Name: optimizing the growth of endothiapepsin crystals for serial crystallography experiments
Uploaded: 2021-02-04T12:30:00
Duration: 9 min 52 s
Description: Watch this Scientific Journal Video about Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments at JoVE.com

This project has received funding from the European Union&#39;s Horizon 2020 research and innovation program under the Marie Sk&#322;odowska-Curie grant agreement No 701647.&#160;Many thanks for the assistance and support of beamline scientists at the Swiss Light Source beamline X10SA-PXII.

The authors have no conflicts of interest to disclose.

The method presented shows how to optimize the crystallization of endothiapepsin from large crystals (&#8805;&#160;100 &#181;m longest dimension), grown in sparse-matrix 96-well screens, to micro-crystals, grown in centrifuge tubes (300 &#181;L volume) via batch. The idea behind the protocol is that the steps taken to optimize endothiapepsin could also be used for other proteins. Ultimately, answering the problem of creating large volumes (&#62;100 &#181;L) of micro-crystals (10-20 &#181;m) for serial crystallography experiments at XFELs and synchrotrons.
The protocol divides the task of large volume micro-crystallization into three steps: (1)&#160;Optimizing crystal morphology, (2)&#160;Transitioning to batch, and (3)&#160;Scaling. In Step 1, the range of crystal forms that a protein can create should be explored in vapor diffusion plates. Conditions that give rise to single, box-like crystals that diffract to the required resolution should be the goal. In Step 2, selected conditions can then be transformed from vapor diffusion into batch. Here, the optimization criterion is crystal growth time and to find conditions that give rise to protein crystals within 24 h. A morphogram can also be plotted giving the experimenter an idea of the location of the solubility line and nucleation zone boundaries. This morphogram is of great utility in Step 3, Scaling. The morphogram will give an indication of the whether nucleation alone can increase Xn and drive down Xs. As the volume of the experiment is increased, Xn and Xs can be continually assessed as the key criteria of scaling success.
In the case of Endothiapepsin, Step 1 unearthed what potentially was a previously unknown crystal morphology for endothiapepsin. This morphology had the same spacegroup as those previously reported but, importantly for serial crystallography, a more box-like shape. Single crystals also seemed to grow from single nucleation points, unlike the fans created from other conditions (Figure 4). For the selected condition, Step 2 was already partially satisfied as crystal growth occurred in &#60; 24 h. The morphogram indicated that both a straight or seeded-batch protocol might be successful when scaling in Step 3. Initial scaling in straight batch, created a condition that produced crystals with an Xn and Xs range of 3.6 &#177; 1.2 x 106 crystals&#183;mL-1 and 42 &#177; 4.1 &#181;m, respectively. These crystals, although acceptable for some serial crystallography experiments, were deemed too large. So additional optimizations were performed. The final protocol produced crystals with a concentration and size range of 3.1 x 106 crystals&#183;mL-1 and 15 &#177; 3.9 &#181;m, respectively. This was more than ideal for the planned experiments.
The method focuses on the transformation of &#39;soluble&#39; protein crystals grown in vapor diffusion plates to batch. The reason for this focus is that the vast majority of soluble protein crystals are grown via vapor diffusion26. However, the concepts presented could also be applied to soluble protein crystals grown using other methods, such as micro-batch. The concepts may also be applicable to membrane protein crystals grown in LCP; as this too is a batch crystallization process.
A key aspect of the protocol is the process of transforming the conditions of crystals grown in vapor diffusion plates such that they can be grown in batch. For this transformation, the method uses the criterion proposed by Beale et al. (2019)26. Crystals grown via a batch process, even in vapor diffusion plates, will form rapidly (&#60; 24 h). This criterion is an approximation based on the speed of vapor diffusion drop equilibration, and is most true for PEG-based precipitant conditions. However, crystallization conditions will contain a wide variety of compounds that will influence the equilibration time. The equilibration of salt-based crystallization conditions, e.g. highly concentrated ammonium chloride, can happen in 1-2 days. Therefore, the 24 h criterion may not be true for salt-based conditions. Salt-based conditions also can have more complex phase diagrams26, 30 that may not conform to the archetype presented in this protocol. A reduction in the time criterion for salt-based conditions to 12 or 6 h may be necessary if scaling into larger volumes proves impossible.
