Name: microcrystallography of protein crystals and in cellulo diffraction
Uploaded: 2017-07-21T14:00:00
Description: Watch this Scientific Journal Video about Microcrystallography of Protein Crystals and In Cellulo Diffraction at JoVE.com

The authors would like to acknowledge Chan-Sien Lay for providing pictures of purified microcrystals, Daniel Eriksson and Tom Caradoc-Davies for support at the MX2 beamline of the Australian Synchrotron, and Kathryn Flanagan and Andrew Fryga from the FlowCore facility at Monash University for their invaluable assistance.

Note: In vivo crystallization has been reported in many organisms including in bacteria, yeast, plants, insects and mammals (reviewed in reference 21). Crystallization of recombinant proteins has also been achieved in the laboratory using transient transfection of mammalian cells and baculovirus infection of insect cells. The following protocol has been developed using the Bombyx mori cypovirus 1 (BmCPV1) polyhedrin gene cloned in a recombinant baculovirus under the baculovirus p...

<ol>
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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=In+vivo+crystallography+at+X-ray+free-electron+lasers:+the+next+generation+of+structural+biology.">In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology.</a> Phil Trans R Soc B. 369 (1647), 20130497 (2014).</li><li>Duszenko, 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=In+vivo+protein+crystallization+in+combination+with+highly+brilliant+radiation+sources+offers+novel+opportunities+for+the+structural+analysis+of+post-translationally+modified+eukaryotic+proteins.">In vivo protein crystallization in combination with highly brilliant radiation sources offers novel opportunities for the structural analysis of post-translationally modified eukaryotic proteins.</a> Acta Cryst. 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This protocol provides two approaches to analyze microcrystals with the aim of facilitating the analysis of very small crystals that would have been overlooked in the past.
Critical steps for microcrystal purification 
The presented protocol has been optimized using Bombyx mori CPV1 polyhedrin expressed in Sf9 cells as a model system. However, in vivo microcrystals display a great variability in mechanical resistance. For instance, cathepsin B needle-lik...

The use of X-ray crystallography for the determination of high-resolution structures of biological macromolecules has experienced a steady progression over the last two decades. The growing uptake of X-ray crystallography by non-expert researchers exemplifies the democratization of this approach in many fields of life sciences1.
Historically, crystals with dimensions below ~10 &#181;m have been considered as challenging, if not unusable, for structure determination. The increasing availability of dedicated microfocus beamlinesat synchrotron radiation sources worldwide and technological advances, such as the development of tools to manipulate microcrystals, have removed much of these barriers that stymied the wide use of X-ray microcrystallography. Advances in serial X-ray microcrystallography2,3 and micro electron diffraction4 have shown that the use of micro- and nanocrystals for structure determination is not only feasible but also sometimes preferable to the use of large crystals5,6,7.
These advances were first applied to the study of peptides8 and natural crystals produced by insect viruses9,10. They are now used for a diverse range of biological macromolecules including the most difficult systems such as membrane proteins and large complexes11. To facilitate the analysis of these microcrystals, they have been analyzed in meso, particularly membrane proteins12 and in microfluidics chips13.
The availability of these novel microcrystallography methodologies has raised the possibility of using in vivo crystallization as a new route for structural biology14,15,16 offering an alternative to classical in vitro crystallogenesis. Unfortunately, even when in vivo crystals can be produced, several obstacles remain such as the degradation or loss of ligands during the purification from cells, difficulty in the manipulation and visualization of the crystals at the synchrotron beamline and tedious X-ray diffraction experiments. As an alternative crystals have also been analyzed directly within the cell without any purification step17,18,19. A comparative analysis suggests that such in cellulo approaches may be more efficient than the analysis of purified crystals and yield data of higher resolution20.
This protocol is intended to assist researchers new to protein microcrystallography. It provides methodologies focusing on sample preparation and manipulation for X-ray diffraction experiments at a synchrotron beamline. Two options are proposed using isolated crystals for classical microcrystallography or crystal-containing cells sorted by flow cytometry for in cellulo analysis (Figure 1).

