This work was supported by the National Institutes of Health grants R01 GM108635 and U54 GM094618, Mayo Clinic-ASU Collaborative Seed Grant Award and NSF STC award 1231306. We thank A. Walker for assistance with manuscript preparation.

1. Filtering Solutions and Lipids
Note: All reagents and tools used in this protocol should be as clean as possible to avoid the introduction of particulate contaminants in the samples, which can clog the LCP injector.
<ol>
	<li>Filter all precipitant solutions and molten lipids through 5 &#181;m spin-down filters. Clean syringes thoroughly and apply compressed air to dry. Optionally, use a portable clean-room hood to prevent trapping dust particles or fibers during sample preparation procedures.</li>
</ol>
2. Reconstitution of MP in LCP
Note: The choice of the best LCP host lipid for crystallization depends on the target MP, and is typically identified during host lipid screening crystallization trials14. Monoacylglycerols (MAGs) represent the most common class of lipids used for LCP crystallization15. The protocols are based on using 9.9 MAG (monoolein) as the host lipid, since this is the most successful lipid for in meso crystallization to date16. 9.9 MAG can be substituted by other LCP host lipids or lipid mixtures after taking into account differences in their phase behavior. For instance, in the case of GPCRs, monoolein is typically doped with cholesterol for receptor stabilization. Here, use 9:1 (w/w) 9.9 MAG:cholesterol mixture as the host lipid.
<ol>
	<li>Mix target MP, which is purified in detergent micelle solution, with the appropriate LCP host lipid using two syringes (#1 and #2) and a coupler, as described previously13.</li>
	<li>Briefly, load syringe #1 with the molten lipid and syringe #2 with the MP solution. Use a ratio between lipid and aqueous MP solution corresponding to the level that is just below the maximal hydration capacity of the corresponding LCP host lipid, e.g., 3:2 v/v lipid/MP solution for 9.9 MAG, 1:1 v/v lipid/MP solution for most other shorter chain MAGs (7.9 MAG, 9.7 MAG, etc.).</li>
	<li>Connect the syringes through a syringe coupler and start mixing the substances by depressing the plungers to move the mixture back and forth between the syringes, until the sample becomes homogeneous and transparent. Prepare approximately 50 &#181;l of the LCP sample for the collection of a complete dataset by LCP-SFX. The final sample volume will typically increase by approximately a factor of two (to ~100 &#181;l) after sample consolidation and lipid titration (Section 5).</li>
</ol>
3. Setting up Crystallization in Syringes
<ol>
	<li>After a transparent and homogeneous LCP is formed, move the entire sample into syringe #2. Detach syringe #1, while keeping the coupler connected to syringe #2.</li>
	<li>Connect a removable needle (gauge 26s) to another 100 &#181;l clean syringe (syringe #3) and aspirate approximately 70 &#181;l of the precipitant solution into it. The composition of the precipitant solution that triggers formation of high-density microcrystals is unique for each target MP and should be determined before proceeding to these protocols. Here, use an optimized precipitant solution composition that yields showers of microcrystals of the A2A receptor (40 mM sodium thiocyanate, 100 mM sodium citrate pH 5, 26-28% PEG400).</li>
	<li>Disconnect the needle from syringe #3 while keeping the Teflon ferrule inside the syringe.</li>
	<li>Connect syringes #2 and #3 through the coupler, making sure that Teflon ferrules are correctly in place in both syringes. Carefully screw the coupler tightly into position.</li>
	<li>Orient the coupled syringes vertically with syringe #2 on the bottom and inject the protein-laden LCP sample from syringe #2 into syringe #3 slowly and steadily until the LCP string touches the plunger of syringe #3. The injected LCP volume will amount to about 1/10 of the initial precipitant volume in syringe #3 (~7 &#181;l). Verify the volume by the scale-reading on both syringes (both plungers should move by about 7 &#181;l).</li>