Another limitation of this method is its apparent complexity. The protocol that was followed to optimize the micro-crystallization of endothiapepsin actually changed the original condition from the sparse-matrix screen relatively little. The first hit observed in the PACT screen was 0.1 HEPES pH 7.0, 0.2 M MgCl2, and 20% (w/v) PEG 6,000. The final scaled crystallization buffer was 0.1 Tris-HCl pH 7.0, 0.15 M MgCl2, and 40% (w/v) PEG 6,000. It is also very possible that the change in buffer from HEPES to Tris-HCl, and MgCl2 concentration, contributed little to the success of the process. Leaving the increase in PEG 6,000 concentration the sole optimization, and one that could have been achieved quite simply.
This assessment, however, is also too simplistic. It not only discounts the problems encountered during scaling (i.e., the use of seeds and quenching), but also the fact that just because this protein proved straightforward, there is no guarantee the next will also prove to be. The steps advised in the protocol, were devised because optimizing the scaling of protein crystallization volumes can be very protein expensive. Over the seven endothiapepsin scaling trials that are shown, 100 mg of protein were consumed. Admittedly some of these steps were performed to show their consequences in the light of this protocol. Even so, 100 mg of a protein, plus potentially a further 50 mg for protein consumed during an experiment (Table 1), can be a significant investment in either time or money.
Fortunately, it is not clear that this mass of required sample is ubiquitous across all proteins. Endothiapepsin was highly soluble, and therefore required a large protein concentration to reach supersaturation. In others (currently under optimization), supersaturation can be reached at 10 or even 5 mg/mL. Such variables are protein specific and need to be embraced when they appear.
Other limitations of the method include its reliance on complex equipment such as liquid handling robots for screen and plate creation, and imagers to automatically image plates when required. Alternative routines have been offered to limit the need of some of these pieces of equipment, but the protocol will be more time consuming to follow without them. The protocol also suggests testing the diffraction of optimized crystals. For crystallographers without regular access to a synchrotron, these tests could prove challenging. Controls at every step may not be necessary, but these tests are strongly recommended once a hit has been identified, and pre-and post-scaling. Non-diffracting crystals at an XFEL are, unfortunately, not an uncommon occurrence. Given this, it is better to err on the side of caution regarding assumptions about crystal diffraction.
Ultimately, this protocol and results presented here will offer a guide, ideas, and an example to those struggling with producing samples for serial crystallography experiments. Hopefully, as serial crystallography is further developed, the sample demands of the technique will be reduced such that the need for protocols like this will be reduced. However, even in this event, the strategies presented here will still be useful to those wishing to explore the crystallization space of their protein.

Room temperature (RT) macromolecular crystallography is&#160;popular again within the structural biology community. The development of X-ray Free Electron Laser (XFEL) light sources has spurred the development of RT sample delivery approaches1,2,3,4, and these methods have now been applied to synchrotrons5,6,7,8. Not only do RT methods open up the possibility of pump-probe experimental strageties9,10,11,12, but there is also mounting evidence that they promote alternative conformational states within proteins13,14,15,16,17.
However, the principal reason why cryo-methods gained traction over RT approaches in the late 1990s was the slowing of radiation damage by sub-zero crystal temperatures18. Cryo-methods19 began to allow for the collection of a complete dataset from a single protein crystal. Modern RT methods at XFELs and synchrotrons solved the problem of single-crystal radiation damage by the development of rapid (&#62; 100 Hz) crystal delivery strategies1,2,3,4. These methods allow for the collection of a complete dataset from thousands of individually exposed crystals. These RT delivery approaches therefore require the production of large quantities of solutions containing homogenous micro-crystals (&#62; 100 &#181;L of &#60; 50 &#181;m crystals). However, since cryo-methods tend to only require single crystals, methods to create such micro-crystalline slurries are currently not ubiquitous across protein crystallography laboratories.
