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><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 &amp;micro;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 ...

optimizing-growth-endothiapepsin-crystals-for-serial-crystallography

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

2.2K Views.  Paul Scherrer Institut. 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.

Video: Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

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

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 &#181;m crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

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

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

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 ...

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and IKK&#946;. Upon activation, the IKK complex phosphorylates the I&#954;B molecules leading to NF-&#954;B nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-&#954;B pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray free electron lasers (XFELs). A series of experiments using the light-driven proton pump bacteriorhodopsin have further established these injectors as a preferred option to deliver crystals for time-resolved serial femtosecond crystallography (TR-SFX) to resolve structural changes of proteins after photoactivation. To obtain multiple structural snapshots of high quality, it is essential to collect large amounts of data and ensure clearance of crystals between every pump laser pulse. Here, we describe in detail how we optimized the extrusion of bacteriorhodopsin microcrystals for our recent TR-SFX experiments at the Linac Coherent Light Source (LCLS). The goal of the method is to optimize extrusion for a stable and continuous flow while maintaining a high density of crystals to increase the rate at which data can be collected in a TR-SFX experiment. We achieve this goal by preparing lipidic cubic phase with a homogenous distribution of crystals using a novel three-way syringe coupling device followed by adjusting the sample composition based on measurements of the extrusion stability taken with a high-speed camera setup. The methodology can be adapted to optimize the flow of other microcrystals. The setup will be available for users of the new Swiss Free Electron Laser facility.

The success of a time-resolved serial femtosecond crystallography experiment is dependent on efficient sample delivery. Here, we describe protocols to optimize the extrusion of bacteriorhodopsin microcrystals from a high viscosity micro-extrusion injector. The methodology relies on sample homogenization with a novel three-way coupler and visualization with a high-speed camera.

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray ...

Chemistry

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 ...

High-Throughput Protein Crystallization via Microdialysis

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug discovery. To date, X-ray crystallography remains the most successful method for solving three-dimensional protein structures at atomic resolution. With recent advances in serial crystallography, either using X-ray free electron lasers (XFELs) or synchrotron light sources, protein crystallography has progressed to the next frontier, where the ability to acquire time-resolved data provides important mechanistic insights into the behavior of biological molecules at room temperature. This protocol describes a straightforward high-throughput (HTP) workflow for screening crystallization conditions through the use of a 96-well dialysis plate. These plates follow the Society for Biomolecular Screening (SBS) standard and can be easily set up using any standard crystallization laboratory. Once optimal conditions are identified, large quantities of crystals (hundreds of microcrystals) can be produced using the dialyzer. To validate the robustness and versatility of this approach, four different proteins were crystallized, including two membrane proteins.

High-Throughput Protein Crystallization via Microdialysis

The presented protocol describes a straightforward approach for screening protein crystallization conditions and crystal growth using a 96-well high-throughput dialysis plate. The use of dialyzer tubes for the large-scale growth of microcrystals is also demonstrated for serial crystallography and MicroED applications.

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug ...

Crystallization of ABC Transporter Membrane Proteins in Lipidic Bicelles

ABCG5/G8 Crystallization in a Lipidic Bicelle Environment for X-Ray Crystallography

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an aqueous environment is typically achieved by employing detergents. These detergents disintegrate the lipid bilayer and solubilize the proteins. The intrinsic habitat of membrane proteins within the lipid bilayer poses a challenge in maintaining their stability and uniformity in solution for structural characterization. Bicelles, which comprise a blend of long and short-chain phospholipids and detergents, replicate the natural lipid structure. The utilization of lipid bicelles and detergents serves as a suitable model system for obtaining high-quality diffraction crystals, specifically to determine the high-resolution structure of membrane proteins. Through these synthetic microenvironments, membrane proteins preserve their native conformation and functionality, facilitating the formation of three-dimensional crystals. In this approach, the detergent-solubilized heterodimeric ABCG5/G8 was reintegrated into DMPC/CHAPSO bicelles, supplemented with cholesterol. This setup was employed in the vapor diffusion experimental procedure for protein crystallization.

Author Spotlight: A Bicelle Crystallization Setup for ABC Transporter Membrane Proteins to Advance Drug Development 

This protocol describes a setup for the crystallization of the sterol transporter ABCG5/G8. ABCG5/G8 is reconstituted into bicelles for hanging-drop crystallization. The protocol does not require specialized materials or substrates, making it accessible and easy to adapt in any laboratory for determining the protein structure through X-ray crystallography.

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an ...

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

Summary

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Chapters in this video

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

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

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

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

High-Throughput Protein Crystallization via Microdialysis

ABCG5/G8 Crystallization in a Lipidic Bicelle Environment for X-Ray Crystallography