An efficient clear-native PAGE–based workflow for cryoelectron microscopy sample preparation of large protein complexes

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However, preparing ultra-large protein complexes (e.g., > 700 kDa) for cryo-EM remains challenging, as it requires preserving both structural integrity and the native state. Conventional isolation methods, such as sucrose density gradient centrifugation, require large sample volumes and provide limited separation resolution. In contrast, native PAGE offers higher resolution; however, no established method exists for extracting protein complexes from gels followed by further purification to achieve high purity. Consequently, no standardized native PAGE-based protocol for cryo-EM sample preparation avoids multiple purification steps. Hence, we aimed to develop a rapid and efficient cryo-EM protein sample preparation method using electroelution with an optimized buffer system that preserves complex integrity to recover target protein complexes after sodium deoxycholate (DOC)-based clear-native PAGE (CN-PAGE). Results We developed an agarose–acrylamide composite gel, which is simpler to prepare and mechanically more robust than conventional linear-gradient acrylamide gels commonly used for CN-PAGE, facilitating precise band excision for efficient electroelution. Cryo-EM structural analysis of the photosystem I–light-harvesting complex I (PSI–LHCI) supercomplex from Arabidopsis thaliana achieved high resolution (2.18 Å) after electroelution from this gel, requiring only buffer exchange by ultrafiltration to remove DOC before grid preparation, without additional chromatographic purification. This finding suggests that DOC may be the main inhibitor of successful grid preparation. Conclusion Our results demonstrate the potential of this method for isolating large protein complexes from small sample volumes for cryo-EM structural analysis. This approach significantly broadens the scope of cryo-EM targets to include challenging systems previously hindered by purification difficulties, thereby accelerating structural studies crucial for understanding complex biological processes. Clear-native PAGE Electroelution Cryo-EM Photosystem Protein complex Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Protein complexes play indispensable roles in various cellular processes ranging from signal transduction to metabolic regulation. Their intricate three-dimensional structures enable specific interactions and regulatory mechanisms central to fundamental biological events. Detailed structural insights into these complexes advance the understanding of protein function and enable broader applications, including drug discovery and synthetic biology. Despite major advances in structural biology techniques such as cryoelectron microscopy (cryo-EM) [ 1 – 5 ], protein complexes remain structurally uncharacterized because of technical challenges, including sample heterogeneity, complex instability, and purification difficulties [ 6 , 7 ]. Developing reliable methods to isolate and analyze protein complex structures is therefore pivotal for revealing the molecular basis of key biological phenomena [ 1 , 3 , 6 , 7 ]. Photosystems (PSs) in photosynthetic organisms are particularly challenging targets for structural characterization. These large membrane-embedded complexes, such as the PSI–light-harvesting complex (LHC) I (hereafter referred to as the PSI complex, weighing ~ 700 kDa) and PSII–LHCII (hereafter referred to as the PSII complex, weighing ~ 1400 kDa) [ 8 , 9 ], exhibit properties that complicate conventional purification approaches. Notably, LHC antenna proteins often dissociate during purification [ 8 , 10 ], complicating both isolation and maintenance of intact complexes. These characteristics can compromise the efficiency of purification strategies such as gel filtration and ion-exchange chromatography, often resulting in low success rates. Furthermore, as membrane-embedded complexes, PSs present additional difficulties in solubilization while maintaining their native conformation during structural studies [ 8 , 11 , 12 ]. Collectively, these challenges hinder the structural and functional characterization of large membrane-protein complexes, including PSs. Therefore, new purification strategies capable of maintaining complex stability and native architecture are required. In addition to column chromatography, sucrose density gradient (SDG) centrifugation has been widely used to separate PSs [ 8 , 13 , 14 ] for structural analysis by X-ray crystallography and cryo-EM. However, this method typically requires large amounts of starting material and provides relatively low-resolution separation. In contrast, native polyacrylamide gel electrophoresis (native PAGE) provides higher resolution while requiring smaller protein amounts [ 9 , 15 ]. Because cryo-EM generally requires less protein than that required by X-ray crystallography, extracting protein complexes from native PAGE gels without disturbing their native structures would be an ideal approach for preparing cryo-EM samples. Two major types of native PAGE exist: blue-native PAGE (BN-PAGE) and clear-native PAGE (CN-PAGE) [ 15 ]. BN-PAGE uses Coomassie Brilliant Blue (CBB G-250) dye to impart negative charge to protein complexes and facilitate migration [ 15 , 16 ], whereas CN-PAGE uses sodium deoxycholate (DOC) [ 15 , 17 ]. CN-PAGE is particularly advantageous for studying pigment–protein complexes in PSs because it avoids CBB dye, which can interfere with subsequent analyses, especially spectroscopy. Thus, CN-PAGE serves as a superior alternative to SDG, offering higher-resolution separation with smaller sample volumes and enabling clear distinction between PSI and PSII complexes before cryo-EM analysis [ 18 ]. However, two major challenges remain for applying CN-PAGE to cryo-EM structural analysis. First, establishing a reliable, reproducible, and broadly applicable method for extracting proteins from the gel matrix after separation remains difficult [ 19 ]. A common extraction method relies on passive diffusion, where an excised gel slice containing the protein complex is soaked in buffer for a prolonged period to allow diffusion from the gel [ 18 , 19 ]. This approach is inefficient, time-consuming, and often results in substantial sample loss. Furthermore, CN-PAGE–based cryo-EM sample preparation generally requires multiple additional purification steps after extraction, further increasing sample loss and labor [ 18 ]. Second, preparing gradient gels for CN-PAGE [ 19 ] is more technically demanding than handling sucrose gradients. These limitations hinder cryo-EM studies of PSI and PSII complexes purified by CN-PAGE. Additionally, the pliable nature of low-concentration acrylamide gels makes it difficult to excise bands precisely after electrophoresis. Hence, to address these technical issues, we aimed to develop a rapid and efficient method for preparing PSI and PSII samples suitable for high-resolution cryo-EM analysis. Our method involves separating PSI and PSII complexes using an agarose–acrylamide composite gel without a gradient, extracting them by electroelution with a new buffer system, and purifying the PSs by ultrafiltration to remove DOC, which may interfere with sample adhesion to cryo-EM grids. Using this approach, we demonstrated that PSs extracted from Arabidopsis thaliana retained structural integrity and were successfully analyzed by cryo-EM. Although demonstrated using PSs as challenging targets, this approach is broadly applicable to diverse large membrane protein complexes. This method provides a streamlined workflow for cryo-EM sample preparation, enabling efficient high-resolution structural analysis of various protein complexes beyond PSs. Materials and methods Plant material and growth condition A. thaliana (ecotype Columbia) plants were grown in fertilized soil for 6 weeks at 23°C under a 14 h light/10 h dark photoperiod with a light intensity of 70 µmol photons m − 2 s − 1 . Fully expanded rosette leaves were harvested from pre-bolting vegetative-stage plants. Agarose–acrylamide composite gel All chemical ratios used for the agarose–acrylamide composite gel (separation gel) and sample gel are listed in Table 1 . Agarose powder (Lonza, Switzerland) was dissolved in a solution containing Milli-Q water and 3× gel buffer. After cooling to approximately 50°C, the remaining components listed in Table 1 were immediately added, followed by thorough mixing. The mixture was poured into a preheated, preassembled gel cassette, leaving sufficient space for the sample gel to solidify at room temperature (20–25°C). Milli-Q water was added after solidification, and the gel was stored at 4°C overnight before proceeding. Experimental procedures followed the protocol detailed in Additional file 1. Sample preparation and separation of thylakoids by DOC-based CN-PAGE Thylakoid membranes from A. thaliana were isolated according to Ye et al. (2025)[ 20 ] with minor modifications. Fresh leaves (approximately 80 plants) were ground in 200 mL of prechilled grinding buffer (20 mM Tricine/KOH, pH 7.6; 0.4 M sorbitol; 10% polyethylene glycol (PEG)-6000; 10 mM ethylenediaminetetraacetic acid [EDTA]-2Na) using a prechilled blender (Twinbird, Japan). The homogenate, filtered through four layers of Miracloth (Merck Millipore, USA), was centrifuged at 10,000 × g for 5 min at 4°C. The pellet was resuspended in 5 mL of wash buffer (10 mM Tricine/KOH, pH 7.6; 0.4 M sorbitol; 2.5 mM MgCl₂; 1.25 mM EDTA-2Na) and recentrifuged under the same conditions. The final pellet was resuspended in a solubilization buffer (50 mM imidazole/HCl, pH 7.0; 20% (v/v) glycerol) to a chlorophyll concentration of 1 mg mL − 1 . The thylakoid suspension was solubilized with 1% (w/v) α-DDM (n-Dodecyl α-D-maltoside) for 2 min on ice in solubilization buffer containing 10 mM sodium fluoride as a phosphatase inhibitor and a plant protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Samples were centrifuged at 22,500 × g for 5 min at 4°C. The resulting supernatant was loaded onto an agarose–acrylamide gel. Electrophoresis was performed at 4°C using an anode buffer (50 mM imidazole/HCl, pH 7.0) and a cathode buffer (50 mM Tricine; 15 mM imidazole/HCl, pH 7.0) containing 0.05% DOC and 0.02% α-DDM, as described by Järvi et al. [ 9 ]. Details are provided in Additional file 1. 