There are examples in the literature of how to do parts of the micro-crystallization optimization procedure for serial crystallography samples. Here, a distinction should be made between membrane and soluble proteins. Protocols to optimize the growth of micro-membrane protein crystals grown in monoolein (or some other lipid), for lipidic cubic phase (LCP), have been well described20,21,22. However, methods for the micro-crystallization of soluble proteins, including membrane proteins grown in non-LCP&#160;conditions, are generally lacking. Previous studies have focused on specific parts of the process, such as micro-crystal screening23,24, enhancing nucleation24, and scaling using free-interface diffusion25, but not a complete method.
However, a method was recently described26 that attempts to offer a complete protocol. Like many aspects of protein crystallography, it is not new. Many of the ideas proposed were already described by Rayment (2002)27. The method aims to show crystallographers how to perform the conversion from a single, crystal grown using vapor diffusion, to a batch methodology to grow thousands of micro-crystals. The method focuses on vapor diffusion as a common starting point, as 95% of all Protein Data Bank (PDB) depositions come from crystals grown in vapor diffusion plates26. Vapor diffusion is, however, not the ideal method for micro-crystallization26, so a methodology is described to convert vapor diffusion to batch crystallization. Once crystals can be grown in batch, scaling routes to larger volumes become more practicable. Given the vagaries of protein crystallization, the authors would stress that this method is not failsafe. However, the protocol should, at least, provide an insight into the &#39;crystallization space&#39; of a protein.
This method relies on the protein crystallization phase diagram and how an understanding of that diagram can act as a guide during micro-crystallization optimization. A protein phase diagram is commonly depicted as an x/y plot with precipitant and protein concentrations on the x and y axes, respectively (Figure 1A). From the pure water point (bottom left corner - Figure 1A), the concentration of both protein and precipitant increases until the solubility line is reached. The solubility line marks the point of supersaturation (purple line - Figure 1A). When a protein is supersaturated, the solution becomes thermodynamically unstable and will begin separating into two phases: &#39;protein-rich&#39; and a stable saturated solution. This separation can occur anywhere beyond the solubility line and its kinetics are dependent upon the properties of the protein and the components of the solution.
When the protein and precipitant concentrations are too great, the protein will decompose unstably out of solution and result in amorphous precipitate (pink region - Figure 1A). However, ordered phase separation can occur in the nucleation region [see Garcia-Ruiz (2003)28 for detailed description] and crystal nucleants have the propensity to form (green region - Figure 1A). Nucleation and growth removes protein from the solution and moves the drop into the metastable region where growth can continue until the solubility line is reached [see McPherson and Kuznetsov (2014)29 for detailed discussion]. The diagram is, for the vast majority of crystallization conditions, a gross oversimplification30. Regardless of this however, the diagram is still of great utility for micro-crystallographers as the mapping of the diagram allows for the solubility line and the kinetics of nucleation to be determined.
In terms of creating micro-crystals, the two factors during crystallization that need to be optimized are the number of crystals (Xn) and their mean, longest dimension (Xs). Xn will be proportional to the number of nucleation events (n) (Eq. 1).
<img alt="Equation 1" src="/files/ftp_upload/61896/61896fig01v2.jpg" />&#160; &#160; &#160;Eq. 1
Xs is proportional to the concentration of free protein above the solubility line (Ps) divided by Xn (Eq. 2).
<img alt="Equation 2" src="/files/ftp_upload/61896/61896eq02.jpg" />&#160; &#160; &#160;Eq. 2
In a perfect situation, every nucleation event would yield a possible crystal and every one of these crystals, would have equal access to the available protein in solution. Figure 2 is a graphical representation from an ideal scenario of the relationship between Xn and Xs. Practically, the principal control a crystallographer has over Xn and Xs is by influencing the amount of nucleation or by the addition of seed crystals. The micro-crystallographer must judge how to increase Xn such that a suitable crystal concentration and crystal size can both be created.
