Systems comprising lithographic elements for protein crystallization

Abstract

The present disclosure provides novel systems and methods for membrane protein crystallization. In particular, the systems of the present disclosure utilize a lithographic electric circuit that provides improved features for membrane protein crystallization and analysis. For instance, the system comprising a lithographic electric circuit is capable of delivering voltage-relaxation protocols into a crystallization chamber containing a lipid matrix with the membrane protein (MP). Further, a user-programmable computer-controlled multi-output variable voltage source can be included as part of the system and can permit the application of different voltage relaxation protocols to multiple samples, simultaneously.

Description

71900-429346-1- SYSTEMS COMPRISING LITHOGRAPHIC ELEMENTS FOR PROTEINCRYSTALLIZATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Serial No. 63 / 700,640, filed on September 28, 2024 and also claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Serial No. 63 / 729,601. filed on December 9. 2024. The entire disclosures of both provisional applications are incorporated herein by reference.GOVERNMENT RIGHTS

[0002] This invention was made with government support under grant numbers P20GM103642, R01GM098343, and GM149367 awarded by National Institutes of Health. The government has certain rights in the invention.BACKGROUND AND SUMMARY

[0003] In the past decade, there has been an increasing number of attempts to elucidate three-dimensional structures of proteins, including membrane proteins. Among the various methodologies utilized for this purpose, cryo-electron microscopy (cryo-EM) and X-ray crystallography studies have shown promise. For instance, nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that play a key role in neurotransmission modulation. The human a402 nAChR subtype is actively expressed in cholinergic neurons and is widely distributed throughout the brain, making it the focus of pharmaceutical research that seeks to develop therapeutic compounds for smoking cessation and other neurodegenerative diseases. However, to study receptor-ligand interactions at an atomic level, a high-resolution three-dimensional (3D) structure of the receptor and its bound ligands is required.

[0004] In this regard, the highest resolutions achieved for the a402 nAChR have been obtained through Cryo-EM technology, owing the latest high-resolution structure (2.35 A) to the study of calcium potentiation in variable stoichiometries ((a4)s(02)2 and (0.4)2(02)3) of the receptor. However, no further advancements in the crystallization of this receptor and its stoichiometries have been made since it was initially crystalized. Moreover, there is a longstanding bottleneck in the field of structural biology for preparing large amounts of pure nAChRs suitable for crystallization. Since nAChRs are integral membrane proteins, their structural integrity is intrinsically associated with their immediate lipid environment. Thus,71900-429346-2- there exists a need for new systems and methods for forming and evaluating three-dimensional structures of membrane proteins such as crystalline structure.

[0005] Accordingly, the present disclosure provides novel systems and methods for protein cry stallization, including membrane proteins and peripheral proteins. In particular, the systems of the present disclosure utilize a lithographic electric circuit that provides improved features for membrane protein crystallization and analysis. For instance, the system comprising a lithographic electric circuit is capable of delivering voltage-relaxation protocols into a cry stallization chamber containing a lipid matrix with the membrane protein (MP). Further, a user-programmable computer-controlled multi-output variable voltage source can be included as part of the system and can permit the application of different voltage relaxation protocols to multiple samples, simultaneously.

[0006] Importantly, the system can utilize Indium Tin Oxide (ITO) glass and can enhance the crystallization of MPs that incorporates the physiological principle of the resting membrane potential (RMP). The systems comprising lithographic elements (e g., device V2.0 and device V3.0) can provide advantages to prepare, improve, reproduce, and isolate / crystallize voltage-dependent conformations of biologically relevant MPs.

[0007] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTIONS OF THE DRAWINGSThe detailed description particularly refers to the accompanying figures in which:

[0008] Figure 1 shows a diagram for a RMP@LMx high-throughput cry stallization screening (RMP-HTCS) system with various crystallization chamber configurations. An embodiment of the "V2.0” device of the system and an embodiment of the “V3.0” device of the system are shown at the bottom of Fig. 1.

[0009] Figure 2A shows the progress of nucleation and crystal growth in LCP. Once an MP is incorporated into the LCP, the diffusion of the protein occurs in bicontinuous cubic-to- lamellar phases. The formation of the MP crystal in the LCP occurs in the lamellar phase (planar bilayer).

[0010] Figure 2B shows application of a protocol of 100 mV w ith a decay time of 0. 10 msec and a frequency of 1.0 Hz for 14 days restructured the LCP / LMx into a planar (lamellar)71900-429346-3- bilayer. Application of a voltage protocol produced a remarkable change in structure of the lipid matrix LCP / LMx. A confocal image of the LCP sample containing a cholesterol fluorescent analog (25-NBD-Cholesterol) was taken immediately after loading the sample in the RMP@LMx V.2.0 chamber and a Z-stack image before applying the voltage protocol (left image). The thickness of the control LCP / LMx sample is ~200mm (Z-section shown inside the rectangle). The image on the right shows the same sample after the application of voltage step protocols. The thickness of the lipid matrix in the center of the LCP / LMx after the voltage step protocol is ~70mm (Z-section shown inside red rectangle on the right). In summary, application of a voltage step protocol to the LCP matrix using the system comprising lithographic elements produced a lamellar planar lipid bilayer. This formation of a planar lipid matrix can enhance the crystallization of MPs.

[0011] Figure 2C shows that using polarized microcopy, lipid phase changes at day 5 of the voltage relaxation experiment were detected.

[0012] Figure 3 A show s LCP Experiments with Monoolein and H2O using a Nikon E400 polarized light microscope to examine the lipid phase changes during the application of repetitive voltage relaxation pulses or fixed voltage protocol. Images shown include control and with voltages pulses. Figure 3B shows graphs comparing the length, width, and area measurements of the samples.

[0013] Figure 4A shows LCP Experiments with Monoolein, Cholesterol, and H2O using a Nikon E400 polarized light microscope to examine the lipid phase changes during the application of repetitive voltage relaxation pulses or fixed voltage protocol. Images shown include control and with voltages pulses. Fig. 4B shows graphs comparing the length, width, and area measurements of the samples.

[0014] Figures 5A-5J display the mobility / diffusion of the MP in the lipid matrix (FRAP experiments). Fig. 5A shows voltage supply board using a Raspberry Pi microcontroller and Fig. 5B show s delivery7of voltage steps or repetitive voltage relaxation pulse protocols. Fig. 5C shows thermal images can be taken to monitor the temperature of the MP samples during crystallization. Fig. 5D shows monitoring of a lithographic crystallization chamber and Fig. 5E show s a DIC image magnification was used to determine the distance between electrodes (0.35mm). In Fig. 5F, a crystallization chamber is coupled to a portable raspberry' controller that delivers voltage steps or voltage pulses during imaging using a confocal microscope and FRAP experiment are performed during crystallization. Figs. 5G and 5H show mobile fraction of a fluorescent cholesterol analog was estimated during a 25 day period. Fig. 51 shows confocal images of the lipid matrix (LCP) including Z-stack reconstructions were taken to evaluate the integrity of the lipid matrix. Finally, Fig. 5J shows71900-429346-4- that voltage variations have a capacitance component that introduces a time-dependent behavior of the single well circuit. The microcircuits used to fabricate the various systems comprising lithographic elements display full spatial and temporal control of voltage during a cry stallization experiment.

[0015] Figure 6 shows EDS spectra of an exemplary' system comprising lithographic elements (i.e. , Lithographic RMP@LMx 2.0 device).

[0016] Figures 7A-7C exhibit diffusion across the lipid matrix and single crystal obtained from LCP crystallization of the human a4|B2 nAChR using the RMP@LMx V. 1.5 device. FIG. 7A shows a SDS-PAGE of the purified sample, used to produce the cry stal. FIG. 7B shows the LCP-FRAP purified a4 2 nAChR-DCs. FIG. 7C illustrates a micrograph of a single crystal of the human a.4|32 nAChR taken at the time of X-ray diffraction.