2D-CN/Sodium dodecyl sulfate (SDS)-PAGE followed by silver staining For second-dimensional electrophoresis, a gel strip corresponding to a single lane was excised from the first-dimensional CN-PAGE gel. The proteins in this strip were then denatured by incubation in SDS solubilization buffer (1% SDS, 50 mM DTT) for 30 min at room temperature. Proteins were separated on a 14% acrylamide gel containing 6 M urea using the Laemmli buffer system [ 21 ]. Precision Plus Protein Unstained Standards (Bio-Rad, Hercules, CA, USA) served as molecular weight markers. After electrophoresis, the separated proteins were visualized using a Pierce Silver Stain Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Bands were identified as previously described [ 9 , 22 ]. PsbB (~ 47 kDa), PsbC (~ 43 kDa), and D1/D2 (~ 28 kDa) were used as PSII markers, whereas PsaD (~ 20 kDa), PsaF (~ 18 kDa), and PsaL (~ 17 kDa) served as PSI indicators. Low-temperature (77 K) chlorophyll fluorescence spectroscopy The fluorescence emission spectra were measured at 77 K using a fluorescence spectrophotometer (F-2700; Hitachi, Japan). An excitation wavelength of 440 nm, which preferentially excites chlorophyll a (Chl a ), was used to record overall emissions from the PSs. Fluorescence intensity of each spectrum was normalized to 683 nm (PSI–PSII and PSII complexes) and 729 nm (PSI complex). Chlorophyll concentration measurement Chlorophyll concentrations of the photosystems were calculated according to the method of Porra et al. (1989)[ 23 ]. The sample solution was diluted in 80% (v/v) acetone, and its absorbance was determined using a Shimadzu UV-2600i UV–Vis spectrophotometer (Shimadzu Corp., Kyoto, Japan). PS sample preparation using electroelution for transmission electron microscopy (TEM) and Cryo-EM PSI and PSII were excised from the agarose–acrylamide gel and extracted by electroelution at 4°C using an Electro-Eluter (Bio-Rad, Hercules, CA, USA) following the manufacturer’s protocol. Buffers for electroelution were based on the DOC-based CN-PAGE system (Fig. 1 a). The anode buffer was used without modification, whereas the cathode buffer was supplemented with 0.05% DOC and 0.02% α-DDM. To enhance protein solubility and prevent aggregation, the cathode chamber between the frit and membrane cap was filled with cathode buffer containing 0.05% α-DDM. Electroelution conditions for two glass tubes were approximately 110 V and 5 mA, with total run time under 3 h when the gel band filled in the glass tube height was less than 1 cm. PSI and PSII were collected from the membrane cap and concentrated using Apollo 7 mL centrifugal concentrators (150 kDa MW cutoff; Orbital Biosciences, Topsfield, MA, USA). The purified proteins were washed three times with 25 mM Bis-Tris buffer (pH 7.5) containing 0.05% α-DDM, concentrated to 20 µL, and stored at − 80°C in the same buffer. Negative-stain electron microscopy (EM) The purified solution was diluted 1:20 or 1:100, and 3.5 µL of diluted sample was applied to glow-discharged continuous carbon film–coated copper grids (Nisshin EM, Tokyo, Japan). After staining with 3.5 µL of 2% uranyl acetate, excess solution was blotted off and grids were air-dried. Samples were examined using an H-7650 Hitachi transmission electron microscope operating at 80 kV and equipped with a 1 × 1 K Tietz FastScan-F114 CCD camera. Cryo-EM grid preparation and data acquisition of A. thaliana PSI-LHCI A 2.6 µL aliquot of PSI–LHCI solution was applied to a glow-discharged Quantifoil holey carbon grid (R1.2/1.3, Cu, 200 mesh) coated with a 5 nm carbon film, blotted for 3.0 s at 4°C, and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Grids were loaded into a Titan Krios G2 (Thermo Fisher Scientific) operating at an acceleration voltage of 300 kV and equipped with a Cs corrector (CEOS GmbH). Images were recorded using a K3 direct electron detector (Gatan, USA) in CDS mode with an energy filter (slit width 20 eV). Data were collected automatically using SerialEM v4.0 [ 24 ] at a physical pixel size of 0.675 Å with 40 frames (1 e − /Å 2 per frame), an exposure time of 1.651 s per movie, and a defocus range of − 0.5 to − 1.0 µm. A total of 8,764 movies were collected. Cryo-EM data processing and model building Movie frames were corrected for beam-induced motion using RELION’s implementation of MotionCor2 [ 25 ], and the contrast transfer function (CTF) was estimated using CTFFIND4 [ 26 ]. Both steps were performed in RELION 4.0 [ 27 ]. Approximately 170,000 particles were automatically selected from almost 1,100 micrographs using a template-free Laplacian-of-Gaussian (LoG) filter and extracted with a 92-pixel box size and 4× binning for two-dimensional (2D) classification. A good 2D class-averaged image was used for Topaz training, after which 1,629,453 particles were automatically picked from all micrographs using Topaz [ 28 ] and three rounds of 2D classification were conducted in RELION 4.0. A total of 361,636 particles were selected to build the 3D initial model of A. thaliana PSI using cryoSPARC2 [ 29 ] and classified into three 3D classes in RELION 4.0. Selected particles were re-extracted at 0.675 Å per pixel and subjected to four 3D refinements, three CTF refinements, and Bayesian polishing. The final 3D refinement and postprocessing yielded maps with a global resolution of 2.18 Å according to the 0.143 Fourier shell correlation criterion. Local resolution was estimated using RELION 4.0. Processing details are provided in Additional file 2. A previously published A. thaliana PSI model (PDB ID: 8J7B) was docked into the EM density map using UCSF ChimeraX [ 30 ]. Each domain and ligands (e.g., Chl a, Chl b) were manually remodeled and refined iteratively using COOT [ 31 ], Phenix [ 32 ], and Servalcat [ 33 ]. Chlorophylls that could not be unambiguously assigned as Chl a or Chl b were defined as unknown ligands (UNLs). All figures were prepared using UCSF ChimeraX. Statistics of 3D reconstruction and model refinement are summarized in Additional file 3. Results An agarose–acrylamide composite gel enables efficient separation of PSI and PSII complexes In this study, we developed an agarose-based composite gel as a simple and rapid alternative to conventional polyacrylamide gradient gels for CN-PAGE. The addition of acrylamide improved gel adhesion to the glass cassette and significantly enhanced the separation of PSI and PSII complexes (Fig. 2 ; Additional file 4a, b).Our agarose–acrylamide composite gel yielded PSI and PSII banding patterns identical to those obtained with a conventional 4%–13% acrylamide gradient gel (Fig. 2 ). Band identities were confirmed by 2D-CN/SDS-PAGE followed by silver staining (Fig. 3 ). To evaluate whether the separated PSs retained their structural integrity for downstream analyses, we performed low-temperature (77 K) chlorophyll fluorescence spectroscopy. The fluorescence spectra showed characteristic peaks for PSII and PSI [ 34 ], without detectable signals from dissociated LHCs or free chlorophyll, indicating that both complexes remained intact within the composite gel (Fig. 3 ). A minor PSII core dimer peak (689 nm) appeared in the PSI band slice, consistent with the overlap arising because of similar molecular sizes—a phenomenon also observed in conventional linear-gradient acrylamide gels. Together, these findings demonstrate that the agarose–acrylamide composite gel provides PS separation comparable to that of conventional acrylamide gradient gels. Protein complexes successfully extracted from agarose–acrylamide composite gel via electroelution The extraction efficiency, as indicated by the residual green color of the gel slices, was lower in the agarose–acrylamide composite gel than in the acrylamide gradient gel. To overcome this limitation, we employed electroelution using a commercial electroeluter to achieve higher recovery efficiency. The apparatus was assembled and operated as shown schematically in Fig. 1 a. The complete workflow for separating and purifying A. thaliana PSs from the composite gel is illustrated in Fig. 1 b. As no buffer system had been established for electroelution of CN-PAGE gels, we adapted the DOC-based CN-PAGE buffer system for this purpose. Electroelution under these optimized conditions efficiently extracted the protein complexes, rendering the gel slices nearly transparent within 2–3 h (Additional file 4c)—a substantial improvement over the conventional soaking method, which typically requires overnight incubation. Optimization of sample preparation after the electroelution for cryo-EM After electroelution, the recovered PSs were concentrated by ultrafiltration (Fig. 1 b). Negative-stain electron microscopy confirmed successful recovery of both PSI and PSII complexes (Fig. 4 ). However, initial attempts to prepare cryo-EM grids using these samples failed to yield resolvable particles, suggesting interference from residual DOC in the electroelution buffer. To test this hypothesis, the samples were subjected to three additional buffer exchange cycles to remove DOC completely. Subsequent grid preparation focused on PSI, which was more abundant than PSII. Following DOC removal, well-dispersed and intact PSI particles were obtained at suitable concentrations for high-resolution cryo-EM analysis. This optimized protocol yielded approximately 20 µL of concentrated PSI sample with a chlorophyll concentration of 400 µg mL − 1 from a starting thylakoid sample containing 200 µg of chlorophyll. Quality assessment of the purified PSI complex by cryo-EM To investigate whether the structure