	<li>Disconnect syringe #2. The coupler now is only connected to syringe #3 that contains the sample immersed in the precipitant solution.</li>
	<li>Use Parafilm to completely seal syringe #3, including the plunger syringe interface, the opening end of the coupler and the needle nut. Incomplete sealing during this step can cause the precipitant conditions to change and the sample to dehydrate.</li>
	<li>Repeat steps 3.3-3.7 to set up crystallization in 6 additional syringes (#4-#9) utilizing a total of ~50 &#181;l of the LCP sample (~7 &#181;l of LCP per each syringe).</li>
	<li>Store sealed syringes in a plastic sealable bag with one or two fiber-free cleaning tissues pre-soaked with water to protect against sample dehydration. Seal the bag and store syringes in a 20 &#176;C incubator during crystal growth.</li>
</ol>
4. Crystal Detection
<ol>
	<li>Remove the syringes from the incubator to image the LCP samples directly inside the syringes (#3-#9) every 12-24 hr under a stereomicroscope with cross-polarized light. Identify crystals as tightly packed shiny particles or, in the case of smaller crystal size as a uniform glow from the LCP filament.</li>
	<li>Return the sealed syringes to the bag, and store at 20 &#176;C for future use.</li>
</ol>
5. Sample Consolidation and Titration with 7.9 MAG
Note: The lipid titration step described here serves two purposes: a) to absorb the excess precipitant solution and b) to prevent lipid freezing upon injection into the XFEL beam. When an LCP sample composed of 9.9 MAG is injected into a vacuum chamber for data collection, intensive evaporation can cool the sample down to temperatures below the equilibrium phase transition temperature (~18 &#176;C), converting parts of the sample into a lamellar crystalline phase (Lc). These patches of Lc phase, when hit by the beam, produce intense powder diffraction rings that can damage a sensitive detector6, such as the Cornell-SLAC pixel array detector (CSPAD). Titration of 9.9 MAG sample with a shorter chain lipid (9.7 MAG or 7.9 MAG) alleviates this problem, as such mixtures have lower phase transition temperatures. Here, we describe titration of a LCP composed of 9.9 MAG with a 7.9 MAG. When shorter chain MAGs (9.7 MAG, 7.9 MAG, etc.) are used for crystallization or when serial crystallography data are collected at ambient pressure the titration in step 5.11 is still required, but it can be performed using the original LCP host lipid employed for crystallization.
<ol>
	<li>About 1 hr before the beginning of data collection, remove syringes with samples from the 20 &#176;C incubator.</li>
	<li>Select 2-4 syringes for sample consolidation based on a similar crystal appearance and similarity of crystallization conditions.</li>
	<li>Carefully remove the Parafilm sealing from the selected syringes.</li>
	<li>Remove the syringe coupler and attach a clean removable needle (gauge 26s) to the first selected syringe. Gently and slowly push the plunger forward to squeeze the precipitant out through the needle into a microcentrifuge tube. Exercise caution at this step because applying a high pressure on the plunger in this step could eject some of the crystal laden LCP along with the precipitant solution, leading to a partial or complete loss of the sample.</li>
	<li>Stop the plunger when most of the precipitant has been removed and the LCP has accumulated at the needle entrance.</li>
	<li>Repeat step 5.4-5.5 with the other selected syringes.</li>
	<li>To consolidate the resultant LCP samples, connect two syringes together through a clean coupler.</li>
	<li>Depress the plunger on one syringe to transfer the entire sample from one of the syringes to another.</li>
	<li>Disconnect the empty syringe.</li>
	<li>Repeat steps 5.7-5.9 to consolidate all of the crystal laden LCP material from the two to four pre-selected syringes into one syringe. Remove as much precipitant as possible.</li>