[0017] Figures 8A-8D show LCP samples containing the purified a4p2 nAChR-DCs during incubation in the RMP@LMx V.1.5 device. Each of FIG. 8A, FIG. 8B, FIG. 8C, and FIG. 8D shows a fluorescent microscopy image of a ‘"nucleation spots"’ characterized by the detection of GFP fluorescence belonging to the a4(32 nAChR-DCs. Every sample is dispensed in the capacitor area of the device to maximize its exposure to the determined voltage. All samples represent crystallization screenings with a constant voltage of 70 mV, ensuring minimal evaporation of the precipitants. At 0 mV (non-polarized control), no crystal formation was observed.

[0018] Figures 9A-9F exhibit synchrotron X-ray diffraction patterns of the human a4p2 nAChR from a single crystal. FIG. 9A-FIG. 9F are reflections resulting from the diffraction of a single cry stal and another "‘crystal-like” sample harvested from the purified a4p2 nAChR solubilized using the detergent LFC-16. The diffracted samples were grown from a purified sample of a4P2 nAChR at 2.0 mg / mL, within the 70-mV slot of the RMP@LMx V. 1.5 device. The reservoir solution used was Pi-PEG # 30 (50 mM ADA pH 6.8, 12.5% w / v PEG 1500 and 10% w / v PEG 1000).

[0019] Figure 10 shows lipid matrix (LCP / LMx) phase changes induced by VRP over a 10-day period. The lipid phase changes Z-stack images using D1C confocal imaging indicates a remarkable structural change from lipidic cubic phase (days 0-3) to a lamellar phase (days 4- 10).

[0020] Figures 11 A-l 1C show the design evolution of the sample holding layer (RMP@LMx) plates. Fig. 11 A displays the initial concept, featuring a “slide-in” assembly mechanism. Fig. 11 B shows a revised design using a modular assembly approach. Fig. 11C shows the final version suitable for manufacturing.71900-429346-5-

[0021] Figure 12A-12K show exemplary designs of tissue culture plates. Figs. 12A- 12F display the dimensional features of an exemplary 12-well tissue culture plate. Figs. 12G- 12K show the corresponding dimensional features of a lid designed to fit the tissue culture plates.

[0022] Figure 13A-13H show exemplary' designs of a protein crystallization plate. Fig. 13A shows the proposed exemplary plate, which has previously been fabricated using resinbased methods. Figs. 13B-13D illustrate the dimensional features of the plate and Figs. 13E- 13G correspond to the lid design. Fig. 13H displays a cross-sectional view of the plate, highlighting the well geometry'.

[0023] Figure 14 shows an embodiment of the “V4.0” device of the system. A circuit board comprising 1TP and the lithographic electric circuit (e.g.. electrodes) is placed into the sample holding layer as a flat slab. After the circuit board is inserted, the sample holding layer can be closed in order to avoid solution leaking.

[0024] Figure 15 shows an embodiment of the “V4.0"’ device of the system. The sample holding layer comprises a plurality of wells and, upon closing, each well is separated. The circuit board aligns a plurality' of electrodes with the plurality of wells. The circuit board can be made of Plexiglas and then sealed with silicon at the bottom of each well.

[0025] Figure 16 shows an embodiment of a system comprising 8 tissue culture wells coupled to one or more holding wells.

[0026] Figure 17 shows an embodiment of a system comprising 12 tissue culture wells coupled to one or more holding wells.

[0027] Figure 18 shows an embodiment of a system comprising 36 tissue culture wells coupled to one or more holding wells.DETAILED DESCRIPTION

[0028] Various embodiments of the invention are described herein as follow s. In an illustrative aspect, a system for membrane protein crystallization is provided. The system for membrane protein crystallization comprises i) a sample holding layer comprising one or more holding wells and ii) a lithographic electric circuit, wherein the sample unit is in contact with an external voltage supply circuit.

[0029] The holding wells present in the sy stem are not limited in any specific volume and the embodiments provided herein can include any volume amount of a holding well. For instance, a holding well can have a volume of between about 1 pl to about 50 pl, or a volume between about 1 pl to about 5 pl, or a volume between about 5 pl to about 10 pl, or a volume between about 10 pl to about 15 pl, or a volume between about 15 pl to about 20 pl, or a71900-429346-6- volume between about 20 pl to about 25 pl, or a volume between about 25 pl to about 30 pl, or a volume between about 30 pl to about 35 pl, or a volume between about 35 pl to about 40 pl, or a volume between about 40 pl to about 45 pl, or a volume between about 45 pl to about 50 Ml-

[0030] In some embodiments, the system for membrane protein cry stallization is exemplified as shown in Figure 1. For instance, an exemplaryL‘V2.0?’ device for the system and an exemplaryc'V3.0” device for the system are shown near the bottom of Figure 1. In some embodiments, the system for membrane protein crystallization is exemplified as a "‘V4.0” device as shown in Figures 14 and 15.

[0031] The various systems of the present disclosure can provide a more versatile crystallization chamber in the sample holding layer that permits for new approaches to membrane protein (MP) crystallization. For instance, a circuit / crystallization chamber comprising Indium Tin Oxide (ITO) can be utilized. Further, the circuit can be customized to a desired number of electrodes (e.g., a crystallization bolus). In addition, applications of Voltage Relaxation Protocols with variable amplitude, frequency, and decay time kinetics can be performed as well as application of DC Protocols with variable amplitude and frequency. The provided systems can also undergo lipid phase manipulations prior or during MPs crystallization as w ell as high throughput screening (HTS) of MP crystallization conditions. Moreover, enabled dynamic imaging studies can be performed, including but not limited to crystal formation (e.g., in real time), FRAP experiments (e.g., diffusion of membrane proteins) during crystal formation, lipid phase monitoring (e.g., polarized light microscopy), and imaging of the interaction of MPs with fluorescent antibodies.

[0032] The various systems of the present disclosure can further provide for tw o-phase MP crystallization. For instance, HTS of drug delivery into the MP during crystallization experiments can be realized. The open crystallization chamber allows for the addition of small molecules, antibodies, or other pharmacological therapies during MP cry stallization screening. This can enable salt, pH and precipitant gradients during crystal formation as well as enable replacing crystallization conditions during experiments. Further, two-phase MP crystallization experiments can be achieved. The MP can be loaded in the lipid matrix or in the buffer chamber phase during a crystallization experiment. The system can monitor the incorporation of the MP in the lipid matrix during a crystallization experiment. Alternatively, the MP can be loaded after a phase change has been detected in the lipid matrix.

[0033] The various systems of the present disclosure can utilize a voltage relaxation concept in Membrane Protein (MP) crystallization, as the kinetics of voltage decay can be used to increase the life-time of a MP conformation. For example, charge residues such as amino71900-429346-7- acids or glycans can create voltage-dependent structural domains in a MP. The presence of charged domains in a MP can led to discrete or major conformational changes in the presence of an electric field. In addition, the kinetics of decay of a voltage relaxation protocol (VRP) that is delivered to the lipid matrix containing the MP can increase or decrease the lifetime of a given conformation of a MP. A VRP can also be combined with a DC protocol to increase the life-time of a conformation of a MP.

[0034] As described herein, the systems of the present disclosure can deliver an array of MP conformations in a single experiment. The VRP delivered into the lipid matrix containing the MP can produce an array of multiple conformations in a single experiment. The kinetics of decay of a voltage relaxation protocol (VRP) can be adjusted during the experiments to reduce or increase the lifetime of the conformation(s) of a MP. Therefore, the VRP protocol can increase or reduce the number of MP conformations during an experiment.

[0035] In an embodiment, the one or more holding wells comprise a lipid matrix. In an embodiment, the lipid matrix comprises a membrane protein.

[0036] In an embodiment, the lipid matrix is present in the one or more holding wells at a volume between about 100 nl to about 300 nl. In an embodiment, the lipid matrix is present in the one or more holding wells at a volume of about 100 nl. In an embodiment, the lipid matrix is present in the one or more holding wells at a volume of about 150 nl. In an embodiment, the lipid matrix is present in the one or more holding wells at a volume of about 200 nl. In an embodiment, the lipid matrix is present in the one or more holding wells at a volume of about 250 nl. In an embodiment, the lipid matrix is present in the one or more holding wells at a volume of about 300 nl.