and arrangement of subunits and cofactors such as chlorophylls and carotenoids were preserved, we performed single-particle cryo-EM of the PSI complex. Using grids coated with a 5 nm carbon film for optimal particle distribution, we obtained an EM density map at 2.18 Å resolution, which enabled atomic model construction (Fig. 5 ). This structure comprised twelve subunits (PsaA–PsaL) and four LHCs and included residues 87–136 of PsaN. PsaN, located on the lumenal side, positioned to interact with PsaA, PsaF, PsaJ, and Lhca2 (Fig. 5 ). Discussion In this study, we obtained a high-resolution A. thaliana PSI cryo-EM structure (Fig. 5 ) by developing a two-part methodological pipeline: separation using an agarose–acrylamide composite gel and recovery by efficient electroelution. Below, we discuss the key characteristics of each component. Our composite gel is a powerful alternative to traditional gradient gels for isolating large protein complexes. Because agarose adheres poorly to glass cassettes, the gels were cast onto a GelBond Film (Lonza, Switzerland) to ensure firm adhesion during vertical electrophoresis. Initial experiments using pure agarose gels (up to 1%) failed to resolve PSI and PSII complexes from the model plant A. thaliana ; both complexes migrated together at the gel front (Additional file 4a). This poor resolution likely resulted from the larger pore size of agarose than that of acrylamide gels. Incorporating a small amount of acrylamide (3.6%) into the agarose matrix overcame this limitation. Although the composite gel did not resolve smaller proteins such as free or dissociated LHCs, it separated complexes larger than PSI (≈ 700 kDa) more rapidly than conventional methods (Fig. 2 ). Thus, our electroelution method is broadly applicable for recovering proteins from gel matrices and is particularly effective for rapid preparation of intact, native-state complexes for demanding applications such as cryo-EM. Advantages and future challenges of the agarose–acrylamide composite gel Our composite gel separates PSI and PSII, comparable to that of a conventional 4%–13% acrylamide gradient gel (Fig. 2 ), while using a single concentration. In standard DOC-based CN-PAGE [ 9 , 10 ], an acrylamide gradient gel separates PSII complexes by size, typically resolving three distinct forms—C 2 S 2 M 2 , C 2 S 2 M, and C 2 S 2 . This size variation corresponds to the progressive dissociation of LHCII antenna complexes, as the largest form (C 2 S 2 M 2 ) transitions to C 2 S 2 M and then to C 2 S 2 [ 8 , 35 – 37 ]. Higher agarose concentrations in the composite gel further improved resolution and band sharpness, particularly for PSI bands. The separated PSs retained functional and structural integrity according to 77 K fluorescence spectroscopy and cryo-EM analysis, respectively. This performance was achieved with a simpler gel preparation that eliminates the need for a gradient mixer and peristaltic pump, saving time and resources. The agarose scaffold confers improved mechanical robustness relative to low-concentration polyacrylamide gels (e.g., < 4%), which are often sticky and fragile and thus difficult to handle. This enhanced stability was critical for precise band excision required for downstream analyses. The primary challenge in gel preparation is temperature control. Agarose requires high temperatures to dissolve fully, whereas acrylamide is prone to degradation at high temperatures. Conversely, lowering the temperature to ensure acrylamide stability and agarose solidification complicates the casting process. Through our optimization, we also found it best to avoid loading water onto the separation gel surface because this can produce irregularities; minor surface unevenness did not compromise final separation quality. Despite these constraints, we established an optimized protocol (Materials and Methods) that reproducibly yields high-quality gels. Future improvements could involve exploring lower-melting-point agarose or additives that enhance acrylamide thermal stability, which would simplify casting and broaden applications. Optimization of the electroelution buffer As previously described that agarose gels have larger pores, we expected high extraction efficiency for PSs during recovery. However, the conventional passive diffusion method optimized for acrylamide gradient gels [ 18 ] was unsuitable for this composite matrix. Even after 24 h of soaking in 1% digitonin, a visible amount of the green-colored complexes remained trapped within the gel slices (Additional file 4c). Buffer selection is crucial for successful electroelution. We used the CN-PAGE buffer with additional detergent supplementation in the membrane cap space to prevent protein aggregation. Aggregation reduces image quality and compromises resolution in subsequent cryo-EM analyses. In our study, a detergent concentration of 0.05% α-DDM was optimal; higher concentrations tend to excessively disperse protein–detergent complexes, increasing retention within frit pores and causing protein loss during electroelution. Electroelution typically completes within 3 h using this buffer system. Despite the composite gel’s greater rigidity, extraction proceeded at a competitive rate. The only subsequent processing step was DOC removal by buffer exchange using ultrafiltration, which enabled high-resolution cryo-EM. This rapid, streamlined workflow likely contributed to the high-resolution results and allowed samples recovered by electroelution to be used directly for cryo-EM without additional chromatographic purification steps. The A. thaliana PSI structure including PsaN We resolved the A. thaliana PSI structure and successfully modeled the PsaN subunit (Fig. 5 ), which had not been reported in previous studies [ 18 ]. This result indicates that our purification approach effectively preserves labile protein–protein interactions that are often lost during conventional multistep purification procedures. PsaN is the only membrane-extrinsic PSI subunit on the lumenal side and is found exclusively in green plants and red algae [ 38 , 39 ]. Because PsaN dissociates readily under salt washing [ 40 ], it was absent from many plant PSI-LHCI cryo-EM structures [ 18 ] or inaccurately localized in some crystal structures [ 41 ]. To date, a clear PsaN structure was reported only in maize PSI with an LHCII trimer purified using a gentle, low-ionic-strength method [ 42 ]. Using our protocol, we resolved PsaN except for its highly mobile N- and C-termini. Retention of this labile subunit suggests our method effectively preserves native supercomplex integrity and offers a valuable alternative to existing protocols. Conclusion In this study, we developed and validated a novel pipeline for purifying large membrane-protein complexes that enables high-resolution cryo-EM structural determination. Our workflow, which combines a single-concentration composite gel with efficient electroelution reduces purification time and minimizes sample degradation. The approach was validated using a 2.18 Å structure of plant PSI that retained the labile PsaN subunit, supporting preservation of native integrity. This work provides both a detailed structural view of plant PSI and a practical, broadly applicable methodology for structural biology. The in vitro purification pipeline complements in situ approaches such as cryo-electron tomography [ 43 , 44 ]. It offers a robust route for analyzing challenging complexes, especially those with small extramembrane domains that are difficult to identify in cells. This streamlined workflow promises to facilitate structural analysis of other large assemblies and to advance understanding of complex biological machinery. Abbreviations CN-PAGE Clear-native polyacrylamide gel electrophoresis CTF Contrast transfer function Cryo-EM Cryoelectron microscopy DDM n-Dodecyl α-D-maltoside DOC Sodium deoxycholate EDTA Ethylenediaminetetraacetic acid EM Electron microscopy LHC Light-harvesting complex PEG Polyethylene glycol PS Photosystem TEM Transmission electron microscopy Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials Cryo-EM density maps of the Arabidopsis thaliana PSI complex, isolated using DOC-based CN-PAGE, have been deposited in the Electron Microscopy Data Bank under the accession code EMD-66073. The atomic coordinates of the PSI complex of A. thaliana have been deposited in the Protein Data Bank under the accession code 9WLS. The raw cryo-EM data (EMPIAR-13077) used to generate the density map are available from the Electron Microscopy Public Image Archive. All other data are available from the corresponding author upon request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI under grant numbers 23H04958 (awarded to G.K.), 23H04960 (awarded to R.T.), and 24K09496 (awarded to A.T.). Authors' contributions Z.Y., S.K., and A.T. conceptualized the study. All the authors contributed to the study design. Z.Y., S.K., and A.T. performed sample preparation and associated procedures, including CN-PAGE, electroelution, and ultrafiltration. A.K. performed the cryo-EM structural analysis. Z.Y., A.T., and A.K. prepared original drafts of the manuscript. All authors have reviewed, revised, and approved the final manuscript. Acknowledgements We thank Nobuyuki Endo (University of Osaka) for assistance with TEM observations. References Bai XC, McMullan G, Scheres SHW. How cryo-EM is revolutionizing structural biology. 