	<li>Add ~5 &#181;l of 7.9 MAG or the original shorter-chain MAG host lipid to an empty syringe. Connect this syringe to the syringe with the consolidated sample through a syringe coupler and mix by alternatively depressing syringe plungers. Repeat until all residual solution is absorbed and a homogeneous and transparent LCP is formed. The final amount of LCP after titration can vary from 20 to 35 &#181;l&#160;depending on the volume of the excess precipitant and its composition.</li>
	<li>Move the entire mixed LCP sample into one syringe and disconnect the empty syringe.</li>
</ol>
6. Characterization of Microcrystals
<ol>
	<li>Attach an LCP-injector loading needle (point style 3, gauge 22, one inch in length) to the syringe with consolidated sample.</li>
	<li>Carefully eject ~1 &#181;l of the LCP sample on a glass slide and cover it with a glass cover slip. Gently press on the cover slip to sandwich the sample.</li>
	<li>Take images of the LCP sample under a stereo microscope at the highest possible magnification (typically 100X) using a bright-field illumination and cross-polarizers. If possible, take additional images using a UV-fluorescence microscope and a SONICC (Second Order Nonlinear Imaging of Chiral Crystals)17 imager to confirm the existence of protein microcrystals in the sample.</li>
	<li>Estimate crystal size and density10. The ideal crystal density for a data collection experiment will depend on the crystal size, the diameter of the X-ray beam and the diameter of the LCP stream, and should result in a crystal hit rate of about 10-40%.</li>
</ol>
7. Adjustment of Crystal Density
Note: If the crystal density found in Step 6.4 is too high, resulting in a large percentage of multiple crystal hits, the crystals should be diluted following the steps outlined below to optimize the sample for data collection. If the crystal density is too low for efficient data collection in all prepared samples, then the crystal growth conditions should be re-optimized, since there is no reliable method for concentrating MP crystals in LCP.
<ol>
	<li>Prepare the required amount of neat LCP to be used for dilution by mimicking the LCP composition (same lipid and precipitant) of the original sample with crystals that needs to be diluted.</li>
	<li>Move all neat LCP into one syringe. Detach empty syringe but leave the coupler connected.</li>
	<li>Remove the needle from the syringe that contains the LCP sample with microcrystals. Connect the sample syringe to the syringe containing neat LCP through the coupler.</li>
	<li>Mix the content of the syringes by pushing it back and forth through the coupler until homogeneity is achieved.</li>
	<li>Repeat Section 6 to re-evaluate the microcrystal density in the adjusted sample.</li>
</ol>
8. LCP Injector Loading and LCP-SFX Data Collection
<ol>
	<li>Disconnect the coupler from the syringe with the sample and attach a 1&#34; loading needle to the syringe.</li>
	<li>Transfer 20-40 &#181;l of the sample into the reservoir of an LCP injector.</li>
	<li>Insert the LCP injector into the sample chamber18, start the injector, adjust the flow rate and collect LCP-SFX data.</li>
</ol>
9. Incorporation of Soluble Protein Crystals in LCP
Note: Using highly viscous media, such as LCP, for delivery of crystals of soluble proteins allows one to dramatically decrease protein consumption in a serial crystallography experiment. In these steps we describe how to incorporate crystals of soluble proteins in LCP. Crystals can be obtained by any technique, consolidated together in the form of a crystal suspension and filtered if necessary.
<ol>
	<li>Adjust crystal density in suspension to ensure crystal hit rates of approximately 10-40%.</li>
	<li>Test the compatibility of the precipitant solution with LCP using 7.9 MAG, 9.7 MAG or a 1:1 mixture of 7.9 MAG and 9.9 MAG as the host lipid.</li>
	<li>Mix ~25 &#181;l of the crystal suspension with ~25 &#181;l of the host lipid using a syringe mixer until a homogeneous LCP forms.</li>
	<li>Evaluate crystal size and density as described in Section 6. Load the sample in the LCP injector and collect data as described in Section 8.</li>
</ol>

