft herauszudrücken. Sichern Sie das Gerät, indem Sie einen Sicherungsring in den Außenring schrauben. Verwenden Sie bei Bedarf ein Anzugswerkzeug.Vermeiden Sie ein übermäßiges Anziehen, da sich empfindliche Quarzwafer dadurch verformen oder reißen können. Lagern Sie die montierten Geräte in einer Box bei Raumtemperatur oder in einem temperaturgesteuerten Inkubator. Nach einigen Stunden oder Tagen legen Sie das Kristallisationsgerät unter ein Mikroskop und überwachen Sie das Kristallwachstum.Optimieren Sie ggf. die Kristallisationsbedingungen wie im Manuskript beschrieben. Installieren Sie zur Kalibrierung einen dünnen YAG-Kristall auf dem Chiphalter und installieren Sie dann den Strahlanschlag. Öffnen Sie als Nächstes die Insitux-Software und führen Sie das angegebene Programm aus, um Röntgenfluoreszenzbilder des direkten Strahls aufzunehmen, wobei das Gerät ein vom Benutzer ausgewählter Name für das Kristallisationsgerät und das Gerät ist.param ist der Dateiname, der gerätespezifische Steuerungsparameter enthält. Finden Sie dann die genaue Position des direkten Röntgenstrahls, indem Sie das Strahlprofilanpassungsprogramm ausführen, wobei das Brennbild der Dateiname des Röntgenfluoreszenzbildes ist. Für das optische Scannen eine Kristallisationsvorrichtung in den Chiphalter legen und mit einer Rändelschraube befestigen.Montieren Sie dann den Chiphalter über einen kinematischen Mechanismus auf dem Translationstisch des Diffraktometers. Abhängig von der Lichtempfindlichkeit der Proteinprobe und dem Zweck des Experiments installieren Sie eine weiße oder infrarote Lichtquelle, um Mikroaufnahmen aus dem optischen Fenster des Geräts aufzunehmen. Sobald das Setup fertig ist, führen Sie das Scanprogramm aus, indem Sie den angegebenen Befehl für das Scannen in Bewegung an der Strahllinie eingeben.Führen Sie dann das Kachelprogramm auf einem Benutzercomputer aus, wobei x der Anfangswert für die Spalte und y der Anfangswert für Schurkenverschiebungen von Mikrobildern ist. Das Programm fügt alle Mikroaufnahmen in eine Montage von ein bis drei Mikrometern Pixelauflösung. Nachdem Sie die Mikrobilder zusammengefügt haben, geben Sie den angegebenen Befehl ein, um das Kristallsuchprogramm auszuführen.Dieses Programm führt Kristallerkennung und Schussplanung durch und die Schlüsselparameter in diesem Programm ermöglichen eine spezifische Kristallauswahl und Zielplanung. Entfernen Sie die Lichtquelle und positionieren Sie den Strahlstopp. Führen Sie dann das Datenerfassungsprogramm für die serielle Beugung aus.Der vorgeschlagene Befehl löst die Datenerfassung aus, indem die geplanten Aufnahmen in einer vorprogrammierten Reihenfolge besucht werden. Jeder Zielkristall wird in die Strahlposition transloziert. Bei jedem Stopp erfolgt die Röntgenbelichtung entweder mit oder ohne Laserbeleuchtung in einer geplanten Zeitverzögerung.In der Studie wurden die Kristallisationsbedingungen zwischen Dampfdiffusion und einer On-Chip-Charge verglichen. Vier Fallstudien zur On-Chip-Kristallisation und repräsentative Datensätze, die direkt von Quarzgeräten gesammelt wurden, werden hier demonstriert. Die dynamischen Kristallographie-Experimente zeigten lichtinduzierte Veränderungen im fernroten Photorezeptorprotein, indem sie Daten von 4.352 Kristallen im Dunkeln und 8.287 Kristallen nach Lichtbeleuchtung verglichen.Der dunkle Datensatz aus der seriellen In-situ-Laue-Beugung führte zu besser aufgelösten Elektronendichten, was eine zuverlässige Modellbildung eines Bilin-Chromophors in einer All-Z-Vorzeichenkonformation ermöglichte. Die lichtinduzierten Differenzkarten haben konzertierte Bewegungen im zentralen Beta-Blatt gezeigt, was auf die Bedeutung der Pi-Pi-Stapelung zwischen den Pyrrolringen des Chromophors und mehreren aromatischen Resten hindeutet. Diese Plattform heißt Insitux.Der einzigartige Vorteil dieser Plattform ist, dass man keine Kristallmanipulation durchführen muss, sobald die Kristallisation abgeschlossen ist. Es ist notwendig, viele Datensätze unter wechselnden Bedingungen zu sammeln, um die Proteinbewegungen zu erfassen. Und das wird mit Insitux möglich, weil es eine großflächige Datenerfassung von Tausenden von Proteinkristallen bei Raumtemperatur ermöglicht.Mit dieser neuen Fähigkeit, einschließlich der Kristallographie, können die lichtempfindlichen Systeme im Inneren des Geräts ausgelöst und das lichtinerte System untersucht werden, wenn das Kristall-auf-Kristall-Gerät in Ausflüsse umgewandelt wird.

Research

JoVE Journal

Biochemistry

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

Herstellung und Lieferung von Protein Microcrystals in Lipidische Cubic Phase für Serial Femtosekunden Kristallographie

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

Forschung

Biochemie

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

Automatische Protokolle für makromolekulare Kristallisation am 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 ...

Microcrystallography of Protein Crystals and In Cellulo Diffraction

Microcrystallography of Protein Crystals and In Cellulo Diffraction

Mikrokristallographie von Proteinkristallen und<em&gt; In Cellulo</em&gt; Beugung

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

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

Platte-basierte Large-Scale Anbau von Caenorhabditis Elegans: Probenvorbereitung für die Studie von Stoffwechselveränderungen bei 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 ...

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

Verwendung der Elektron-paramagnetische Resonanz in biologischen Proben bei Umgebungstemperatur und 77 K

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

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

Großtechnische Produktion von rekombinanten RNAs auf einem kreisförmigen Gerüst mit einem Viroid-abgeleitete System bei Escherichia coli

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

Etikettenfreier Immunpräzipitations-Massenspektrometrie-Workflow für großangelegtes Nuklear-Interactome-Profiling

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

Kristallisation und strukturelle Bestimmung eines Enzyms: Substratkomplex durch serielle Kristallisation in einem vielseitigen mikrofluidischen Chip

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

Feste serielle Zieldatenerfassung an der 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 ...

Microcrystal Electron Diffraction of Small Molecules

Mikrokristall-Elektronenbeugung kleiner Moleküle

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