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Tables Table 1 Composition of separation and sample gels for CN-PAGE Separation gel Agarose concentration 0% 0.25% 0.5% 0.75% 1% Agarose (mg) 45 90 135 180 30% Acrylamide 2.25 mL 3× Gel Buffer 6 mL Milli-Q 10 mL 10% APS 60 µL TEMED 15 µL Total 18 mL Sample gel Acrylamide concentration 3.75% 30% Acrylamide 0.72 mL 3× Gel Buffer 2 mL Milli-Q 3.28 mL 10% APS 50 µL TEMED 5 µL Total 6 mL Additional Declarations No competing interests reported. Supplementary Files Additionalfiles14.pdf Additional file 1.pdf Detailed protocols for the preparation of agarose–acrylamide composite gels and the electroelution procedure. Additional file 2.pdf Cryo-EM data collection and refinement statistics. Additional file 3.pdf Summary of Cryo-EM data acquisition and image processing. (a) Cryo-EM image of purified photosystem (PS)I complex of Arabidopsis thaliana isolated using DOC-based clear-native polyacrylamide gel electrophoresis (CN-PAGE) method. (b) 2D class images of the PSI complex. (c, d) Local resolution and fourier shell correlation curve of the final cryo-EM map of the PSI complex. (E) Flow chart of cryo-EM image processing. Additional file 4.pdf CN-PAGE separation of Arabidopsis thaliana thylakoid membrane complexes and comparison of protein yields obtained by soaking and electroelution. (a) Arabidopsis thaliana thylakoid membrane complexes were separated by clear-native polyacrylamide gel electrophoresis (CN-PAGE) on 0.5% and 1% agarose gels. (b) CN-PAGE and (c) chlorophyll fluorescence images obtained at room temperature. Notably, the chlorophyll yield from photosystem (PS)I was much lower than that from PSII and was not clearly visible. (d) Chlorophyll pigments remaining after extraction using the conventional soaking method or electroelution. 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The cathode buffer contained 0.05% DOC and 0.02% α-DDM, whereas the tank was filled with anode buffer. The membrane cap space was filled with cathode buffer supplemented with an additional 0.05% α-DDM. \u003cstrong\u003e(b) \u003c/strong\u003eWorkflow for sample preparation from DOC-based CN-PAGE to transmission electron microscopy (TEM) or cryoelectron microscopy (cryo-EM) analysis. Gel slices containing the protein of interest were excised and placed in a glass tube for electroelution. Afterward, the target protein was collected from the membrane cap, concentrated, and buffer-exchanged by ultrafiltration. To remove residual DOC, the sample was washed three times with 25 mM Bis-Tris (pH 7.5) containing 0.05% α-DDM. The final concentrated protein was used for TEM or cryo-EM analysis.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8130027/v1/9eb16378df89c4e12f66e428.png"},{"id":96251023,"identity":"002b729c-6d0f-4ab4-85f7-df3258a77bb1","added_by":"auto","created_at":"2025-11-19 07:39:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":190518,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of separation patterns from DOC-based CN-PAGE using an agarose–acrylamide composite gel versus a conventional 4–13% gradient polyacrylamide gel.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThylakoid membrane protein complexes from \u003cem\u003eA. thaliana\u003c/em\u003e were solubilized with 2% α-DDM, and a sample amount equivalent to 8.3 μg of chlorophyll was loaded per lane. In both gels, the photosystem (PS)II–light-harvesting complex (LHC)II and PSI–LHCI complexes were well resolved, although the LHCII trimer and LHC monomers (free LHCs) were resolved only in the gradient gel.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8130027/v1/bb199de8df6f178a7818270f.png"},{"id":96249137,"identity":"14bb9413-a15c-47e9-9650-7bec5c3bbb39","added_by":"auto","created_at":"2025-11-19 07:30:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":185648,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e2D-CN/sodium dodecyl sulfate (SDS)-PAGE analysis and 77 K chlorophyll fluorescence emission spectra of separated photosynthetic complexes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eSilver-stained 2D-CN/SDS-PAGE gel showing the subunit compositions of the PSI–PSII megacomplex, PSII-LHCIIcomplexes, and PSI–LHCI (PSI). \u003cstrong\u003e(b) \u003c/strong\u003eChlorophyll fluorescence emission spectra (77 K) of the indicated complexes isolated from a CN-PAGE gel. The PSI sample contained a minor amount of PSII dimer contamination. The fluorescence intensity of each spectrum was normalized to 683 nm (PSI–PSII and PSII complexes) and 729 nm (PSI complex).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8130027/v1/a582075ce5c415bf24d2c126.png"},{"id":96136427,"identity":"d0cd560b-58fe-4664-9327-64f8d1ddf2b6","added_by":"auto","created_at":"2025-11-18 04:03:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":262964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTEM images of negatively stained PSI and PSII complexes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePSI \u003cstrong\u003e(a) \u003c/strong\u003eand PSII \u003cstrong\u003e(b) \u003c/strong\u003ecomplexes, purified as described in this study, were observed using TEM.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8130027/v1/277225ba2bcbce6fdfa022d2.png"},{"id":96207873,"identity":"0c9f1e40-a96a-4555-a7a4-8dee7d1d78cf","added_by":"auto","created_at":"2025-11-18 17:47:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":565768,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverall architecture of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e PSI complex.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Cryo-EM density map obtained by single-particle analysis of the \u003cem\u003eA. thaliana\u003c/em\u003e PSI complex, viewed from the stromal side. \u003cstrong\u003e(b) \u003c/strong\u003eOverall structure as viewed from the lumenal side. The magnified view of the region surrounding the PsaN subunit is shown within the yellow circles. The PsaN subunit is shown in light blue color. \u003cstrong\u003e(c)\u003c/strong\u003e Atomic model of the residues 87–136 of the PsaN subunit, containing two α-helices.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-8130027/v1/cc93a380f2c7b8e02b0eded5.png"},{"id":104739633,"identity":"ddd51096-bdbd-41e1-9a0a-829d16e888f7","added_by":"auto","created_at":"2026-03-16 16:11:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2669305,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8130027/v1/e8749b4c-4afc-400e-940a-176f7e439e6f.pdf"},{"id":96136437,"identity":"e85a67fa-3ce8-4746-af17-b4ed1601ed2d","added_by":"auto","created_at":"2025-11-18 04:03:17","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6666134,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1.pdf\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetailed protocols for the preparation of agarose–acrylamide composite gels and the electroelution procedure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 2.pdf\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM data collection and refinement statistics.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 3.pdf\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSummary of Cryo-EM data acquisition and image processing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Cryo-EM image of purified photosystem (PS)I complex of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e isolated using DOC-based clear-native polyacrylamide gel electrophoresis (CN-PAGE) method. (b) 2D class images of the PSI complex. (c, d) Local resolution and fourier shell correlation curve of the final cryo-EM map of the PSI complex. (E) Flow chart of cryo-EM image processing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 4.pdf\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCN-PAGE separation of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e thylakoid membrane complexes and comparison of protein yields obtained by soaking and electroelution.\u003c/p\u003e\n\u003cp\u003e(a) \u003cem\u003eArabidopsis thaliana\u003c/em\u003e thylakoid membrane complexes were separated by clear-native polyacrylamide gel electrophoresis (CN-PAGE) on 0.5% and 1% agarose gels. (b) CN-PAGE and (c) chlorophyll fluorescence images obtained at room temperature. Notably, the chlorophyll yield from photosystem (PS)I was much lower than that from PSII and was not clearly visible. (d) Chlorophyll pigments remaining after extraction using the conventional soaking method or electroelution.\u003c/p\u003e","description":"","filename":"Additionalfiles14.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8130027/v1/8234e4a3838066e182fabf8b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"An efficient clear-native PAGE–based workflow for cryoelectron microscopy sample preparation of large protein complexes","fulltext":[{"header":"Background","content":"\u003cp\u003eProtein complexes play indispensable roles in various cellular processes ranging from signal transduction to metabolic regulation. Their intricate three-dimensional structures enable specific interactions and regulatory mechanisms central to fundamental biological events. Detailed structural insights into these complexes advance the understanding of protein function and enable broader applications, including drug discovery and synthetic biology. Despite major advances in structural biology techniques such as cryoelectron microscopy (cryo-EM) [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], protein complexes remain structurally uncharacterized because of technical challenges, including sample heterogeneity, complex instability, and purification difficulties [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Developing reliable methods to isolate and analyze protein complex structures is therefore pivotal for revealing the molecular basis of key biological phenomena [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePhotosystems (PSs) in photosynthetic organisms are particularly challenging targets for structural characterization. These large membrane-embedded complexes, such as the PSI\u0026ndash;light-harvesting complex (LHC) I (hereafter referred to as the PSI complex, weighing\u0026thinsp;~\u0026thinsp;700 kDa) and PSII\u0026ndash;LHCII (hereafter referred to as the PSII complex, weighing\u0026thinsp;~\u0026thinsp;1400 kDa) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], exhibit properties that complicate conventional purification approaches. Notably, LHC antenna proteins often dissociate during purification [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], complicating both isolation and maintenance of intact complexes. These characteristics can compromise the efficiency of purification strategies such as gel filtration and ion-exchange chromatography, often resulting in low success rates. Furthermore, as membrane-embedded complexes, PSs present additional difficulties in solubilization while maintaining their native conformation during structural studies [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Collectively, these challenges hinder the structural and functional characterization of large membrane-protein complexes, including PSs. Therefore, new purification strategies capable of maintaining complex stability and native architecture are required.