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<li>Sugahara, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Grease+matrix+as+a+versatile+carrier+of+proteins+for+serial+crystallography%5BTitle%5D)+AND+%22Nat+Meth%22%5BJournal%5D)">Grease matrix as a versatile carrier of proteins for serial crystallography.</a> Nat Meth. 12 (1), 61-63 (2014).</li>
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The authors have no conflicts of interest to disclose.

The protocols described here provide a general outline of the sample preparation procedure for a standard LCP-SFX experiment with an LCP injector. Based on our experience, the total amount of the final microcrystal-laden LCP sample required for collecting a full dataset is typically 50-100 &#181;l (Table 1; 25-50 &#181;l of initial protein-laden LCP sample), depending on the microcrystal size, quality and density. Since each 100 &#181;l gas-tight glass syringe can accommodate about 7 &#181;l of LCP in the form of a fully extended string to ensure proper and even diffusion of the precipitant solution into the LCP, at least four to seven samples in syringes should be prepared for each LCP-SFX experiment.
Since the sample is extruded through a narrow 20-50 &#181;m in diameter capillary, it is crucial to avoid any foreign particulate material in the sample. Dusts and fibers that could be occasionally introduced in the LCP sample or precipitant solutions may clog the injector capillary and increase the down-time during the experiment. Therefore, it is important to filter the lipid and all solutions through a 5 &#181;m pore filter before preparing samples. Optionally, a clean room or a portable clean room hood can be used for sample preparation.
These protocols are based on using 9.9 MAG (monoolein) as the host lipid for MP crystallization. They, however, are not limited to 9.9 MAG and can be easily adapted to any other LCP host lipids or lipid mixtures after taking into account the differences in the lipid phase behavior. Most of the structures of MPs crystallized in LCP were obtained using one of the MAGs, with 9.9 MAG being the most successful representative so far15. The main drawback of using 9.9 MAG for SFX is its transition to lamellar crystal phase upon cooling below 18 &#176;C, which often occurs when the data acquisition is performed in vacuum. The titration with 7.9 MAG described in Section 5 of this protocol provides a good solution to this problem. In our experience, crystals withstand such a titration without any adverse effects. If, however, the addition of 7.9 MAG or another short-chained MAG is not desirable, the titration can be performed with 9.9 MAG. In this case, the gas that stabilizes the flow of LCP during injection into the vacuum should be switched from helium to nitrogen, which can prevent the formation of a crystalline lipid phase in most samples, at the expense of having a less stable sample flow.
The gel-like consistency of LCP has a great advantage for use as a crystal carrier medium, since it allows adjustment of the crystal flow rate across a wide range, suitable for serial crystallography data collection at modern XFEL and synchrotron sources. Matching the crystal flow rate with the XFEL pulse repetition rate results in dramatic reduction in crystal consumption by orders of magnitude compared to liquid injection. LCP is a suitable carrier medium for delivery not only of MP crystals grown in it, but also for crystals of soluble proteins21. Several alternative viscous crystal carrier media have recently been introduced22-24, extending the arsenal of tools and reagents for conducting serial crystallography experiments. Moreover, serial crystallography with LCP as a crystal delivery medium has been demonstrated at conventional synchrotron sources, at least for well-diffracting crystals24,25. Future upgrades of synchrotron sources that boost X-ray intensity by orders of magnitude, as well as improvements in detector technologies, will make serial crystallography an even more accessible and attractive method for structure determination.
LCP-SFX has demonstrated its potency for MP structural determination. For challenging biological systems (such as GPCRs or macromolecular complexes) where large, diffraction-quality crystals are difficult to grow, LCP-SFX may provide an attractive, if not the only, viable option to solve an atomic resolution structure. With all its advantages, i.e., using LCP as the matrix for MP crystallization and crystal delivery, low protein consumption, absence of radiation damage, room temperature data collection, possibilities of time-resolved studies26,27 and no crystal harvesting requirement, LCP-SFX should play an important role in the future of structural biology.