[0037] In an embodiment, the lipid matrix is present in the one or more holding wells at a volume between about 10 nl to about 30 nl. In an embodiment, the lipid matrix is present in the one or more holding wells at a volume of about 10 nl. In an embodiment, the lipid matrix is present in the one or more holding wells at a volume of about 15 nl. In an embodiment, the lipid matrix is present in the one or more holding wells at a volume of about 20 nl. In an embodiment, the lipid matrix is present in the one or more holding wells at a volume of about 25 nl. In an embodiment, the lipid matrix is present in the one or more holding wells at a volume of about 30 nl.

[0038] In an embodiment, the sample unit is a sample board.

[0039] In an embodiment, the lithographic electric circuit comprises one or more electrodes. In an embodiment, the one or more electrodes are configured to correspond to the one or more holding wells.71900-429346-8-

[0040] In an embodiment, the one or more electrodes are separated by a distance of between about 50 pm and 100 pm. In an embodiment, the one or more electrodes are separated by a distance of between about 100 pm and 150 pm. In an embodiment, the one or more electrodes are separated by a distance of betw een about 150 pm and 200 pm. In an embodiment, the one or more electrodes are separated by a distance of between about 200 pm and 250 pm. In an embodiment, the one or more electrodes are separated by a distance of between about 250 pm and 300 pm. In an embodiment, the one or more electrodes are separated by a distance of between about 300 pm and 350 pm. In an embodiment, the one or more electrodes are separated by a distance of between about 350 pm and 400 pm.

[0041] In an embodiment, the one or more electrodes are separated by a distance of about 50 pm. In an embodiment, the one or more electrodes are separated by a distance of about 60 pm. In an embodiment, the one or more electrodes are separated by a distance of about 70 pm. In an embodiment, the one or more electrodes are separated by a distance of about 80 pm. In an embodiment, the one or more electrodes are separated by a distance of about 90 pm. In an embodiment, the one or more electrodes are separated by a distance of about 100 pm.

[0042] In an embodiment, the one or more electrodes are separated by a distance of about 110 pm. In an embodiment, the one or more electrodes are separated by a distance of about 120 pm. In an embodiment, the one or more electrodes are separated by a distance of about 130 pm. In an embodiment, the one or more electrodes are separated by a distance of about 140 pm. In an embodiment, the one or more electrodes are separated by a distance of about 150 pm. In an embodiment, the one or more electrodes are separated by a distance of about 160 pm. In an embodiment, the one or more electrodes are separated by a distance of about 170 pm. In an embodiment, the one or more electrodes are separated by a distance of about 180 pm. In an embodiment, the one or more electrodes are separated by a distance of about 190 pm. In an embodiment, the one or more electrodes are separated by a distance of about 200 pm.

[0043] In an embodiment, the one or more electrodes are separated by a distance of about 210 pm. In an embodiment, the one or more electrodes are separated by a distance of about 220 pm. In an embodiment, the one or more electrodes are separated by a distance of about 230 pm. In an embodiment, the one or more electrodes are separated by a distance of about 240 pm. In an embodiment, the one or more electrodes are separated by a distance of about 250 pm. In an embodiment, the one or more electrodes are separated by a distance of about 260 pm. In an embodiment, the one or more electrodes are separated by a distance of about 270 pm. In an embodiment, the one or more electrodes are separated by a distance of71900-429346-9- about 280 pm. In an embodiment, the one or more electrodes are separated by a distance of about 290 pm. In an embodiment, the one or more electrodes are separated by a distance of about 300 pm.

[0044] In an embodiment, the one or more electrodes are separated by a distance of about 310 pun. In an embodiment, the one or more electrodes are separated by a distance of about 320 pm. In an embodiment, the one or more electrodes are separated by a distance of about 330 pm. In an embodiment, the one or more electrodes are separated by a distance of about 340 (rm. In an embodiment, the one or more electrodes are separated by a distance of about 350 pun. In an embodiment, the one or more electrodes are separated by a distance of about 360 pm. In an embodiment, the one or more electrodes are separated by a distance of about 370 pm. In an embodiment, the one or more electrodes are separated by a distance of about 380 pm. In an embodiment, the one or more electrodes are separated by a distance of about 390 pun. In an embodiment, the one or more electrodes are separated by a distance of about 400 pirn.

[0045] In an embodiment, the lithographic electric circuit is configured to deliver one or more voltage relaxation pulses. In an embodiment, the one or more voltage relaxation pulses are delivered to a membrane protein sample. In an embodiment, the one or more voltage relaxation pulses are delivered to a lipid matrix comprising a membrane protein.

[0046] In an embodiment, the lithographic electric circuit is printed on the sample unit. In an embodiment, the lithographic electric circuit is etched on the sample unit. In an embodiment, the lithographic electric circuit is present on a glass plate. In an embodiment, the glass plate is coated with indium tin oxide (ITO).

[0047] In an embodiment, the external voltage supply circuit is configured to apply a plurality of voltage relaxation protocols to a plurality of the one or more holding wells, In an embodiment, each of the holding wells comprises a lipid matrix. In an embodiment, the plurality of voltage relaxation protocols are applied to the plurality of the one or more holding wells simultaneously.

[0048] In an embodiment, the external voltage supply circuit is configured to alter intensity of a potential field. In an embodiment, the external voltage supply circuit is configured to alter direction of a potential field.

[0049] In an embodiment, the system further comprises a cover slip. In an embodiment, the cover slip is a glass cover slip.

[0050] In an embodiment, the system further comprises a spacer. In an embodiment, the spacer is a silicone spacer. In an embodiment, the spacer is a polystyrene spacer.71900-429346-10-

[0051] In an embodiment, the system further comprises a lid layer configured for placement over the sample holding layer. In an embodiment, the lid layer is a locking lid layer. In an embodiment, the lid layer is configured to seal contents of the sample holding layer.

[0052] In an embodiment, the system further comprises an incubator receiving the sample unit. In an embodiment, the system further comprises a temperature unit monitoring and controlling a temperature inside of the incubator. In an embodiment, the system further comprises a humidity unit monitoring and controlling a humidity inside of the incubator.

[0053] In an illustrative aspect, a system for peripheral protein crystallization is provided. The system for membrane protein cry stallization comprises i) a sample holding layer comprising one or more holding wells and ii) a lithographic electric circuit, wherein the sample unit is in contact with an external voltage supply circuit. The previously described embodiments of the system for membrane protein crystallization are applicable to the system for peripheral protein crystallization, except that a peripheral protein is the subject of the alternate system instead of a membrane protein. In an embodiment, the peripheral protein is an antigen.

[0054] In an illustrative aspect, a use of the system for membrane protein cry stallization is provided. In an embodiment, the system provides for harvesting of one or more cry stals from a membrane protein. In an embodiment, the system provides for screening of cry stallization conditions a membrane protein. In an embodiment, the screening is a high throughput screening. In an embodiment, the system provides for imaging crystal formation from a membrane protein. In an embodiment, the system provides for in situ crystal diffraction from a membrane protein. In an embodiment, the system provides for in situ cry stal data collection of a resting membrane potential (RMP).

[0055] In an embodiment, the use comprises a light imaging experiment. In an embodiment, the use comprises fluorescence recovery after photobleaching (FRAP). In any of the embodiments, the use comprises real time monitoring and / or analysis.

[0056] In an illustrative aspect, a further system is provided. The system comprises a) a sample unit comprising i) a sample holding layer comprising one or more holding wells, ii) a lithographic electric circuit, and iii) one or more tissue culture wells coupled to one or more holding wells, wherein the sample unit is in contact yvith an external voltage supply circuit.

[0057] The previously described embodiments of the system composition are applicable to the further system comprising the one or more tissue culture wells described herein.

[0058] The tissue culture yvells present in the further system are not limited in any number and the embodiments provided herein can include any number of tissue culture wells. In an embodiment, the further system comprises at least 8 tissue culture yvells. This71900-429346-11- embodiment is exemplified as shown in Figure 16. In an embodiment, the further system comprises at least 12 tissue culture wells. This embodiment is exemplified as shown in Figure 17. In an embodiment, the further system comprises at least 36 tissue culture wells. This embodiment is exemplified as shown in Figure 18.