The majority of crystallization techniques require a &#39;transitionary period&#39; (Figure 1B). For example, in a vapor diffusion experiment, upon mixing the protein and precipitant solutions, the concentrations of each will change as the drop equilibrates with the well solution. One hopes that these changes will gradually transition the drop into the nucleation zone where the propensity for crystallization will increase. As crystals begin to nucleate and grow, the amount of protein in solution will begin to fall, decreasing the probability of further nucleation. The ultimate amount of nucleation will be protein and condition specific, and also dependent upon the depth of penetration of into the nucleation zone. Given the limited nucleation zone penetration of methods that require a transitionary step, the level of nucleation will ultimately be limited to the rate of nucleation at the metastable-nucleation region boundary.
Due to the importance of being able to enhance the level of nucleation for a micro-crystallographer, it is important to move to a batch crystallization methodology. Batch can take greater advantage of the whole nucleation region (Figure 1C). In batch methods, the idea is to mix the protein and precipitant together such that a supersaturated solution is created without the need of any changes in component concentrations. Nucleation should be possible immediately upon mixing. Batch methods therefore allow for the entire nucleation zone to be theoretically reached. Any increase in nucleation kinetics beyond the metastable-nucleation boundary can then be utilized.
If the basal-level of crystal nucleation is not enough to generate a large Xn, micro-seeding methods can be used. In micro-seeding, pre-grown crystals are broken up to create a slurry of crystalline fragments which can act as a scaffold for fresh crystal growth31,32. Micro-seeding has been widely used in serial crystallographic sample preparation as a way to increase Xn without the need of increasing crystal nucleation (Figure 1C).
The transition from vapor diffusion to batch can be visualized on a phase diagram as moving the experimental starting point from either the non-supersaturated or metastable regions to the nucleation zone. This can be done by increasing the protein and/or precipitant concentrations, and/or the ratio of the two within the drop (Figure 1D), and observing which conditions yield crystals appearing rapidly (&#60; 24 h)26. Complete vapor diffusion drop equilibration can take days or weeks33. Therefore, by looking for conditions that show rapidly appearing crystals, batch conditions can be found without having to move to alternative crystallization screening formats such as micro-batch34,35,36,37.
Once the nucleation zone has been found, a batch condition has been found and a morphogram - here, a rough phase diagram - can be created. The morphogram is of great utility when contemplating whether to use a seeded-batch or straight batch protocol. By plotting the Xn as a function of the protein and precipitant concentration, an assessment of the nucleation kinetics can be made26. If Xn remains low across the whole nucleation region, seeded-batch may be required to make Xn large enough to limit crystal growth. This assessment is the first step in the process of scaling to larger volumes (&#62; 100 &#181;L).
This method was designed such that it could be conducted in the majority of crystallization laboratories by using standard vapor diffusion crystallization equipment. Many studies have also been conducted which describe techniques to facilitate many parts of this process, should the equipment be available. These include, but are not limited to, dynamic light scattering (DLS)25,27, non-linear imaging20,24,25, powder diffraction20,24,27, and electron microscopy26 [see Cheng et al. (2020)40 for a nice review].
The aim of this work is to provide a visual demonstration of the method to transition from small volume (&#60; 500 nL) vapor diffusion crystallization to large volume (&#62; 100 &#181;L) batch crystallization. Endothiapepsin from Cryphonectria parasitica has been used as an example system for demonstrating this translation. The type of experiment and sample delivery method that the micro-crystals are required for will influence the ideal Xs output26. For mixing experiments requiring a millisecond time resolution41 or gas-dynamic virtual nozzles42, a final Xs of &#60; 5 &#181;m may be desirable. In this case, the goal was to produce protein crystals that diffract to approximately 1.5 &#197;, for a photon-activated pump-probe experiment, and using a fixed-target delivery approach.