X-ray crystallography is the most successful technique to date for solving atomic-resolution structures of membrane proteins (MPs). Crystallization from lipidic mesophases, also known as lipidic cubic phase (LCP), or in meso crystallization, represents one of the major developments in MP crystallography that has enabled the high-resolution structure determination of challenging targets such as G protein-coupled receptors (GPCRs)1. Recent advancements in LCP tools and technologies have made this technique available to a large community of structural biologists around the world2. However, in many cases the MP crystals that form in LCP are too small to be used even at most advanced microfocus synchrotron beamlines, where the samples suffer from severe radiation damage and deterioration before sufficient signal at high resolution can be obtained3.
A new approach to crystallographic data collection was enabled with the commissioning of the first X-ray free-electron laser (XFEL). The method is based on the principle of &#34;diffraction before destruction&#34;, first introduced theoretically4 and then confirmed experimentally on biological samples at the Linac Coherent Light Source (LCLS)3, the world&#39;s pioneer in hard XFELs. An XFEL generates high-brightness X-ray pulses within few tens of femtosecond duration that diffract from a pristine crystal before the protein atoms can move in response to radiation damage. In the initial experiments, microcrystals were continuously supplied to the intersection with the XFEL beam in an aqueous stream produced by a liquid injector5. This experimental setup is known as serial femtosecond crystallography (SFX). The main drawback to using liquid injectors for SFX is their high flow rate, which consequently requires tens to hundreds of milligrams of purified protein for collection of a complete dataset. By employing a special injector that can handle the gel-like LCP samples (LCP microextrusion injector)6, we successfully delivered microcrystals of several different human GPCRs grown in an LCP matrix and obtained their high-resolution structures6-9, with a significantly reduced protein consumption to under 0.3 mg per dataset. The injector consists of a reservoir that can hold up to 20, 40 or 100 &#181;l of crystal-laden LCP and a narrow capillary (10-50 &#181;m in diameter) through which the sample is extruded by application of a high pressure (up to 10,000 psi) from a hydraulic plunger with a pressure amplified stage, driven by a liquid from an HPLC (high-performance liquid chromatography) pump. The stream of LCP exiting the capillary nozzle is stabilized by a parallel flow of nonreactive gas (typically helium or nitrogen).
Here we provide visual demonstrations of the steps required to prepare and characterize samples for an LCP-SFX experiment. Additional details can be found in the published protocols10. As an example, we will use adenosine A2A receptor11 and prepare crystals of this protein in LCP for the X-ray data collection using SFX approach. While our protocols are compatible with any injector capable of streaming LCP for data collection by serial crystallography at XFEL and synchrotron sources, for illustration purposes, we will use the LCP injector described in Ref. 6. Optimized precipitant conditions that produce high-density microcrystals in LCP should be identified by high-throughput crystallization screening12,13 before proceeding to this protocol. A typical flowchart for this protocol is shown in Figure 1.