[0059] The tissue culture wells present in the further system are not limited in any specific volume and the embodiments provided herein can include any volume amount of a tissue culture well. For instance, tissue culture well can have a volume of between about 50 pl to about 1000 pl, or a volume between about 50 pl to about 100 pl, or a volume between about 100 pl to about 150 pl, or a volume between about 150 pl to about 200 pl, or a volume between about 200 pl to about 250 pl, or a volume between about 250 pl to about 300 pl, or a volume between about 300 pl to about 350 pl. or a volume between about 350 pl to about 400 pl, or a volume between about 400 pl to about 450 pl, or a volume between about 450 pl to about 500 pl, or a volume between about 500 pl to about 550 pl, or a volume between about 550 pl to about 600 pl, or a volume between about 600 pl to about 650 pl, or a volume between about 650 pl to about 700 pl, or a volume between about 700 pl to about 750 pl, or a volume between about 750 pl to about 800 pl, or a volume between about 800 pl to about 850 pl, or a volume betw een about 850 pl to about 900 pl, or a volume between about 900 pl to about 950 pl, or a volume between about 950 pl to about 1000 pl.

[0060] In any of the embodiments of the further system comprising the one or more tissue culture wells, the system can further comprise a lid for the portion of the system comprising the tissue culture wells. In any of the embodiments of the further system comprising the one or more tissue culture wells, the system can further comprise a case for the portion of the system comprising the tissue culture wells.

[0061] Materials and methods described in U.S. Patent No. 11,717.819 are known to the skilled artisan and the patent is hereby incorporated herein in its entirety.

[0062] The following numbered embodiments are contemplated and are non-limiting:1. A system for membrane protein crystallization comprising a sample unit comprising i) a sample holding layer comprising one or more holding wells and ii) a lithographic electric circuit, wherein the sample unit is in contact with an external voltage supply circuit.2. The system of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the one or more holding wells comprise a lipid matrix.3. The system of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the lipid matrix comprises a membrane protein.71900-429346-12-4. The system of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the lipid matrix is present in the one or more holding wells at a volume between about 100 nl to about 300 nl.5. The system of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the lipid matrix is present in the one or more holding wells at a volume of about 100 nl.6. The system of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the lipid matrix is present in the one or more holding wells at a volume of about 150 nl.7. The system of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the lipid matrix is present in the one or more holding wells at a volume of about 200 nl.8. The system of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the lipid matrix is present in the one or more holding wells at a volume of about 250 nl.9. The system of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the lipid matrix is present in the one or more holding wells at a volume of about 300 nl.10. The system of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the lipid matrix is present in the one or more holding wells at a volume between about 10 nl to about 30 nl.11. The system of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the lipid matrix is present in the one or more holding wells at a volume of about 10 nl.12. The system of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the lipid matrix is present in the one or more holding wells at a volume of about 15 nl.13. The system of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the lipid matrix is present in the one or more holding wells at a volume of about 20 nl.14. The system of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the lipid matrix is present in the one or more holding wells at a volume of about 25 nl.71900-429346-13-15. The system of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the lipid matrix is present in the one or more holding wells at a volume of about 30 nl.16. The system of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the sample unit is a sample board.17. The system of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the lithographic electric circuit comprises one or more electrodes.18. The system of clause 17, any other suitable clause, or any combination of suitable clauses, wherein the one or more electrodes are configured to correspond to the one or more holding wells.19. The system of clause 17. any other suitable clause, or any combination of suitable clauses, wherein the one or more electrodes are separated by a distance of between about 50 pm and 100 pm.20. The system of clause 17. any other suitable clause, or any combination of suitable clauses, wherein the one or more electrodes are separated by a distance of between about 100 pm and 150 pm.21. The system of clause 17, any other suitable clause, or any combination of suitable clauses, wherein the one or more electrodes are separated by a distance of between about 150 pm and 200 pm.22. The system of clause 17, any other suitable clause, or any combination of suitable clauses, wherein the one or more electrodes are separated by a distance of between about 200 pm and 250 pm.23. The system of clause 17. any other suitable clause, or any combination of suitable clauses, wherein the one or more electrodes are separated by a distance of between about 250 pm and 300 pm.24. The system of clause 17, any other suitable clause, or any combination of suitable clauses, wherein the one or more electrodes are separated by a distance of between about 300 pm and 350 pm.25. The system of clause 17, any other suitable clause, or any combination of suitable clauses, wherein the one or more electrodes are separated by a distance of between about 350 pm and 400 pm.26. The system of clause 17. any other suitable clause, or any combination of suitable clauses, wherein the one or more electrodes are separated by a distance of about 50 pm, or a distance of about 60 pm, or a distance of about 70 pm, or a distance of about 80 pm, or a distance of about 90 pm, or a distance of about 100 pm.71900-429346-14-27. The system of clause 17. any other suitable clause, or any combination of suitable clauses, wherein the one or more electrodes are separated by a distance of about 110 pm, or a distance of about 120 pm, or a distance of about 130 pm, or a distance of about 140 pm, or a distance of about 150 pm.28. The system of clause 17, any other suitable clause, or any combination of suitable clauses, wherein the one or more electrodes are separated by a distance of about 160 pm, or a distance of about 170 pm, or a distance of about 180 pm, or a distance of about 190 pm, or a distance of about 200 pm.29. The system of clause 17, any other suitable clause, or any combination of suitable clauses, wherein the one or more electrodes are separated by a distance of about 210 pm, or a distance of about 220 pm. or a distance of about 230 pm. or a distance of about 240 pm. or a distance of about 250 pm.30. The system of clause 17, any other suitable clause, or any combination of suitable clauses, wherein the one or more electrodes are separated by a distance of about 260 pm, or a distance of about 270 pm, or a distance of about 280 pm. or a distance of about 290 pm. or a distance of about 300 pm.31. The system of clause 17, any other suitable clause, or any combination of suitable clauses, wherein the one or more electrodes are separated by a distance of about 310 pm, or a distance of about 320 pm, or a distance of about 330 pm, or a distance of about 340 pm. or a distance of about 350 pm.32. The system of clause 17, any other suitable clause, or any combination of suitable clauses, wherein the one or more electrodes are separated by a distance of about 360 pm, or a distance of about 370 pm, or a distance of about 380 pm, or a distance of about 390 pm. or a distance of about 400 pm.33. The system of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the lithographic electric circuit is configured to deliver one or more voltage relaxation pulses.34. The system of clause 33. any other suitable clause, or any combination of suitable clauses, wherein the one or more voltage relaxation pulses are delivered to a membrane protein sample.35. The system of clause 33, any other suitable clause, or any combination of suitable clauses, wherein the one or more voltage relaxation pulses are delivered to a lipid matrix comprising a membrane protein.36. The system of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the lithographic electric circuit is printed on the sample unit.71900-429346-15-37. The system of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the lithographic electric circuit is etched on the sample unit.38. The system of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the lithographic electric circuit is present on a glass plate.39. The system of clause 38, any other suitable clause, or any combination of suitable clauses, wherein wherein the glass plate is coated with indium tin oxide (ITO).40. The system of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the external voltage supply circuit is configured to apply a plurality of relaxation protocols to a plurality of the one or more holding wells, wherein each of the holding wells comprises a lipid matrix.41. The system of clause 40. any other suitable clause, or any combination of suitable clauses, wherein wherein the plurality of voltage relaxation protocols are applied to the plurality of the one or more holding w ells simultaneously.42. The system of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the external voltage supply circuit is configured to alter intensity of a potential field.43. The system of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the external voltage supply circuit is configured to alter direction of a potential field.44. The system of clause 1, any other suitable clause, or any combination of suitable clauses, further comprising a cover slip.45. The system of clause 44, any other suitable clause, or any combination of suitable clauses, wherein the cover slip is a glass cover slip.46. The system of clause 1, any other suitable clause, or any combination of suitable clauses, further comprising a spacer.47. The system of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the spacer is a silicone spacer or wherein the spacer is a polystyrene spacer.48. The system of clause 1, any other suitable clause, or any combination of suitable clauses, further comprising a lid layer configured for placement over the sample holding layer.49. The system of clause 48, any other suitable clause, or any combination of suitable clauses, wherein the lid layer is a locking lid layer.50. The system of clause 48. any other suitable clause, or any combination of suitable clauses, wherein the lid layer is configured to seal contents of the sample holding layer.51. The system of clause 1, any other suitable clause, or any combination of suitable clauses, further comprising an incubator receiving the sample unit.71900-429346-16-52. The system of clause 33. any other suitable clause, or any combination of suitable clauses, further comprising a temperature unit monitoring and controlling a temperature inside of the incubator.53. The system of clause 33, any other suitable clause, or any combination of suitable clauses, further comprising a humidity unit monitoring and controlling a humidity inside of the incubator.54. Use of the system of any one of clauses 1 to 53 for membrane protein crystallization.55. The use of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the system provides for harvesting of one or more cry stals from a membrane protein.56. The use of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the system provides for screening of crystallization conditions of a membrane protein.57. The use of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the screening is a high throughput screening.58. The use of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the system provides for imaging crystal formation from a membrane protein.59. The use of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the system provides for in situ crystal diffraction from a membrane protein.60. The use of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the system provides for in situ crystal data collection of a resting membrane potential (RMP).61. The use of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the use comprises a light imaging experiment.62. The use of clause 54, any other suitable clause, or any combination of suitable clauses, wherein the use comprises fluorescence recovery after photobleaching (FRAP).63. The use of any one of clauses 54 to 62, wherein the use comprises real time monitoring and / or analysis.64. A system comprising a) a sample unit comprising i) a sample holding layer comprising one or more holding wells, ii) a lithographic electric circuit, and iii) one or more tissue culture wells coupled to one or more holding wells, wherein the sample unit is in contact with an external voltage supply circuit.65. The system of clause 64, any other suitable clause, or any combination of suitable clauses, wherein the one or more tissue culture wells comprise at least 8 tissue culture wells.66. The system of clause 64. any other suitable clause, or any combination of suitable clauses, wherein the one or more tissue culture wells comprise at least 12 tissue culture wells.71900-429346-17-67. The system of clause 64. any other suitable clause, or any combination of suitable clauses, wherein the one or more tissue culture wells comprise at least 36 tissue culture wells.68. The system of clause 64, any other suitable clause, or any combination of suitable clauses, wherein the system further comprises a lid for the one or more tissue culture wells.69. The system of clause 64, any other suitable clause, or any combination of suitable clauses, wherein the system further comprises a case for the one or more tissue culture wells.EXAMPLESEXAMPLE 1Fabrication of Systems for Membrane Protein Crystallization