To give an illustration of the sample requirements of such a serial crystallography experiment using endothiapepsin, Table 1 shows the experimental parameters of a hypothetical experiment. The sample information was based upon the protocol described below. Given some conservative estimates on hit rates and data collection requirements, 50 mg is the total sample consumption estimate for the whole experiment.
Figure 3 shows a flow-chart of the complete optimization process from initial small volume vapor diffusion crystallization to large scale batch. For the majority of serial crystallography projects, this protocol will begin at Step 2: &#39;transitioning to batch&#39;, since the target protein will already have been crystallized. However, Step 1 has been included for completeness and to remind readers of its importance. Finding a condition that gives rise to a well diffracting, single, large crystal is the best starting point for micro-crystal optimization. In Step 2, this condition can then be optimized from vapor diffusion to batch, and a morphogram of the nucleation and metastable regions can be plotted. Once this has been done, scaling the batch condition to larger volumes can be performed in Step 3. By the end of the flow-chart, a crystallographer will have created a repeatable, large-volume (&#62; 100 &#181;L), micro-crystallization, batch protocol for endothiapepsin. This method can then be applied to their particular protein of interest.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Swissci 96-well 2-Drop plates</td>
			<td>Molecular Dimensions</td>
			<td>MD11-002</td>
			<td>96-well 2-drop crystallisation plate</td>
		</tr>
		<tr>
			<td>Swissci 96-well 3-Drop plates</td>
			<td>Molecular Dimensions</td>
			<td>MD11-003</td>
			<td>96-well 3-drop crystallisation plate</td>
		</tr>
		<tr>
			<td>mosquito LCP liquid handling robot</td>
			<td>sptlabtech</td>
			<td>mosquito LCP</td>
			<td>Crystallisation robot</td>
		</tr>
		<tr>
			<td>ClearVue Sheets</td>
			<td>Molecular Dimensions</td>
			<td>MD6-015</td>
			<td>96-well crystallization plate seals</td>
		</tr>
		<tr>
			<td>Safe-Tube 1.5 mL</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td>1.5 mL centrifuge tubes</td>
		</tr>
		<tr>
			<td>Scaple</td>
			<td>Swan and Morton</td>
			<td>No. 3 scalple and No. 3 handle</td>
			<td>Scalple for cutting open plate seals</td>
		</tr>
		<tr>
			<td>MS 3 Vortex</td>
			<td>IKA</td>
			<td>3319000</td>
			<td>Vortex for mixing solution and making seed stocks</td>
		</tr>
		<tr>
			<td>24-well XRL Plate</td>
			<td>Molecular Dimensions</td>
			<td>MD3-11</td>
			<td>24-well hanging-drop plates</td>
		</tr>
		<tr>
			<td>Tube revolver/rotator</td>
			<td>Thermo Fischer Scientific</td>
			<td>88881001</td>
			<td>Tube revolver for mixing solution during scaling</td>
		</tr>
		<tr>
			<td>Eppendorf Research plus pipettes</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Range of manual pipettes, 0.5-10, 1-20, 10-100, 100-1000 &#181;L</td>
		</tr>
		<tr>
			<td>Eppendorf pipette tips</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Range of tip sizes for manual pipettes</td>
		</tr>
		<tr>
			<td>Suparen 600</td>
			<td>Prochem AG</td>
			<td>Suparen 600</td>
			<td>Endothiapepsin solution</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>241245-1KG</td>
			<td>Sodium Acetate</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Merck</td>
			<td>8382T014</td>