<table><tbody><tr><td>LCP host lipid monoolein (9.9 MAG)</td><td>Nu Chek Prep</td><td>M239</td><td>Hosting lipid for the protein to be crystallized</td></tr><tr><td>LCP host lipid monoolein (9.9 MAG)</td><td>Sigma</td><td>M7765&amp;nbsp;</td><td>Hosting lipid for the protein to be crystallized</td></tr><tr><td>LCP host lipid monopalmitolein (9.7 MAG)</td><td>Nu Chek Prep</td><td>M219</td><td>Hosting lipid for the protein to be crystallized</td></tr><tr><td>LCP host lipid 7.9 MAG</td><td>Avanti Polar Lipids</td><td>850534</td><td>Hosting lipid for tiration to avoid lamellar crystal phase (Lc) formation during sample injection</td></tr><tr><td>Purified protein solution concentrated to 20-50 mg/ml</td><td></td><td></td><td>Target protein</td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>C606-1</td><td>Used to dissolve lipids</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>179337-500ML</td><td>Used to wash syringes</td></tr><tr><td>Eleven 100 &amp;#956;l Hamilton gas-tight syringes without needles</td><td>Hamilton</td><td>7656-01</td><td>For LCP preparation</td></tr><tr><td>Eight syringe couplers</td><td>Formulatrix</td><td>SKU 209526</td><td>For LCP preparation</td></tr><tr><td>LCP-injector loading needle</td><td>Hamilton</td><td>7804-01 point style 3, length 1 inch</td><td>For LCP dispensing and injector loading</td></tr><tr><td>Removable needle</td><td>Hamilton</td><td>7768-01</td><td>For removing precipitant solution</td></tr><tr><td>Parafilm M</td><td>Parafilm</td><td>PM992</td><td>Used for syringe sealing</td></tr><tr><td>Lint free lens cleaning tissues</td><td>Fisher</td><td>NC9592151</td><td></td></tr><tr><td>Centrifugal filter tubes, Ultrafree-MC, 0.5 ml, Durapore 5 &amp;micro;m</td><td>Millipore</td><td>UFC3 0SV 00</td><td>Used for filtering of precipitant solutions and lipids</td></tr><tr><td>Microscope slides, 1 inch x 3 inch&amp;nbsp;</td><td>GoldSeal</td><td>3010</td><td></td></tr><tr><td>Two wash bottles with fine tips. One filled with milli-Q or distilled water, the other with methanol</td><td>VWR</td><td>89094-640</td><td>These are used to clean the syringes, needles and coupler.&amp;nbsp; Methanol is used first, then water.</td></tr><tr><td>Gloves</td><td>Microflex</td><td>XC-310-L</td><td>For blow drying syringes, needles, ferrules, and couplers</td></tr><tr><td>Safety glasses</td><td></td><td></td><td></td></tr><tr><td>Pressurized air cans</td><td>Office Depot (Dust-Off)</td><td>527494</td><td>For syringe cleaning</td></tr><tr><td>Dry block heater with a digital temperature controller</td><td>Fisher</td><td>11-720-10BQ&amp;nbsp;</td><td>Used for thawing lipids</td></tr><tr><td>Benchtop Centrifuge</td><td>Fisher</td><td>05-413-340</td><td>Spinning down solutions</td></tr><tr><td>Clean Room Mini - 12 inch Portable Positive Pressure Hood with HEPA filter</td><td>Sentry Air Systems, Inc.</td><td>SS-112-PCR</td><td>Cleaning sample preparation</td></tr><tr><td>Stereo zoom microscope equipped with a linear polarizer and rotating analyzer&amp;nbsp;</td><td>Nikon</td><td>SMZ1500</td><td>Crystal density check</td></tr><tr><td>UV microscope</td><td>JAN Scientific</td><td>UVEX-P</td><td>Optional, for crystal imaging purposes</td></tr><tr><td>SHG imager</td><td>Formulatrix</td><td>SONICC</td><td>Optional, for crystal imaging purposes</td></tr></tbody></table>