\u003c/p\u003e\u003cp\u003eIn addition to column chromatography, sucrose density gradient (SDG) centrifugation has been widely used to separate PSs [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] for structural analysis by X-ray crystallography and cryo-EM. However, this method typically requires large amounts of starting material and provides relatively low-resolution separation. In contrast, native polyacrylamide gel electrophoresis (native PAGE) provides higher resolution while requiring smaller protein amounts [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Because cryo-EM generally requires less protein than that required by X-ray crystallography, extracting protein complexes from native PAGE gels without disturbing their native structures would be an ideal approach for preparing cryo-EM samples.\u003c/p\u003e\u003cp\u003eTwo major types of native PAGE exist: blue-native PAGE (BN-PAGE) and clear-native PAGE (CN-PAGE) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. BN-PAGE uses Coomassie Brilliant Blue (CBB G-250) dye to impart negative charge to protein complexes and facilitate migration [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], whereas CN-PAGE uses sodium deoxycholate (DOC) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. CN-PAGE is particularly advantageous for studying pigment\u0026ndash;protein complexes in PSs because it avoids CBB dye, which can interfere with subsequent analyses, especially spectroscopy. Thus, CN-PAGE serves as a superior alternative to SDG, offering higher-resolution separation with smaller sample volumes and enabling clear distinction between PSI and PSII complexes before cryo-EM analysis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, two major challenges remain for applying CN-PAGE to cryo-EM structural analysis. First, establishing a reliable, reproducible, and broadly applicable method for extracting proteins from the gel matrix after separation remains difficult [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. A common extraction method relies on passive diffusion, where an excised gel slice containing the protein complex is soaked in buffer for a prolonged period to allow diffusion from the gel [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This approach is inefficient, time-consuming, and often results in substantial sample loss. Furthermore, CN-PAGE\u0026ndash;based cryo-EM sample preparation generally requires multiple additional purification steps after extraction, further increasing sample loss and labor [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Second, preparing gradient gels for CN-PAGE [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] is more technically demanding than handling sucrose gradients. These limitations hinder cryo-EM studies of PSI and PSII complexes purified by CN-PAGE. Additionally, the pliable nature of low-concentration acrylamide gels makes it difficult to excise bands precisely after electrophoresis.\u003c/p\u003e\u003cp\u003eHence, to address these technical issues, we aimed to develop a rapid and efficient method for preparing PSI and PSII samples suitable for high-resolution cryo-EM analysis. Our method involves separating PSI and PSII complexes using an agarose\u0026ndash;acrylamide composite gel without a gradient, extracting them by electroelution with a new buffer system, and purifying the PSs by ultrafiltration to remove DOC, which may interfere with sample adhesion to cryo-EM grids. Using this approach, we demonstrated that PSs extracted from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e retained structural integrity and were successfully analyzed by cryo-EM. Although demonstrated using PSs as challenging targets, this approach is broadly applicable to diverse large membrane protein complexes. This method provides a streamlined workflow for cryo-EM sample preparation, enabling efficient high-resolution structural analysis of various protein complexes beyond PSs.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003ePlant material and growth condition\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eA. thaliana\u003c/em\u003e (ecotype Columbia) plants were grown in fertilized soil for 6 weeks at 23\u0026deg;C under a 14 h light/10 h dark photoperiod with a light intensity of 70 \u0026micro;mol photons m \u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Fully expanded rosette leaves were harvested from pre-bolting vegetative-stage plants.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAgarose\u0026ndash;acrylamide composite gel\u003c/h2\u003e\u003cp\u003eAll chemical ratios used for the agarose\u0026ndash;acrylamide composite gel (separation gel) and sample gel are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Agarose powder (Lonza, Switzerland) was dissolved in a solution containing Milli-Q water and 3\u0026times; gel buffer. After cooling to approximately 50\u0026deg;C, the remaining components listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e were immediately added, followed by thorough mixing. The mixture was poured into a preheated, preassembled gel cassette, leaving sufficient space for the sample gel to solidify at room temperature (20\u0026ndash;25\u0026deg;C). Milli-Q water was added after solidification, and the gel was stored at 4\u0026deg;C overnight before proceeding. Experimental procedures followed the protocol detailed in Additional file 1.\u003c/p\u003e\n\u003ch3\u003eSample preparation and separation of thylakoids by DOC-based CN-PAGE\u003c/h3\u003e\n\u003cp\u003eThylakoid membranes from \u003cem\u003eA. thaliana\u003c/em\u003e were isolated according to Ye et al. (2025)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] with minor modifications. Fresh leaves (approximately 80 plants) were ground in 200 mL of prechilled grinding buffer (20 mM Tricine/KOH, pH 7.6; 0.4 M sorbitol; 10% polyethylene glycol (PEG)-6000; 10 mM ethylenediaminetetraacetic acid [EDTA]-2Na) using a prechilled blender (Twinbird, Japan). The homogenate, filtered through four layers of Miracloth (Merck Millipore, USA), was centrifuged at 10,000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 5 min at 4\u0026deg;C. The pellet was resuspended in 5 mL of wash buffer (10 mM Tricine/KOH, pH 7.6; 0.4 M sorbitol; 2.5 mM MgCl₂; 1.25 mM EDTA-2Na) and recentrifuged under the same conditions. The final pellet was resuspended in a solubilization buffer (50 mM imidazole/HCl, pH 7.0; 20% (v/v) glycerol) to a chlorophyll concentration of 1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The thylakoid suspension was solubilized with 1% (w/v) α-DDM (n-Dodecyl α-D-maltoside) for 2 min on ice in solubilization buffer containing 10 mM sodium fluoride as a phosphatase inhibitor and a plant protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Samples were centrifuged at 22,500 \u0026times;\u003cem\u003eg\u003c/em\u003e for 5 min at 4\u0026deg;C.\u003c/p\u003e\u003cp\u003eThe resulting supernatant was loaded onto an agarose\u0026ndash;acrylamide gel. Electrophoresis was performed at 4\u0026deg;C using an anode buffer (50 mM imidazole/HCl, pH 7.0) and a cathode buffer (50 mM Tricine; 15 mM imidazole/HCl, pH 7.0) containing 0.05% DOC and 0.02% α-DDM, as described by J\u0026auml;rvi et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Details are provided in Additional file 1.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2D-CN/Sodium dodecyl sulfate (SDS)-PAGE followed by silver staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor second-dimensional electrophoresis, a gel strip corresponding to a single lane was excised from the first-dimensional CN-PAGE gel. The proteins in this strip were then denatured by incubation in SDS solubilization buffer (1% SDS, 50 mM DTT) for 30 min at room temperature. Proteins were separated on a 14% acrylamide gel containing 6 M urea using the Laemmli buffer system [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Precision Plus Protein Unstained Standards (Bio-Rad, Hercules, CA, USA) served as molecular weight markers.\u003c/p\u003e\u003cp\u003eAfter electrophoresis, the separated proteins were visualized using a Pierce Silver Stain Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer\u0026rsquo;s protocol. Bands were identified as previously described [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. PsbB (~\u0026thinsp;47 kDa), PsbC (~\u0026thinsp;43 kDa), and D1/D2 (~\u0026thinsp;28 kDa) were used as PSII markers, whereas PsaD (~\u0026thinsp;20 kDa), PsaF (~\u0026thinsp;18 kDa), and PsaL (~\u0026thinsp;17 kDa) served as PSI indicators.\u003c/p\u003e\n\u003ch3\u003eLow-temperature (77 K) chlorophyll fluorescence spectroscopy\u003c/h3\u003e\n\u003cp\u003eThe fluorescence emission spectra were measured at 77 K using a fluorescence spectrophotometer (F-2700; Hitachi, Japan). An excitation wavelength of 440 nm, which preferentially excites chlorophyll \u003cem\u003ea\u003c/em\u003e (Chl \u003cem\u003ea\u003c/em\u003e), was used to record overall emissions from the PSs. Fluorescence intensity of each spectrum was normalized to 683 nm (PSI\u0026ndash;PSII and PSII complexes) and 729 nm (PSI complex).