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>
	<li>Tari, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+structural+biology+in+drug+discovery.">The utility of structural biology in drug discovery.</a> Methods Mol Bio (Clifton, N.J). 841, 1-27 (2012).</li><li>Gati, C., 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=Serial+crystallography+on+in+vivo+grown+microcrystals+using+synchrotron+radiation.">Serial crystallography on in vivo grown microcrystals using synchrotron radiation.</a> IUCrJ. 1 (2), (2014).</li><li>Redecke, 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=Natively+inhibited+Trypanosoma+brucei+cathepsin+B+structure+determined+by+using+an+X-ray+laser.">Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser.</a> Science. 339 (2013), 227-230 (2013).</li><li>Shi, D., Nannenga, B. L., Iadanza, M. G., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+electron+crystallography+of+protein+microcrystals.">Three-dimensional electron crystallography of protein microcrystals.</a> eLife. 2, e01345 (2013).</li><li>Evans, G., Axford, D., Waterman, D., Owen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+microcrystallography.">Macromolecular microcrystallography.</a> Crystallog. Rev. 17 (2), 105-142 (2011).</li><li>Smith, J. L., Fischetti, R. 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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).

<table border="0" cellpadding="0" cellspacing="0" width="629">
	<colgroup>
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		<col />
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	</colgroup>
	<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>
		</tr>
		<tr height="42">
			<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.

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Biochemistry

8.9K visualizações.  Monash University. 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...