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.

Below we describe representative results obtained with samples prepared by following the above protocols, which allowed us to collect SFX data and solve high-resolution structures of five human GPCRs: serotonin receptor 5-HT2B in complex with an agonist ergotamine8 (PDB ID 4NC3), smoothened receptor (SMO) in complex with an antagonist cyclopamine6 (PDB ID 4O9R), &#948;-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH27(PDB ID 4RWD), angiotensin receptor in complex with a blocker ZD71559 (PDB ID 4YAY), and a complex between rhodopsin and arrestin19 (PDB ID 4ZWJ); and two test soluble proteins: lysozyme (PDB ID 4ZIX) and phycocyanin (PDB ID 4ZIZ).
5-HT2B mediates various central and peripheral physiologic functions of the neurotransmitter serotonin. This receptor was used to establish and validate the LCP-SFX method, by comparing the room temperature structure obtained at LCLS with the cryocooled structure solved by traditional microcrystallography at the Advanced Photon Source (APS). A total of ~100 &#181;l LCP sample with protein microcrystals (average size about 5 &#181;m) was prepared and used for SFX data collection, and the LCP-SFX structure of 5HT2B was successfully solved at 2.8 &#197; (Figure 2)8. Additional useful details on sample preparation and data collection are provided in Table 1.
Smoothened receptor (SMO) is a member of class F GPCRs and the molecular target of the teratogen cyclopamine, with well-demonstrated functions in embryonic development and tumor growth. Initially, relatively large crystals of SMO/cyclopamine with an average size of 120&#215;10&#215;5 &#181;m were obtained and then used for data collection at a synchrotron source using conventional goniometer-based crystallography. However, these large crystals produced poor diffraction at a micro-focus beamline (10 &#181;m in diameter) suffering from large mosaicity (above 2-3 degrees), probably due to the accumulation of crystal growth defects, or from effects related to cryocooling. LCP-SFX, however, enabled us to collect quality diffraction data at LCLS from 5 &#181;m-sized crystals at room temperature. The structure was solved by molecular replacement at an anisotropic 3.4, 3.2 and 4.0 &#197; resolution along the three principal axes, clearly identifying the location of cyclopamine in the binding pocket6 (Figure 3).
Alkaloid opiates, such as morphine, targeting &#181;-opioid receptor (&#181;-OR) are widely used for severe pain management. However, their extensive usage leads to acquired tolerance and addiction. Co-administration of morphines with &#948;-opioid receptor (&#948;-OR) antagonists has been shown to prevent the abovementioned side effects, thus promoting the search for compounds with a mixed &#948;-OR-antagonist and &#181;-OR-agonist function. We obtained the initial diffraction data on &#948;-OR in a complex with a bi-functional tetra-peptide DIPP-NH2 using cryocooled crystals that diffracted to 3.3 &#197; at a synchrotron X-ray source employing conventional goniometer-based data collection strategy. These data revealed a partially ambiguous electron density for the peptide ligand. Subsequently, XFEL diffraction data at room temperature were obtained and the structure was determined at 2.7 &#197; resolution showing a clear density for the ligand and the receptor7. This structure offered an opportunity for further understanding opioid receptor function and selectivity and provided valuable insights for the development of new analgesics.
Angiotensin II type 1 receptor (AT1R) is a GPCR serving as a primary regulator of blood pressure. We crystallized AT1R in complex with an antagonist ZD7155 in LCP. Optimized crystals reached a maximum size of 40&#215;4&#215;4 &#181;m3 with the best diffraction reaching only ~4 &#197; at a synchrotron source. By changing crystallization conditions, we obtained showers of smaller crystals (10&#215;2&#215;2 &#181;m3), which were used to obtain the room temperature structure at 2.9 &#197; resolution using XFEL radiation. A total of 2,764,739 detector images were collected to make a complete dataset from about 65 &#181;l of crystal-laden LCP corresponding to about 0.29 mg of protein9. Of the total number of frames, 457,275 were identified as crystal hits, corresponding to a hit rate of 17%, of which 73,130 frames (16% of hits) were successfully indexed and integrated.
GPCRs signal through two main pathways mediated by either G proteins or arrestins. The structure of the &#946;2-adrenergic receptor bound to a heterotrimeric Gs protein was solved a few years ago20, whereas the structure of a GPCR in complex with arrestin had remained elusive. We obtained small crystals of a rhodopsin-arrestin fusion protein in LCP that reached 25-30 &#181;m in the longest dimension, but despite extensive optimization, diffracted only to ~7 &#197; resolution at synchrotron sources. By using the LCP-SFX method, within 12 hours of XFEL beamtime, we collected 22,262 crystal hits, out of which 18,874 patterns were successfully indexed and integrated to anisotropic resolution limits of 3.8 &#197; / 3.8 &#197; / 3.3 &#197;19. Rhodopsin-arrestin is a very challenging protein complex that resisted structure determination using traditional approaches. The successful determination of this structure has demonstrated the huge potential of the LCP-SFX method for tackling difficult problems and provided a unique opportunity to examine the mechanism of arrestin-biased signaling in GPCRs.
Finally, in addition to membrane protein crystals grown in the lipidic phase, our sample preparation and delivery method was successfully adapted for soluble protein crystals, where LCP is used as a carrier medium for the delivery of crystals, allowing us to dramatically decrease the protein consumption required for structure determination. Structures of two model proteins, lysozyme and phycocyanin, were solved at 1.89 &#197; and 1.75 &#197; resolution respectively by this method, using less than 0.1&#8197;mg of each protein21.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Serotonin receptor 2B</td>