[0063] The instant example provides an exemplary system comprising three main components: (1) a small sample-holding cry stallization circuit board with electrodes to apply voltage, (2) a motherboard to which the sample boards can attach and which contains the voltage supply circuits, and (3) a computer that controls the user-programmable voltage outputs via a custom made graphical user interface. A diagram of the system with various cry stallization chamber configurations is shown in Fig. 1.

[0064] Printed circuit board (PCB) schematics and layouts can be designed in Autodesk Eagle, and AutoCAD and the circuit boards can be fabricated with different PCB finishes, including Electroless Nickel-Immersion Gold and Immersion Silver. A graphical user interface can be programmed in C++ using the Qt cross-platform application framework, to ensure widespread compatibility' in the future. This software will allow a user to program DC outputs for multiple voltage outputs simultaneously, as well as voltage ramps, pulses, and other waveforms.EXAMPLE 2Fabrication of Systems for Membrane Protein Crystallization Comprising Lithographic Electric Circuits

[0065] In the instant example, an exemplary system comprising a lithographic electric circuit capable of delivering voltage-relaxation protocols into a crystallization chamber is provided. The lithographic electric circuits are fabricated in a micrometer scale and are capable of delivering voltage relaxation pulses into the lipid matrix containing the membrane protein (MP). A user-programmable computer-controlled multi-output variable voltage source can be included as part of the system and can permit the application of different voltage relaxation protocols to multiple samples, simultaneously.71900-429346-18-

[0066] The system comprising lithographic elements utilize Indium Tin Oxide (ITO) glass and can allow for membrane protein MP crystallization experiments, for instance by producing crystals that can be frozen. These systems can enhance the crystallization of MPs that incorporates the physiological principle of the resting membrane potential (RMP). The systems comprising lithographic elements (e.g., device V2.0 and device V3.0) can provide advantages to prepare, improve, reproduce, and isolate / crystallize voltage-dependent conformations of biologically relevant MPs.

[0067] For instance, an exemplar}' system comprising a lithographic electric circuit is capable of delivering voltage-relaxation protocols into the crystallization chamber using Indium Tin Oxide (ITO) glass is provided. The system can allow membrane protein crystallization experiments, voltage relaxation experiments in the crystallization of membrane proteins, confocal microscopy observation of crystal growth, and the application of voltage pulses in vivo while performing the FRAP experiments.

[0068] The system can include a voltage pulse generator that can change the amplitude, frequency, and duty cycle of the voltage pulse. This pulse generator can be used with the lithographic device to apply voltage relaxation protocols to the LCP lipid matrix. As part of the programming of the pulse generator, time cycles can be included for the block application of pulse patterns while the samples are within a controlled environment.EXAMPLE 3Lipid Phase Change Analysis

[0069] By applying a protocol of 100 mV with a decay time of 0.10 msec and a frequency of 1.0 Hz for 14 days, significant structural (lipid phase) changes can be observed in an LCP / LMx matrix. As used in the instant example. “LCP / LMx” indicates that once a voltage protocol is applied to the LCP, a mixture of lipid phases will be present in the LCP / LMx.

[0070] In order to enhance the crystallization of the MP, the LCP must diffuse from cubic phase into lamellar (planar) bilayer domains (see Fig. 2A). Thus, it is important to emphasize that in the LCP, the MP crystal forms in the lamellar (planar) bilayer.

[0071] Confocal images of the lipid matrix (or LCP) including Z-stack reconstructions were taken to evaluate the integrity of the lipid matrix before and after a protocol of lOOmV relaxation pulses of 0.10 msec’1decay time with a frequency of 1Hz for 14 days. As shown in Fig. 2B, the diameter of the LCP / LMx was remarkably reduced by -35%. Application of lOOmV relaxation pulses of 0. 10 msec-1decay time with a frequency of 1Hz for 14 days restructured the lipid matrix LPC / LMx into a planar (lamellar) bilayer. This structural change is not caused by temperature changes (or dehydration of the LMx), given that an infrared71900-429346-19- camera was used to monitor temperature and also monitored the humidity during the experiment.

[0072] The analyses demonstrated that the system comprising lithographic elements can be used to promote the planar lipid bilayer that facilitates the crystallization of MPs.

[0073] Further, Polarized Light Microscopy (PLM) has been used to quickly determine lipidic phases. For the instant example, a conductive indium tin oxide (ITO) coated glass "sandw ich" can be used with a polarized light microscope to probe the effects of the applied voltage protocols on the lipid phase via the system comprising lithographic elements. Lipid phase changes can be examined via polarized light microscopy while different voltage protocols are applied to the LCP / LMx containing the MP.

[0074] For instance, a Nikon E400 polarized light microscope can examine the lipid phase changes during the application of repetitive voltage relaxation pulses or fixed voltage protocol (see, e.g., Figs. 3A-3B and 4A-4B). As shown in Fig. 3A and Fig. 4A, the lipid matrix turns black at day 5, indicating a remarkable structural change to a lamellar phase. In contrast, the control lipid matrix remains in a cubic phase. A Scanning Electron Microscopy can be used to characterize these phase changes.EXAMPLE 4Analysis of Mobility / Diffusion of the MP in the Lipid Matrix

[0075] Fluorescence recovery after photobleaching (FRAP) has been effectively used to study the diffusion of MPs such as bacteriorhodopsin in lipidic cubic phase (LCP). In general, the diffusion properties within the LCP / LMx have been shown to strongly depend on the protein construct and applied screening conditions. A high protein mobile fraction and a fast diffusion rate correlate well with good crystallization conditions. Thus, a mobile fraction of 0.8 or higher is generally taken as an indication of significant diffusion of the MP with high potential for crystallization.