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sf9 cells</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SF900-SFM insect medium</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1L cell culture flask</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Shaking incubator for insect cell culture</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50mL conical tubes</td>
			<td style="width:127px;">Falcon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge with swing buckets for 50mL tubes</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sonicator equiped with a 19mm probe</td>
			<td>MSE Soniprep 150&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Glass slides</td>
			<td style="width:127px;">Hampton Research</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemacytometer</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Propidium iodide</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD Influx cell sorter&#160;</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hampton Heavy atom screens</td>
			<td style="width:127px;">Hampton Research</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Micromesh</td>
			<td style="width:127px;">Mitigen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">700/25 meshes offer a larger surface. Indexed meshes can be purchased for systematic studies.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Paper wick</td>
			<td style="width:127px;">Mitigen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">The size of the paper wick can be varied for optimal flow. This will largely depend on the nature of the crystals and cryoprotectant used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethylene glycol</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan blue</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:216px;">MX2 microfocus beamline</td>
			<td style="width:127px;">Australian Synchrotron</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">A list of available microfocus beamlines can be found in Boudes et al. (2014) Reflections on the Many Facets of Protein 
			Microcrystallography. Australian Journal of Chemistry 67 (12), 1793&#8211;1806, 
			doi:10.1071/CH14455.</td>
		</tr>
	</tbody>
</table>

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.

An overview of both alternative methods for structure determination using in vivo microcrystals is presented (Figure 1). Polyhedra can easily be purified by sonication and centrifugation. Due to their density, they form a layer at the bottom of the tube underneath a layer of debris that can be removed by pipetting (Figure 3a and 3b). The sample is then subjected to several rounds of sonication and washes...

Watch this Scientific Journal Video about Microcrystallography of Protein Crystals and In Cellulo Diffraction at JoVE.com

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.

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 ...

The overall goal of this protocol is to introduce X-ray crystallography methodologies applied to microcrystals and particularly to those produced in cells which are considered in vivo crystals. The main advantage of this approach is that it provides a simple and efficient process to produce, detect, and analyze microcrystals. This method aims at facilitating the use of microcrystals by using crystals grown inside living cells which removes the need for complex purification process and Aprocox sedation trials.In this video we will be focusing on simple preparation and manipulation for X-ray deflection experiments at a synchrotron beamline. We show the steps that differ the most from classical crystallography. In this demonstration we will be using crystals from the vital polyhedron protein grown in success but the method can also be applied to proteins forming crystals in any bacterial, insect, mammalian, or yeast cells.First, incubate 30 milliliters of Sf9 cells over expressing the cypovirus polyhedron protein for three days at 27 degrees celsius on a rotating platform. Transfer 500 microliters of Sf9 cell culture into a micro centrifuge tube using a sterile serological pipette, taking care to ensure that the sterility of the main culture is maintained by using a class II bio safety cabinet and following standard aseptic techniques. Pipette five microliters of cells from the micro centrifuge tube onto a glass slide.Then, gently angle a glass cover slip over the sample with one edge touching the drop and carefully lower the coverslip onto the liquid, avoiding the formation of air bubbles. Next, image the slide with an inverted microscope. Carefully examine the cells, starting at 200x magnification and zooming in at maximal magnification when detecting a potential crystal.Look for sharp edges and changes in refractivity. If crystals are identified, examine the culture daily to monitor crystal growth by repeating the previous steps. For Sf9 cells, harvest by transferring the culture to a centrifuge tube.Place the tube on ice and proceed to the next step as soon as possible. For isolation of crystal-containing cells, pipette four milliliters of infected Sf9 cells in a tube adapted to flow cytometry analysis. Prepare a second tube with an equivalent number of Sf9 cells from a mock infected culture as a control.Next, add propidium iodide to both samples to a final concentration of one microgram per milliliter, just prior to flow cytometric evaluation. Now, apply control sample cells to a standard flow cytometer capable of cell sorting according to FSC and SSC. Analyze at least 20, 000 events.After discarding dead cells, cell clumps, and debris from the analysis, generate the FSC to SSC plot. Following this, analyze at least 20, 000 events of the crystal-containing sample. Compare the FSC to SSC plot with the control and look for the appearance of a distinct population corresponding to crystal-containing cells.Define the gates around the crystal-containing cell population, usually appearing at higher SSC or FSC. Using this gating, sort about 150, 000 to 200, 000 of the crystal-containing cells into a 1.5 milliliter micro centrifuge tube prefilled with 50 microliters of PBS buffer. Image the sorted samples by light microscopy as previously described to confirm which population is associated with crystal-containing cells.At this stage cells can be incubating with heavy-atom solutions if experimental phasing is required. This approach follows screening of various compounds and incubation times as described in the text and in other reviews. Set up the tools for mounting the sample by first attaching a micro mesh mounted on a pin to the end of a magnetic wand.Place the wand sideways into the cleft of a dialysis float buoy. Then, pick up a paper wick with a pair of tweezers and lock them. Double check that the set up is complete before proceeding.Now, mix the cells with an equal volume of 0.4%trypan blue solution to a final concentration of 0.2%After resuspending the cells, pipette 0.5 microliters of the sorted cells onto the micro mesh. After allowing the cells to deposit on the micro mesh surface, remove most of the excess liquid by blotting with a paper wick. If the micro mesh is not sufficiently blotted, the cells will be at different depths within the excess liquid, which will complicate their alignment to the X-ray beam at the synchrotron.In addition, the excess liquid will increase the background X-ray scattering. If the micro mesh is blotted in excess, the liquid will be completely removed and the sample will not be held in the grid openings. With optimal blotting, only a thin layer of liquid remains on the micro mesh, protecting the sample and forcing it into the grid openings.Some crystals will require the use of a cryoprotectant. This is not required in our case, but the procedure would be the same as for applying the sample and then blotting. Ensure that only a thin layer of liquid is remaining.Immediately flash-cool the micro mesh in liquid nitrogen and transfer it to a robot puck for transport to a synchrotron microfocus beamline. Due to the small size of in vivo crystals, collect afraction data on a microfocus crystallography beamline. First, mount the micro mesh on the goniometer.Now, start by centering the micro mesh with the beam path using the face-on and side-on orientations. With the micro mesh face-on, fine tune the alignment by aligning the center of one of the horizontal lanes of the micro mesh. Collect data along this horizontal lane with only minor readjustments to the alignment.Once all the crystals of the lane have been tested, proceed to the next lane and repeat the alignment procedure. Make sure that the lanes that have been processed are clearly identified so that crystals are not missed or shot twice using the trypan blue color change as a guide. An overview of two methods for structure determination using in vivo microcrystals is presented.The top row describes the classical approach involving purification of crystals from cells while the bottom row shows the in cellulo crystallography approach that keeps the crystals in the host cells for data collection. For the in cellulo approach, the flow cytometry profile of non-infected cells is compared with a profile of crystal-containing cells and used to determine which cell population should be selected to sort crystal-containing cells away from the other cells. In this example, cells containing polyhedra have a higher side scattering than non-infected cells.Cells stained with trypan blue can be easily visualized using cameras available at most synchrotron beamlines. While purified crystals are laborious to identify and align with the X-ray beam due to their small size, when collecting data it is best to proceed in a grid pattern in order to minimize centering procedures and avoid exposing the same cell or crystal twice or missing cells and crystals. Once mastered, this flow through takes about half a day from harvest of the crystal-containing cells to data collection.This procedure is compatible with heavy-atom soaks for experimental phasing. In the case of the polyhedron, the normal structure was obtained in eight days from the start of the protein expression. Don't forget that working with liquid nitrogen can be extremely hazardous and that precautions against suffocation and burns must be taken.