\u003c/p\u003e\n\u003ch3\u003eChlorophyll concentration measurement\u003c/h3\u003e\n\u003cp\u003eChlorophyll concentrations of the photosystems were calculated according to the method of Porra et al. (1989)[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The sample solution was diluted in 80% (v/v) acetone, and its absorbance was determined using a Shimadzu UV-2600i UV\u0026ndash;Vis spectrophotometer (Shimadzu Corp., Kyoto, Japan).\u003c/p\u003e\n\u003ch3\u003ePS sample preparation using electroelution for transmission electron microscopy (TEM) and Cryo-EM\u003c/h3\u003e\n\u003cp\u003ePSI and PSII were excised from the agarose\u0026ndash;acrylamide gel and extracted by electroelution at 4\u0026deg;C using an Electro-Eluter (Bio-Rad, Hercules, CA, USA) following the manufacturer\u0026rsquo;s protocol. Buffers for electroelution were based on the DOC-based CN-PAGE system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The anode buffer was used without modification, whereas the cathode buffer was supplemented with 0.05% DOC and 0.02% α-DDM. To enhance protein solubility and prevent aggregation, the cathode chamber between the frit and membrane cap was filled with cathode buffer containing 0.05% α-DDM. Electroelution conditions for two glass tubes were approximately 110 V and 5 mA, with total run time under 3 h when the gel band filled in the glass tube height was less than 1 cm.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePSI and PSII were collected from the membrane cap and concentrated using Apollo 7 mL centrifugal concentrators (150 kDa MW cutoff; Orbital Biosciences, Topsfield, MA, USA). The purified proteins were washed three times with 25 mM Bis-Tris buffer (pH 7.5) containing 0.05% α-DDM, concentrated to 20 \u0026micro;L, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C in the same buffer.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eNegative-stain electron microscopy (EM)\u003c/h2\u003e\u003cp\u003eThe purified solution was diluted 1:20 or 1:100, and 3.5 \u0026micro;L of diluted sample was applied to glow-discharged continuous carbon film\u0026ndash;coated copper grids (Nisshin EM, Tokyo, Japan). After staining with 3.5 \u0026micro;L of 2% uranyl acetate, excess solution was blotted off and grids were air-dried. Samples were examined using an H-7650 Hitachi transmission electron microscope operating at 80 kV and equipped with a 1 \u0026times; 1 K Tietz FastScan-F114 CCD camera.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCryo-EM grid preparation and data acquisition of\u003c/b\u003e \u003cb\u003eA. thaliana\u003c/b\u003e \u003cb\u003ePSI-LHCI\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA 2.6 \u0026micro;L aliquot of PSI\u0026ndash;LHCI solution was applied to a glow-discharged Quantifoil holey carbon grid (R1.2/1.3, Cu, 200 mesh) coated with a 5 nm carbon film, blotted for 3.0 s at 4\u0026deg;C, and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Grids were loaded into a Titan Krios G2 (Thermo Fisher Scientific) operating at an acceleration voltage of 300 kV and equipped with a Cs corrector (CEOS GmbH). Images were recorded using a K3 direct electron detector (Gatan, USA) in CDS mode with an energy filter (slit width 20 eV). Data were collected automatically using SerialEM v4.0 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] at a physical pixel size of 0.675 \u0026Aring; with 40 frames (1 e \u003csup\u003e\u0026minus;\u003c/sup\u003e/\u0026Aring;\u003csup\u003e2\u003c/sup\u003e per frame), an exposure time of 1.651 s per movie, and a defocus range of \u0026minus;\u0026thinsp;0.5 to \u0026minus;\u0026thinsp;1.0 \u0026micro;m. A total of 8,764 movies were collected.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCryo-EM data processing and model building\u003c/h3\u003e\n\u003cp\u003eMovie frames were corrected for beam-induced motion using RELION\u0026rsquo;s implementation of MotionCor2 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and the contrast transfer function (CTF) was estimated using CTFFIND4 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Both steps were performed in RELION 4.0 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eApproximately 170,000 particles were automatically selected from almost 1,100 micrographs using a template-free Laplacian-of-Gaussian (LoG) filter and extracted with a 92-pixel box size and 4\u0026times; binning for two-dimensional (2D) classification. A good 2D class-averaged image was used for Topaz training, after which 1,629,453 particles were automatically picked from all micrographs using Topaz [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and three rounds of 2D classification were conducted in RELION 4.0. A total of 361,636 particles were selected to build the 3D initial model of \u003cem\u003eA. thaliana\u003c/em\u003e PSI using cryoSPARC2 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and classified into three 3D classes in RELION 4.0.\u003c/p\u003e\u003cp\u003eSelected particles were re-extracted at 0.675 \u0026Aring; per pixel and subjected to four 3D refinements, three CTF refinements, and Bayesian polishing. The final 3D refinement and postprocessing yielded maps with a global resolution of 2.18 \u0026Aring; according to the 0.143 Fourier shell correlation criterion. Local resolution was estimated using RELION 4.0. Processing details are provided in Additional file 2.\u003c/p\u003e\u003cp\u003eA previously published \u003cem\u003eA. thaliana\u003c/em\u003e PSI model (PDB ID: 8J7B) was docked into the EM density map using UCSF ChimeraX [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Each domain and ligands (e.g., Chl a, Chl b) were manually remodeled and refined iteratively using COOT [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], Phenix [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and Servalcat [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Chlorophylls that could not be unambiguously assigned as Chl a or Chl b were defined as unknown ligands (UNLs). All figures were prepared using UCSF ChimeraX. Statistics of 3D reconstruction and model refinement are summarized in Additional file 3.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAn agarose\u0026ndash;acrylamide composite gel enables efficient separation of PSI and PSII complexes\u003c/h2\u003e\u003cp\u003eIn this study, we developed an agarose-based composite gel as a simple and rapid alternative to conventional polyacrylamide gradient gels for CN-PAGE. The addition of acrylamide improved gel adhesion to the glass cassette and significantly enhanced the separation of PSI and PSII complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Additional file 4a, b).Our agarose\u0026ndash;acrylamide composite gel yielded PSI and PSII banding patterns identical to those obtained with a conventional 4%\u0026ndash;13% acrylamide gradient gel (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Band identities were confirmed by 2D-CN/SDS-PAGE followed by silver staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate whether the separated PSs retained their structural integrity for downstream analyses, we performed low-temperature (77 K) chlorophyll fluorescence spectroscopy. The fluorescence spectra showed characteristic peaks for PSII and PSI [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], without detectable signals from dissociated LHCs or free chlorophyll, indicating that both complexes remained intact within the composite gel (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A minor PSII core dimer peak (689 nm) appeared in the PSI band slice, consistent with the overlap arising because of similar molecular sizes\u0026mdash;a phenomenon also observed in conventional linear-gradient acrylamide gels. Together, these findings demonstrate that the agarose\u0026ndash;acrylamide composite gel provides PS separation comparable to that of conventional acrylamide gradient gels.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eProtein complexes successfully extracted from agarose\u0026ndash;acrylamide composite gel via electroelution\u003c/h2\u003e\u003cp\u003eThe extraction efficiency, as indicated by the residual green color of the gel slices, was lower in the agarose\u0026ndash;acrylamide composite gel than in the acrylamide gradient gel.\u003c/p\u003e\u003cp\u003eTo overcome this limitation, we employed electroelution using a commercial electroeluter to achieve higher recovery efficiency. The apparatus was assembled and operated as shown schematically in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. The complete workflow for separating and purifying \u003cem\u003eA. thaliana\u003c/em\u003e PSs from the composite gel is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb. As no buffer system had been established for electroelution of CN-PAGE gels, we adapted the DOC-based CN-PAGE buffer system for this purpose. Electroelution under these optimized conditions efficiently extracted the protein complexes, rendering the gel slices nearly transparent within 2\u0026ndash;3 h (Additional file 4c)\u0026mdash;a substantial improvement over the conventional soaking method, which typically requires overnight incubation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eOptimization of sample preparation after the electroelution for cryo-EM\u003c/h2\u003e\u003cp\u003eAfter electroelution, the recovered PSs were concentrated by ultrafiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Negative-stain electron microscopy confirmed successful recovery of both PSI and PSII complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, initial attempts to prepare cryo-EM grids using these samples failed to yield resolvable particles, suggesting interference from residual DOC in the electroelution buffer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo test this hypothesis, the samples were subjected to three additional buffer exchange cycles to remove DOC completely. Subsequent grid preparation focused on PSI, which was more abundant than PSII. Following DOC removal, well-dispersed and intact PSI particles were obtained at suitable concentrations for high-resolution cryo-EM analysis. This optimized protocol yielded approximately 20 \u0026micro;L of concentrated PSI sample with a chlorophyll concentration of 400 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e from a starting thylakoid sample containing 200 \u0026micro;g of chlorophyll.