[0076] The applied electric field may cause spatial reorganization of the lipid matrix and could affect the MP diffusion. For the instant example, FRAP experiments can be performed for MPs (and lipids) using the system comprising lithographic elements. Also, because many MPs have a net charge, the electric field could be moving the proteins in the sample tow ard one electrode. Thus, the voltage generating circuit can be modified to periodically change its polarity’ and maintain the proteins between both electrodes.

[0077] Analysis according to the instant example is shown in Figs. 5A-5J.EXAMPLE 571900-429346-20- Analysis of Thermal Effects

[0078] Electric current flowing through the lipid matrix may heat up the sample, potentially leading to disruption of the desired phase. Thus, infrared imaging can be used to determine if the temperature changes in the sample, due to the applied voltage, are significant.

[0079] A small infrared camera such as the FLIR ETS320, which can detect temperature shifts smaller than 0.06°C, can be placed inside the incubator together with boards containing crystallization samples under RMP to assess whether the temperature remains stable during the procedure (see Fig. 5D). If the sample temperatures increase to a level that affects the lipid phase, the incubator temperature may need to be ramped down during cry stallization trials.EXAMPLE 6Analysis for Leaching of Metals

[0080] Printed circuit boards contain many metals, including nickel, gold, silver, palladium, tin, and lead. A Scanning Electron Microscopy / Energy Dispersive Spectroscopy (SEM-EDS) can be utilized to determine if certain metals are leaching into the LCP samples.

[0081] In the SEM-EDS technique, sample materials are irradiated with electrons resulting in the emission of x-rays characteristic to the elements present in the sample. The energy emissions are translated into spectral peaks (EDS spectra) of varying intensity, resulting in a spectrum profile (see Fig. 6), that identifies the different inorganic elements present in the sample.

[0082] A lipid matrix sample was placed on a crystallization board and a protocol of lOOmV relaxation pulses of 0.10 msec'1decay time with a frequency of 1Hz for 14 days. The sample was extracted, prepared, and analyzed using a JEOL-JSM-IT500Hz / LA with a DRY SD30 detector. Analyses of the LCP samples and lithographic chambers were performed in a low vacuum at 20kV to determine if metals had leeched onto it. Backscatter Electron Images (BEI) micrographs were obtained between 75 and 1000X magnification. Infrared Spectra were also taken from the LCP / LMx samples. No traces of metals were observed in LCP / LMx samples following the protocol of lOOmV relaxation pulses of 0.10 msec1decay time with a frequency of 1Hz for 14 days.EXAMPLE 7Crystallization Screening of Purified Human a4B2 nAChR Using a Polarized in meso MethodExpression and purification of the human a-f>2 nAChR71900-429346-21-

[0083] For the instant example, pEZT-BM vectors of each subunit, a4 and [32, were optimized for large-scale expression of the assembled receptor using the BacMam System. The expression of the nicotinic acetylcholine receptor (nAChR) followed a developed protocol. Briefly, suspension cultures of HEK GnTI-cells (ATCC, CRL 3022) were cultivated in FreeStyle 293 (Gibco) media supplemented with 3 mM sodium butyrate and 0.1 mM nicotine to enhance protein overexpression. The cells were transduced with previously amplified baculoviruses encoding each subunit at a multiplicity of infection (MOI) of 0.25:0.5 of a4 and [32, respectively, and then incubated at 30 °C, 8% CO2, and 95% humidity for 72 hours.

[0084] Afterwards, the cells were harvested by centrifugation and resuspended in 20 mM Tris, pH 7.4, 150 mM NaCl (TBS buffer), 1 mM nicotine, and 1 mM of phenyl methane sulfonyl fluoride (PMSF). The resuspended cells were lysed using the Emulsiflex C5 (Avestin) and subjected to a two-step differential centrifugation to isolate the membranes containing the nAChRs. The initial centrifugation settings were 9,800 x g for 15 minutes to remove cellular debris, followed by 186,000 x g for 2 hours at 4 °C to collect the membranes. The pelleted membranes were mechanically homogenized and incubated for 1 hour at 4 °C in a solubilization buffer containing 1 mM nicotine, 40 mM LFC-16, and 0.2 mM cholesteryl hemisuccinate (CHS) (pH 7.4). After solubilization, the samples were centrifuged at 186,000 x g for 1 hour at 4 °C and purified via affinity chromatography using a Strep Trap HP column (GE Healthcare). The binding buffer consisted of 150 mM NaCl, 20 mM Tris (pH 7.4), 1 mM PMSF, 1 mM LFC-16, 1 mM nicotine, 0.2 mM CHS, and 1 mM tris (2-carboxyethyl) phosphine (TCEP). The same binding buffer was used to prepare the elution buffer by adding 5 mM desthiobiotin.In meso crystallization and X-ray diffraction

[0085] Crystallization screenings were performed incorporating the designed lithographic membrane protein crystallization device. To concentrate the a4(32 nAChR- detergent complexes (DC), the samples were centrifuged using Amicon centrifugal filter units (Millipore). Afterward, the concentrated protein fractions were reconstituted into the lipid cubic phase (LCP) monoolein matrix. In brief, the monoolein was melted at 40 °C and loaded from the reservoir using a 250-pL gastight syringe with a coupler. An additional 100-pL gastight syringe was used to load the a4(32 nAChR-DC samples. The syringes were connected through the coupler to mix the monoolein and the protein at a 40 / 60 (v / v) ratio, respectively. The contents of each syringe were mixed gently until the lipid mesophase became transparent and without white aggregates.71900-429346-22-

[0086] After the receptor-detergent complexes were incorporated into the lipid mesophase, the mixture was transferred into a 10-pL gastight syringe attached to a PB600 dispenser and dispensed in 0.2-pL boluses on a 36-capacitor plate. Additionally, 1 pL of precipitant solution composed of Pi-PEG #30 50 mM ADA pH 6.8, 12.5% w / v PEG 1500, and 10% w / v PEG 1000 (Hampton Research) were added to each sample before sealing with a coverslip to avoid sample dehydration. The plates were then connected to a high-throughput crystallographic screening device (RMP@LMx) and incubated at 20 °C inside a Faraday cage to isolate from electromagnetic contaminants. The crystallization screenings were monitored for 1-2 months at varying voltages ranging from 70 to 120 mV. After the incubation period, crystals were harvested using a fluorescence microscope (Olympus) and collected with MiTeGen loops before being flash-frozen in liquid nitrogen.Lipidic cubic phase-fluorescence recovery after photobleaching (LCP-FRAP)

[0087] FRAP experiments according to were performed where the purified a.4[S2- nAChR-LFC-16 complex was reconstituted in the LCP matrix and incubated. Because the modified construct of the a.4|32-nAChR intrinsically expresses green fluorescent protein (GFP), no additional fluorophores were incorporated into the samples. The FRAP assays were conducted at room temperature during a 30-day period at intervals of 5 days using a Zeiss Axio Observer LSM 800 confocal microscope at a 20X total magnification. Five pre-bleach images established the baseline fluorescence with a laser bleaching intensity of 6.97% of the total power, followed by the scanning sequence of 500 images. Consequently, each sample was integrated within three 30.0-pm regions of interest (ROIs). The average integrated intensity of the bleached 30.0-pm ROI was used to correct for photobleaching from irradiation during the image-acquisition. The equations used to calculate fractional fluorescence recovery are well established.Analysis of phospholipid molecular species by ultra-performance liquid chromatography (UPLC) coupled to electrospray ionization tandem mass spectrometry (ESI-MS / MS)