microcrystallography-of-protein-crystals-and-in-cellulo-diffraction

Research

JoVE Journal

Biochemistry

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

This article presents a method to generate protein crystals derivatized with I3C (5-amino-2,4,6-triiodoisophthalic acid) using microseeding to generate new crystallization conditions in sparse matrix screens. The trays can be set up using liquid dispensing robots or by hand.

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial crystallography experiments at both synchrotrons and XFELs. The method is based upon an understanding of the protein crystal phase diagram, and how that knowledge can be utilized. The method is divided into three stages: (1) optimizing crystal morphology, (2) transitioning to batch, and (3) scaling. Stage 1 involves finding well diffracting, single crystals, hopefully but not necessarily, presenting in a cube-like morphology. In Stage 2, the Stage 1 condition is optimized by crystal growth time. This strategy can transform crystals grown by vapor diffusion to batch. Once crystal growth can occur within approximately 24 h, a morphogram of the protein and precipitant mixture can be plotted and used as the basis for a scaling strategy (Stage 3). When crystals can be grown in batch, scaling can be attempted, and the crystal size and concentration optimized as the volume is increased. Endothiapepsin has been used as a demonstration protein for this protocol. Some of the decisions presented are specific to endothiapepsin. However, it is hoped that the way they have been applied will inspire a way of thinking about this procedure that others can adapt to their own projects.

The aim of this article is to give the viewer a solid understanding of how to transform their small-volume, vapor-diffusion protocol, for growing large, single protein crystals, into a large-volume batch micro-crystallization method for serial crystallography.

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial ...

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 ...

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.

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 ...