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eQuality assessment of the purified PSI complex by cryo-EM\u003c/h2\u003e\u003cp\u003eTo investigate whether the structure and arrangement of subunits and cofactors such as chlorophylls and carotenoids were preserved, we performed single-particle cryo-EM of the PSI complex. Using grids coated with a 5 nm carbon film for optimal particle distribution, we obtained an EM density map at 2.18 \u0026Aring; resolution, which enabled atomic model construction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis structure comprised twelve subunits (PsaA\u0026ndash;PsaL) and four LHCs and included residues 87\u0026ndash;136 of PsaN. PsaN, located on the lumenal side, positioned to interact with PsaA, PsaF, PsaJ, and Lhca2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we obtained a high-resolution \u003cem\u003eA. thaliana\u003c/em\u003e PSI cryo-EM structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) by developing a two-part methodological pipeline: separation using an agarose\u0026ndash;acrylamide composite gel and recovery by efficient electroelution. Below, we discuss the key characteristics of each component.\u003c/p\u003e\u003cp\u003eOur composite gel is a powerful alternative to traditional gradient gels for isolating large protein complexes. Because agarose adheres poorly to glass cassettes, the gels were cast onto a GelBond Film (Lonza, Switzerland) to ensure firm adhesion during vertical electrophoresis. Initial experiments using pure agarose gels (up to 1%) failed to resolve PSI and PSII complexes from the model plant \u003cem\u003eA. thaliana\u003c/em\u003e; both complexes migrated together at the gel front (Additional file 4a). This poor resolution likely resulted from the larger pore size of agarose than that of acrylamide gels. Incorporating a small amount of acrylamide (3.6%) into the agarose matrix overcame this limitation.\u003c/p\u003e\u003cp\u003eAlthough the composite gel did not resolve smaller proteins such as free or dissociated LHCs, it separated complexes larger than PSI (\u0026asymp;\u0026thinsp;700 kDa) more rapidly than conventional methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Thus, our electroelution method is broadly applicable for recovering proteins from gel matrices and is particularly effective for rapid preparation of intact, native-state complexes for demanding applications such as cryo-EM.\u003c/p\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eAdvantages and future challenges of the agarose\u0026ndash;acrylamide composite gel\u003c/h2\u003e\u003cp\u003eOur composite gel separates PSI and PSII, comparable to that of a conventional 4%\u0026ndash;13% acrylamide gradient gel (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), while using a single concentration.\u003c/p\u003e\u003cp\u003eIn standard DOC-based CN-PAGE [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], an acrylamide gradient gel separates PSII complexes by size, typically resolving three distinct forms\u0026mdash;C\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eM\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eM, and C\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e. This size variation corresponds to the progressive dissociation of LHCII antenna complexes, as the largest form (C\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eM\u003csub\u003e2\u003c/sub\u003e) transitions to C\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eM and then to C\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Higher agarose concentrations in the composite gel further improved resolution and band sharpness, particularly for PSI bands.\u003c/p\u003e\u003cp\u003eThe separated PSs retained functional and structural integrity according to 77 K fluorescence spectroscopy and cryo-EM analysis, respectively. This performance was achieved with a simpler gel preparation that eliminates the need for a gradient mixer and peristaltic pump, saving time and resources.\u003c/p\u003e\u003cp\u003eThe agarose scaffold confers improved mechanical robustness relative to low-concentration polyacrylamide gels (e.g., \u0026lt;\u0026thinsp;4%), which are often sticky and fragile and thus difficult to handle. This enhanced stability was critical for precise band excision required for downstream analyses.\u003c/p\u003e\u003cp\u003eThe primary challenge in gel preparation is temperature control. Agarose requires high temperatures to dissolve fully, whereas acrylamide is prone to degradation at high temperatures. Conversely, lowering the temperature to ensure acrylamide stability and agarose solidification complicates the casting process. Through our optimization, we also found it best to avoid loading water onto the separation gel surface because this can produce irregularities; minor surface unevenness did not compromise final separation quality. Despite these constraints, we established an optimized protocol (Materials and Methods) that reproducibly yields high-quality gels. Future improvements could involve exploring lower-melting-point agarose or additives that enhance acrylamide thermal stability, which would simplify casting and broaden applications.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eOptimization of the electroelution buffer\u003c/h2\u003e\u003cp\u003eAs previously described that agarose gels have larger pores, we expected high extraction efficiency for PSs during recovery. However, the conventional passive diffusion method optimized for acrylamide gradient gels [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] was unsuitable for this composite matrix. Even after 24 h of soaking in 1% digitonin, a visible amount of the green-colored complexes remained trapped within the gel slices (Additional file 4c).\u003c/p\u003e\u003cp\u003eBuffer selection is crucial for successful electroelution. We used the CN-PAGE buffer with additional detergent supplementation in the membrane cap space to prevent protein aggregation. Aggregation reduces image quality and compromises resolution in subsequent cryo-EM analyses. In our study, a detergent concentration of 0.05% α-DDM was optimal; higher concentrations tend to excessively disperse protein\u0026ndash;detergent complexes, increasing retention within frit pores and causing protein loss during electroelution.\u003c/p\u003e\u003cp\u003eElectroelution typically completes within 3 h using this buffer system. Despite the composite gel\u0026rsquo;s greater rigidity, extraction proceeded at a competitive rate. The only subsequent processing step was DOC removal by buffer exchange using ultrafiltration, which enabled high-resolution cryo-EM. This rapid, streamlined workflow likely contributed to the high-resolution results and allowed samples recovered by electroelution to be used directly for cryo-EM without additional chromatographic purification steps.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe\u003c/b\u003e \u003cb\u003eA. thaliana\u003c/b\u003e \u003cb\u003ePSI structure including PsaN\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe resolved the \u003cem\u003eA. thaliana\u003c/em\u003e PSI structure and successfully modeled the PsaN subunit (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), which had not been reported in previous studies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This result indicates that our purification approach effectively preserves labile protein\u0026ndash;protein interactions that are often lost during conventional multistep purification procedures.\u003c/p\u003e\u003cp\u003ePsaN is the only membrane-extrinsic PSI subunit on the lumenal side and is found exclusively in green plants and red algae [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Because PsaN dissociates readily under salt washing [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], it was absent from many plant PSI-LHCI cryo-EM structures [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] or inaccurately localized in some crystal structures [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. To date, a clear PsaN structure was reported only in maize PSI with an LHCII trimer purified using a gentle, low-ionic-strength method [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Using our protocol, we resolved PsaN except for its highly mobile N- and C-termini. Retention of this labile subunit suggests our method effectively preserves native supercomplex integrity and offers a valuable alternative to existing protocols.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we developed and validated a novel pipeline for purifying large membrane-protein complexes that enables high-resolution cryo-EM structural determination. Our workflow, which combines a single-concentration composite gel with efficient electroelution reduces purification time and minimizes sample degradation. The approach was validated using a 2.18 \u0026Aring; structure of plant PSI that retained the labile PsaN subunit, supporting preservation of native integrity.\u003c/p\u003e\u003cp\u003eThis work provides both a detailed structural view of plant PSI and a practical, broadly applicable methodology for structural biology. The in vitro purification pipeline complements in situ approaches such as cryo-electron tomography [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. It offers a robust route for analyzing challenging complexes, especially those with small extramembrane domains that are difficult to identify in cells. This streamlined workflow promises to facilitate structural analysis of other large assemblies and to advance understanding of complex biological machinery.