[0088] The lipids were extracted using the Bligh and Dyer method with the addition of butylated hydroxy toluene (BHT; 2.9 x | ()5M). To hydrolyze the phospholipids, the samples w ere refluxed for 3.5 hours with MeOH / HCl and dried under nitrogen. For lipid analysis in UPLC-ESI-MS / MS, the samples were sent to CD BioGlyco Company. These were analyzed with Vanquish ultra-high performance liquid phase (UHPLC) coupled with a Thermo Orbitrap Fusion high-resolution mass spectrometer. The chromatography was performed using a Thermo-C30 (2.6 pm, 2.1 mm x 100 mm) column at 40 °C. For mass spectrometry, the mobile71900-429346-23- phase A was a 10 mM ammonium acetate solution in 60:40 of acetonitrile and water. In mobile phase B. the 10 mM ammonium acetate solution was prepared in a 10:90 acetonitrile to isopropanol. The elution gradient was 0-2 min, 30% B, 2-2.1 min, 43% B, 2. 1-12 min, 55% B, 12-15 min, 65% B, 15-19.1 min, 100% B, and 19.1-20 min, 30% B. The injection volume was 5 pL with a flow rate of 0.26 mL / min. Prior to injection in the spectrometer, the samples were reconstituted in 100 pL of isopropanol, acetonitrile, and water (65:30:5). LipidSearch Software (V4.2.28) was used to identify lipids on the raw mass spectra, and for peak alignment and filtering. Total peak normalization was applied to correct the values for magnitude comparison.Lipid analysis of purified human a4fi2 nAChR-DCs

[0089] The UPLC ESI-MS / MS analysis of purified a4|32 nAChR provides an insight into the lipidic profile present within the LFC-16 micelles. As expected, phospholipids were abundantabundant, comprising a total of five species present (LPC. PC, LPMt, PEt, PG), with lysophosphatidylcholine (LPC) being the most abundant (Table 1). Notably, an abundance of LPC 16:0 of molecular weight (MW) 496.3398 m / z was observed, indicating that the micelle is primarily composed of the LFC-16 detergent (MW 495.63).

[0090] Furthermore, these five species displayed acyl-chain lengths ranging from 16:0 to 18:0 carbons, which is consistent with previously published data showing high phospholipid levels in the plasma membrane of HEK cells, among other cell types. Lipids different from the detergent, found in larger quantities, might be closely associated with the 0.4(12 nAChR transmembrane domain (TD) and could play an important role in maintaining its integrity.

[0091] As shown in Table 1, among the species, LPC 16:0 (496.3398 m / z) is found in higher quantities than any other, suggesting that the micelle is composed mostly of the detergent LFC-16.71900-429346-24-a4fi2 nAChR-DCs Diffusion in LCP

[0092] Human a402 nAChR was successfully expressed the using the BacMam system on HEK GnTI- cells. The membranes were isolated through cell lysis and differential centrifugation, followed by solubilization. The solubilized o402-nAChR-LFC-16 complex was then readily purified by affinity chromatography through its strep tag (FIG. 7A). Measuring the fluorescence fractional recovery has proven effective at demonstrating nAChR’s ability to freely diffuse across the LCP matrix. FRAP was employed to evaluate the stability of a.402- nAChR complexes following solubilization with the LFC-16 detergent. The results strongly suggest that the a402 nAChR-DCs were actively diffusing, with a fractional recovery above 80% throughout the entire 30-day incubation period (FIG. 7B).Polarized in meso crystallization screening of the human a4f2 nAChR

[0093] The purified a402-nAChR-LFC-16 complex produced fractions primed for immediate use in crystallization screenings. Within the screening ranges from 70 mV to 120 mV, the samples in the 70 mV group generated crystals that provided valuable diffraction data (FIG. 7C).

[0094] Using the LCP method as the crystallization matrix, since it provides a membrane-like environment for the receptor, LCP samples were prepared with concentrations ranging from 1. 1 to 3. 1 mg / mL of the purified a.402-n AChR to screen for nucleation or protein crystal formation. Notably, nucleation began approximately two weeks after the incubation period at 22 °C (FIG. 8A-FIG. 8D). GFP-labeled a402-nAChR allowed for tracking the protein within the lipid matrix, revealing a diffusion pattern toward nucleation spots that could eventually result in crystal formation. In samples incubated in higher voltages (> 70 mV) dehydration ensued, causing sample loss. Conversely, other samples solubilized using alternative phospholipid analog detergents showed slower nucleation rates and increased aggregation, and they failed to yield protein crystals. Furthermore, additional crystallization screenings using n-dodecyl-0-D-maltoside (DDM), a common detergent used for membrane protein solubilization known to hinder protein function were conducted. No nucleation spots were observed in these samples.71900-429346-25-

[0095] The samples subject to higher voltages underwent dehydration after a two-week incubation period, whereas those exposed to lower voltages remained in their same gel-like state. The evaporation of the precipitant solution in the former might be attributable to an increased temperature caused by the constant voltage exposure. From these first crystallization screenings, a single crystal was harvested from a sample of LCP containing a4p2-nAChR-LFC- 16 complex at 2.0 mg / m, within the 70-mV slot of the RMP@LMx V. 1.5 device. The crystal was promptly stored in liquid nitrogen for further diffraction analysis at the Argonne National Laboratory - Advanced Photon Source (APS) (FIG. 8A-FIG. 8D).Synchrotron x-ray diffraction

[0096] The crystallization conditions that previously yielded diffraction patterns in Tc nAChR studies also resulted in a crystal that enabled the first diffraction patterns of a4(32- nAChR-LFC-16 complex using LCP crystallization setups, as shown in FIGS. 9A-9F. These patterns suggest a consistent degree of organization across all data sets. “Crystal-like” samples were repeatedly obtained that lack diffraction, but were detectable by GFP, indicating that a402-nAChR-LFC-16 compex is in fact diffusing toward nucleation under these conditions. Even though the crystallization condition that had the best results in past experiments were consistently used, additional conditions were tested between screenings. Among these variations, one particular condition resulted in several “crystal-like” formations, including GFP fluorescent crystals. The crystallization condition comprised 0. 1 M sodium cacodylate, 18% (w / v) PEG 8000, 0.2 M calcium acetate, and 0.5 mM DTT; and its diffraction resulted in patterns similar to those obtained in previous experiments.EXAMPLE 8Analysis of Lipid Phase Changes for RMP@LMx

[0097] For the instant example, a polarized light microscope (PLM) was used to examine the lipid phase changes of a lipidic cubic phase (LCP) lipid matrix by the application of a voltage relaxation protocol (VRP) protocol. PLM has been used to quickly determine the lipidic phases / By applying VRP protocols, the system was observed to precisely control the polarization of the lipid bilayer. This capability enables regulation of both the conformational state of the membrane proteins and the phase behavior of the lipid matrix.

[0098] Once an MP is incorporated into the LCP, the diffusion of the protein occurs in bicontinuous cubic-to-lamellar phases. The formation of the MP crystal in the LCP occurs in the lamellar phase domains of the lipid matrix.71900-429346-26-

[0099] Furthermore, the detection of lipid phase changes was also achieved using the systems comprising lithographic elements in conjunction confocal microscopy and real-time polarized microscopy. Analysis indicated that the application of a VRP to the LCP matrix using the described systems produced a lamellar lipid matrix. The formation of a lamellar lipid matrix can enhance the crystallization of MPs. These advancements support high-throughput screening (HTS) for Membrane protein crystallization through two approaches: (1) voltagedependent screening and (2) lipid-phase-based screening for crystal formation. Figure 10 displays the results of the analysis of the instant example.EXAMPLE 9Fabrication of Sample Units and Tissue Culture Embodiments

[0100] A customized sample unit (plate) was developed to enhance the versatility of the RMP@LMx device. The primary' objective was to increase the volume capacity' of each well, transitioning from the previously dispensed 1 microliter to volumes in the milliliter range. This modification was initially intended to prevent sample dehydration during protein crystallization screenings. However, the increased volume capacity also enables expanded functionality, including applications in tissue culturing.

[0101] One of the initial designs featured a "slide-in" assembly mechanism. However, this concept posed challenges in consistently securing the slide, leading to leakage issues (Figure 11A). To address this, the design was modified to improve sealing reliability.Prototy pes were produced using PSA-ty pe materials and replaced the slide-in concept with a modular assembly approach (Figure 1 IB). The plate was designed for single-use and is assembled by adhering the glass slide to the base using a pressure-sensitive adhesive (PSA), followed by placement of the lid.