			<td>Tris</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M2670-1kg</td>
			<td>Magnesium Chloride</td>
		</tr>
		<tr>
			<td>PEG 6,000</td>
			<td>Sigma-Aldrich</td>
			<td>81255-1kg</td>
			<td>PEG 6,000</td>
		</tr>
		<tr>
			<td>Ethelyene glycol</td>
			<td>Sigma-Aldrich</td>
			<td>324558-1L</td>
			<td>Ethelyene glycol for cyro-protecting the crystals</td>
		</tr>
		<tr>
			<td>PACT Premier HT screen</td>
			<td>Molecular Dimensions</td>
			<td>MD1-36</td>
			<td>PACT Premier 96-well crystal screen</td>
		</tr>
		<tr>
			<td>DOW CORNING high vacuum grease</td>
			<td>Molecular Dimensions</td>
			<td>MD6-02</td>
			<td>Grease for sealing 24-well plates</td>
		</tr>
		<tr>
			<td>Hirschmann 22 x 22 mm glaser cover slides</td>
			<td>Hirschmann</td>
			<td>8000104</td>
			<td>Cover slides for sealing 24-well sitting drop plates</td>
		</tr>
		<tr>
			<td>Crystal pins</td>
			<td>PSI</td>
			<td>Manufactured inhouse</td>
			<td>Thin-film supports for micro-crystals.</td>
		</tr>
		<tr>
			<td>1-1.3 mm SiLibeads Type S</td>
			<td>Faust</td>
			<td>6239547</td>
			<td>Glass beads for making mico-seed stocks</td>
		</tr>
		<tr>
			<td>Macbook Pro</td>
			<td>Apple</td>
			<td>Macbook Pro</td>
			<td>Computer for performing data analysis</td>
		</tr>
		<tr>
			<td>CCP4 software suite</td>
			<td>CCP4</td>
			<td></td>
			<td>Diffraction pattern data processing software</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>Microsoft Office</td>
			<td>Plotting tool for phase diagram</td>
		</tr>
		<tr>
			<td>Hausser Scientific Bright-Line counting chamber</td>
			<td>Thermo Fischer Scientific</td>
			<td>02-671-51B</td>
			<td>Tool to calculate crystal concentration</td>
		</tr>
		<tr>
			<td>PACT Premier</td>
			<td>Molecular Dimensions</td>
			<td>MD1-29-ECO</td>
			<td>Sparse-matrix crystallization screen</td>
		</tr>
		<tr>
			<td>Rock Imager</td>
			<td>Formulatrix</td>
			<td>Rock Imager</td>
			<td>Temperature controlled crystal plate storage and imager</td>
		</tr>
		<tr>
			<td>Rock MakerWeb</td>
			<td>Formulatrix</td>
			<td>Rock MakerWeb</td>
			<td>Crystal plate creation and image storage stoftware</td>
		</tr>
		<tr>
			<td>Formulator</td>
			<td>Formulatrix</td>
			<td>Formulator</td>
			<td>96-well crystal screen creation liquid handling robot</td>
		</tr>
		<tr>
			<td>Leica MZ16 Microscope</td>
			<td>Leica</td>
			<td>Leica MZ16</td>
			<td>Light microscope</td>
		</tr>
		<tr>
			<td>LAS V4.6</td>
			<td>Leica</td>
			<td>LAS V4.6</td>
			<td>Software for Leica microscopes</td>
		</tr>
		<tr>
			<td>Spectra/Por 3.5 kDa dialysis tubing</td>
			<td>Spectrumlabs</td>
			<td>Spectra/Por 3 Dialysis Membrane</td>
			<td>3.5 kDa dialysis membrane</td>
		</tr>
		<tr>
			<td>Dialysis tubing closures</td>
			<td>Spectrumlabs</td>
			<td>Spectra/Por 3 Duniversal Closures</td>
			<td>Clips to seal the dialysis tubing ends</td>
		</tr>
		<tr>
			<td>Amicon 10 kDa centrifugal concentrator</td>
			<td>Merck-Millipore</td>
			<td>Amicon Ultra-15 10 kDa centrifugal concentrator</td>
			<td>10 kDa centrifugal filter</td>
		</tr>
		<tr>
			<td>5810 R swing bucket centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R Centrifuge</td>
			<td>Swing bucket centrifuge</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments at JoVE.com

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

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 ...

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