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCN-PAGE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Clear-native polyacrylamide gel electrophoresis\u003c/p\u003e\n\u003cp\u003eCTF\u0026nbsp; \u0026nbsp; \u0026nbsp;Contrast transfer function\u003c/p\u003e\n\u003cp\u003eCryo-EM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cryoelectron microscopy\u003c/p\u003e\n\u003cp\u003eDDM\u0026nbsp; \u0026nbsp;n-Dodecyl \u0026alpha;-D-maltoside\u003c/p\u003e\n\u003cp\u003eDOC\u0026nbsp; \u0026nbsp;\u0026nbsp;Sodium deoxycholate\u003c/p\u003e\n\u003cp\u003eEDTA\u0026nbsp;\u0026nbsp;Ethylenediaminetetraacetic acid\u003c/p\u003e\n\u003cp\u003eEM\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Electron microscopy\u003c/p\u003e\n\u003cp\u003eLHC\u0026nbsp; \u0026nbsp;\u0026nbsp;Light-harvesting complex\u003c/p\u003e\n\u003cp\u003ePEG\u0026nbsp; \u0026nbsp; \u0026nbsp;Polyethylene glycol\u003c/p\u003e\n\u003cp\u003ePS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Photosystem\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTEM \u0026nbsp; \u0026nbsp;Transmission electron microscopy\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCryo-EM density maps of the \u003cem\u003eArabidopsis thaliana\u003c/em\u003e PSI complex, isolated using DOC-based CN-PAGE, have been deposited in the Electron Microscopy Data Bank under the accession code EMD-66073. The atomic coordinates of the PSI complex\u003cem\u003e\u0026nbsp;of A. thaliana\u003c/em\u003e have been deposited in the Protein Data Bank under the accession code 9WLS. The raw cryo-EM data (EMPIAR-13077) used to generate the density map are available from the Electron Microscopy Public Image Archive. All other data are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI under grant numbers 23H04958 (awarded to G.K.), 23H04960 (awarded to R.T.), and 24K09496 (awarded to A.T.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.Y., S.K., and A.T. conceptualized the study. All the authors contributed to the study design. Z.Y., S.K., and A.T. performed sample preparation and associated procedures, including CN-PAGE, electroelution, and ultrafiltration. A.K. performed the cryo-EM structural analysis. Z.Y., A.T., and A.K. prepared original drafts of the manuscript. All authors have reviewed, revised, and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Nobuyuki Endo (University of Osaka) for assistance with TEM observations. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBai XC, McMullan G, Scheres SHW. How cryo-EM is revolutionizing structural biology. Trends Biochem Sci. 2015;40:49\u0026ndash;57. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tibs.2014.10.005\u003c/span\u003e\u003cspan address=\"10.1016/j.tibs.2014.10.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEgelman EH. The current revolution in cryo-EM. 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Nature. 2024;631:232\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41586-024-07488-9\u003c/span\u003e\u003cspan address=\"10.1038/s41586-024-07488-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1 Composition of separation and sample gels for CN-PAGE\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSeparation gel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003eAgarose concentration\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.9375%;\"\u003e\n \u003cp\u003e0.25%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.1919%;\"\u003e\n \u003cp\u003e0.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.75%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003eAgarose (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.9375%;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.1919%;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e135\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e180\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e30% Acrylamide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e2.25 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e3\u0026times; Gel Buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e6 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003eMilli-Q\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e10 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e10% APS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e60\u0026nbsp;\u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003eTEMED\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e15\u0026nbsp;\u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e18 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"left\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample gel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eAcrylamide concentration\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e3.75%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003e30% Acrylamide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e0.72 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003e3\u0026times; Gel Buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e2 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eMilli-Q\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e3.28 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003e10% APS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e50\u0026nbsp;\u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eTEMED\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e5\u0026nbsp;\u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e6 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Clear-native PAGE, Electroelution, Cryo-EM, Photosystem, Protein complex","lastPublishedDoi":"10.21203/rs.3.rs-8130027/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8130027/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eCryoelectron microscopy (cryo-EM) has revolutionized protein research by enabling high-resolution structural analysis. However, preparing ultra-large protein complexes (e.g., \u0026gt;\u0026thinsp;700 kDa) for cryo-EM remains challenging, as it requires preserving both structural integrity and the native state. Conventional isolation methods, such as sucrose density gradient centrifugation, require large sample volumes and provide limited separation resolution. In contrast, native PAGE offers higher resolution; however, no established method exists for extracting protein complexes from gels followed by further purification to achieve high purity. Consequently, no standardized native PAGE-based protocol for cryo-EM sample preparation avoids multiple purification steps. Hence, we aimed to develop a rapid and efficient cryo-EM protein sample preparation method using electroelution with an optimized buffer system that preserves complex integrity to recover target protein complexes after sodium deoxycholate (DOC)-based clear-native PAGE (CN-PAGE).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe developed an agarose\u0026ndash;acrylamide composite gel, which is simpler to prepare and mechanically more robust than conventional linear-gradient acrylamide gels commonly used for CN-PAGE, facilitating precise band excision for efficient electroelution. Cryo-EM structural analysis of the photosystem I\u0026ndash;light-harvesting complex I (PSI\u0026ndash;LHCI) supercomplex from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e achieved high resolution (2.18 \u0026Aring;) after electroelution from this gel, requiring only buffer exchange by ultrafiltration to remove DOC before grid preparation, without additional chromatographic purification. This finding suggests that DOC may be the main inhibitor of successful grid preparation.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eOur results demonstrate the potential of this method for isolating large protein complexes from small sample volumes for cryo-EM structural analysis. This approach significantly broadens the scope of cryo-EM targets to include challenging systems previously hindered by purification difficulties, thereby accelerating structural studies crucial for understanding complex biological processes.\u003c/p\u003e","manuscriptTitle":"An efficient clear-native PAGE–based workflow for cryoelectron microscopy sample preparation of large protein complexes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 04:03:12","doi":"10.21203/rs.3.rs-8130027/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-08T21:03:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-07T13:20:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-03T09:04:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232751586259515624101361590199577760973","date":"2025-11-21T10:40:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-20T23:56:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"132310988808540302882384032056597571179","date":"2025-11-20T01:08:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273688872813695786414027471872744012769","date":"2025-11-19T22:53:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-19T18:08:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-17T04:10:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-17T04:09:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Methods","date":"2025-11-17T01:10:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d7e9c2cd-10d3-448e-a52f-49feb7c6c0b5","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:06:31+00:00","versionOfRecord":{"articleIdentity":"rs-8130027","link":"https://doi.org/10.1186/s13007-026-01513-w","journal":{"identity":"plant-methods","isVorOnly":false,"title":"Plant Methods"},"publishedOn":"2026-03-10 15:58:34","publishedOnDateReadable":"March 10th, 2026"},"versionCreatedAt":"2025-11-18 04:03:12","video":"","vorDoi":"10.1186/s13007-026-01513-w","vorDoiUrl":"https://doi.org/10.1186/s13007-026-01513-w","workflowStages":[]},"version":"v1","identity":"rs-8130027","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8130027","identity":"rs-8130027","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}