[0102] Both the RM P A LM. x device and its associated plate system were designed for scalability7, enabling customization to meet a wide range of experimental needs. The initial prototypes were presented in a standard size range for compatibility' with the current electric board configuration and early-stage testing and validation. However, the system is inherently scalable and can be modified in both dimensions and layout to suit different applications.

[0103] Figures 12A-12K illustrate the design of a 12-well tissue culture plate prototy pe currently in production. Figs. 12A-12F detail the dimensional features of the plate, which measures approximately 56.10 mm x 52.75 mm x 8.70 mm. The plate includes a recessed bottom section with a width of 50.10 mm. specifically engineered to accommodate the insertion of a 50 mm x 60 mm glass slide device and structural ribs to secure the glass in place. Each well features a 0. 1 mm indent and is manufactured with a surface roughness of Ra < 0.01 mm to71900-429346-27- support optimal cell adhesion and imaging clarity, while the remaining surfaces of the plate maintain a roughness of Ra < 0.02 mm. For this model, each well has a volume capacity of about 300 microliters.

[0104] Figs. 12G-12K present the corresponding lid design, which measures approximately 52.52 mm x 55.93 mm x 10.35 mm. The lid includes integrated ribs that facilitate ventilation of the tissue cultures. A raised section at the front of the lid prevents interference with the inserted glass slide device, ensuring a secure fit. The lid also incorporates a locking mechanism along its sides that aligns with the plate edges, preventing accidental detachment during handling or incubation. According to the manufacturing details, burrs do not exceed 0.05 mm and the PSA thickness is about 0.1 mm.

[0105] Figures 13A-13H illustrate the design of a protein crystallization plate prototype intended for manufacturing. Fig. 13A presents the proposed final version, which has been fabricated in-house using resin-based methods and is currently undergoing testing. Fig. 13B- 13D detail the dimensional features of the plate, which measures approximately 57 mm x 50.79 mm x 3.55 mm. The bottom section includes a recessed area with a width of 50.00 mm, specifically designed to accommodate the insertion of the RMP@LMx device. Each w ell features a 0.5 mm indent, designed to facilitate protein crystal harvesting by providing a wider upper surface area. This design allows users to introduce harvesting loops without clashing with the microscope’s objectives, improving usability during crystal collection.

[0106] Figs. 13E-13G depict the lid design, which measures approximately 57. 13 mm x 51.29 mm x 4.20 mm. Unlike the tissue culture plate, the lid does not incorporate ventilation ribs, a deliberate choice to minimize sample dehydration during crystallization. Similar to the tissue culture plates, a raised section at the front of the lid prevents physical interference with the inserted glass slide device. The lid is designed to fit securely over the plate but does not currently incorporate a dedicated locking mechanism, though we are considering integrating latching features to enhance stability during handling. Fig. 13H represents the well geometry and the volume capacity for this specific model is 25 microliters. While manufacturing specifications such as surface roughness and tolerances are pending finalization, the design aims to maintain similar standards to the tissue culture plate, including optical clarity and compatibility with automated systems.

[0107] The RMP@LMx device itself is designed to be scalable. Both the circuit layout and glass dimensions can be customized to match the plate format, allowing seamless integration across different well configurations and experimental workflows. To accommodate diverse experimental needs, the RMP? / LMx plate system was also designed to be both scalable and application-specific. Plate formats can be configured to include anywhere from 1 to 38471900-429346-28- wells, supporting workflows ranging from low-throughput assays to high-throughput screenings. All plate variants are intended for single-use (disposable) and are fabricated from polystyrene (PS) that are RoHS compliant, selected for its optical clarity and compatibility' with biological applications.

[0108] Tissue culture plates are sterilized by the ethylene oxide (EO) method and are individually wrapped in trays to maintain sterility until use. These plates are suitable for cellbased assays, long-term culturing workflows, and live monitoring or microscopy as it is transparent with flat optical surfaces. Protein crystallization plates, while typically not requiring sterilization, may be optionally sterilized depending on specific experimental needs. The well dimensions and geometries are tailored to each application: crystallization plates feature smaller wells optimized for microvolume dispensing and evaporation control, while tissue culture plates incorporate larger wells to support higher media volumes and extended incubation periods. Optional features such as tissue culture surface treatment are available depending on the intended use case.

Claims

71900-429346-29-WHAT IS CLAIMED IS:

1. A system for membrane protein crystallization comprising a sample unit comprising i) a sample holding layer comprising one or more holding wells and ii) a lithographic electric circuit, wherein the sample unit is in contact with an external voltage supply circuit.

2. The system of claim 1, wherein the one or more holding wells comprise a lipid matrix.

3. The system of claim 2, wherein the lipid matrix comprises a membrane protein.

4. The system of claim 2, wherein the lipid matrix is present in the one or more holding wells at a volume between about 100 nl to about 300 nl.

5. The system of claim 2, wherein the lipid matrix is present in the one or more holding wells at a volume between about 10 nl to about 30 nl.

6. The system of claim 1, wherein the lithographic electric circuit comprises one or more electrodes.

7. The system of claim 6, wherein the one or more electrodes are configured to correspond to the one or more holding wells.

8. The system of claim 6, wherein the one or more electrodes are separated by a distance of betw een about 50 pm and 100 pm.

9. The system of claim 6. wherein the one or more electrodes are separated by a distance of between about 100 pm and 150 pm.

10. The system of claim 6, wherein the one or more electrodes are separated by a distance of betw een about 150 pm and 200 pm.

11. The system of claim 1, wherein the lithographic electric circuit is configured to deliver one or more voltage relaxation pulses.

12. The system of claim 11 , wherein the one or more voltage relaxation pulses are delivered to a membrane protein sample.

13. The system of claim 11, wherein the one or more voltage relaxation pulses are delivered to a lipid matrix comprising a membrane protein.

14. The system of claim 1, wherein the lithographic electric circuit is printed on the sample unit.

15. The system of claim 1, wherein the lithographic electric circuit is etched on the sample unit.

16. The system of claim 1. wherein the lithographic electric circuit is present on a glass plate.71900-429346-30-17. The system of claim 16, wherein the glass plate is coated with indium tin oxide (ITO).

18. The system of claim 1 , wherein the external voltage supply circuit is configured to apply a plurality of voltage relaxation protocols to a plurality of the one or more holding wells, wherein each of the holding wells comprises a lipid matrix.

19. The system of claim 18, wherein the plurality of voltage relaxation protocols are applied to the plurality of the one or more holding wells simultaneously.

20. The system of claim 1, wherein the external voltage supply circuit is configured to alter intensity7of a potential field.

21. The system of claim 1, wherein the external voltage supply circuit is configured to alter direction of a potential field.

22. The system of claim 1, further comprising an incubator receiving the sample unit.

23. Use of the system of any one of claims 1 to 22 for membrane protein crystallization.

24. The use of claim 23, wherein the system provides for harvesting of one or more cry stals from a membrane protein.

25. The use of claim 23, wherein the system provides for screening of crystallization conditions a membrane protein.

26. The use of claim 25, wherein the screening is a high throughput screening.

27. The use of claim 23, wherein the system provides for imaging crystal formation from a membrane protein.

28. The use of claim 23, wherein the system provides for in situ cry stal diffraction from a membrane protein.

29. The use of claim 23, wherein the system provides for in situ crystal data collection of a resting membrane potential (RMP).

30. The use of claim 23, wherein the use comprises a light imaging experiment.

31. The use of claim 23, wherein the use comprises fluorescence recovery after photobleaching (FRAP).

32. A system comprising a) a sample unit comprising i) a sample holding layer comprising one or more holding wells, ii) a lithographic electric circuit, and iii) one or more tissue culture wells coupled to one or more holding wells, wherein the sample unit is in contact with an external voltage supply circuit.71900-429346-31-33. The system of claim 32, wherein the one or more tissue culture wells comprise at least 8 tissue culture wells.

34. The system of claim 32, wherein the one or more tissue culture wells comprise at least 12 tissue culture wells.

35. The system of claim 32, wherein the one or more tissue culture wells comprise at least 36 tissue culture wells.

36. The system of claim 32, wherein the system further comprises a lid for the one or more tissue culture wells.

37. The system of claim 32, wherein the system further comprises a case for the one or more tissue culture wells.

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