			<td>Smoothened receptor&#160;</td>
			<td>&#948;-Opioid receptor&#160;</td>
			<td>Angiotensin II receptor type 1</td>
			<td>Rhodopsin-Arrestin&#160;</td>
			<td>Lysozyme</td>
			<td>Phycocyanin</td>
		</tr>
		<tr>
			<td>PDB ID</td>
			<td>4NC3</td>
			<td>4O9R</td>
			<td>4RWD</td>
			<td>4YAY</td>
			<td>4ZWJ</td>
			<td>4ZIX</td>
			<td>4ZIZ</td>
		</tr>
		<tr>
			<td>Total sample used*, &#956;l</td>
			<td>100</td>
			<td>83</td>
			<td>50</td>
			<td>65</td>
			<td>75</td>
			<td>10</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Total protein used, &#956;g</td>
			<td>300</td>
			<td>500</td>
			<td>300</td>
			<td>290</td>
			<td>340</td>
			<td>100</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Average crystal size, &#956;m</td>
			<td>5&#215;5&#215;5</td>
			<td>&#60;5</td>
			<td>5&#215;2&#215;2</td>
			<td>10&#215;2&#215;2</td>
			<td>5-10</td>
			<td>5&#215;2&#215;2</td>
			<td>10&#215;10&#215;5</td>
		</tr>
		<tr>
			<td>Hit rate, %</td>
			<td>3.6</td>
			<td>7.8</td>
			<td>5.9</td>
			<td>17</td>
			<td>0.45</td>
			<td>40</td>
			<td>6.5</td>
		</tr>
		<tr>
			<td>Total data collection time, hr</td>
			<td>10</td>
			<td>8</td>
			<td>4.6</td>
			<td>6.4</td>
			<td>12</td>
			<td>0.75</td>
			<td>0.67</td>
		</tr>
		<tr>
			<td>Number of indexed patterns</td>
			<td>32,819</td>
			<td>61,964</td>
			<td>36,083&#160;</td>
			<td>73,130</td>
			<td>18,874</td>
			<td>54,544</td>
			<td>6,629</td>
		</tr>
		<tr>
			<td>Space group</td>
			<td>C 2 2 21</td>
			<td>P 21</td>
			<td>C 2</td>
			<td>C 2&#160;</td>
			<td>P 21&#160;21&#160;21</td>
			<td>P 43 21 2</td>
			<td>H 32</td>
		</tr>
		<tr>
			<td>Resolution, &#197;</td>
			<td>2.8</td>
			<td>3.2 / 3.4 / 4.0</td>
			<td>2.7</td>
			<td>2.9</td>
			<td>3.3 / 3.8 / 3.8</td>
			<td>1.89</td>
			<td>1.75</td>
		</tr>
	</tbody>
</table>
*After lipid titration
Table 1. Summary of the sample preparation and data collection statistics for representative structures solved using the LCP-SFX method. 
The amount of sample required for an LCP-SFX experiment depends on the crystal diffraction quality, crystal density, the diameter of the injector nozzle, as well as the XFEL beam size, intensity and pulse repetition rate.
<img alt="Figure 1" src="/files/ftp_upload/54463/54463fig1.jpg" /> 
Figure 1. Flowchart of a typical sample preparation procedure for LCP-SFX. Sample preparation starts with LCP crystallization of the target protein in gas-tight glass syringes. After crystals are obtained, the crystal-laden LCP is consolidated in one syringe and titrated with additional lipid, to absorb the excess precipitant solution. Crystals are then characterized using various microscopy methods prior to XFEL data collection. <a href="https://www.jove.com/files/ftp_upload/54463/54463fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54463/54463fig2.jpg" /> 
Figure 2. A representative result, the structure of 5-HT2B in complex with ergotamine. (a) Microcrystals of 5-HT2B/ergotamine imaged using a bright-field microscope mode8. This figure has been reused from reference 8 with copyright permission from Science. (b) Microcrystals of 5-HT2B/ergotamine imaged using a cross-polarized microscope mode8. This figure has been reused from reference 8 with copyright permission from Science. (c) A cartoon representation of the 5-HT2B/ergotamine structure obtained by the LCP-SFX approach. Lipids that were built in the model are shown in stick representation. The ligand ergotamine is shown in spheres representation. Solid lines indicate the approximate membrane boundaries. <a href="https://www.jove.com/files/ftp_upload/54463/54463fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54463/54463fig3.jpg" /> 
Figure 3. A representative result, the structure of SMO in complex with cyclopamine. Left panel, microcrystals of SMO/cyclopamine imaged using a cross-polarized microscope mode6. This figure has been reused from reference 6 with copyright permission from Nature Communications. Right panel, a cartoon representation of the SMO/cyclopamine structure obtained by the LCP-SFX approach. Ligand cyclopamine is shown in spheres representation. Solid lines indicate the approximate membrane boundaries. <a href="https://www.jove.com/files/ftp_upload/54463/54463fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography at JoVE.com

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

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 ...

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Biochemistry

11.5K Views.  University of Southern California. 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.

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

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.
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Microcrystallography of Protein Crystals and In Cellulo Diffraction

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From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

Chemistry

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.

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Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

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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.

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

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

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

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

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

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Biology

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 ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

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

Summary

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Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

High-Throughput Protein Crystallization via Microdialysis

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

Crystallization of Membrane Proteins in Lipidic Mesophases