Cryo-EM structure of the spinach cytochrome b6 f complex at 3.6 Å resolution.

No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Complex purification

Dimeric cyt b 6 f was isolated from dark-adapted market spinach ( S. oleracea ) in a procedure adapted from Dietrich and Kuhlbrandt 32 . In brief, spinach leaves were homogenized in buffer 1 (50 mM Tris-HCl pH 7.5, 200 mM sucrose and 100 mM NaCl). Homogenate was then filtered and centrifuged for 15 min at 4,540 g , 4 °C. Following centrifugation, the supernatant containing cell debris was discarded and the pellet resuspended in buffer 2 (10 mM Tricine-NaOH pH 8 and 150 mM NaCl) before centrifugation again for 15 min (4,540 g , 4 °C). The resultant pellet was resuspended in buffer 3 (2 M NaBr, 10 mM Tricine-NaOH pH 8 and 300 mM sucrose) and incubated on ice for 15 min before diluting twofold with ice-cold milliQ H 2 O and centrifuging (15 min, 4,540 g , 4 °C). The resultant pellet was resuspended in buffer 3 and incubated on ice for 15 min before diluting twofold with ice-cold milliQ H 2 O and centrifuging again (15 min, 4,540 g , 4 °C). The pellet was resuspended in buffer 2 and centrifuged for 15 min, 4,540 g at 4 °C. The final pellet was resuspended in a small volume of buffer 4 (40 mM Tricine pH 8.0, 10 mM MgCl 2 and 10 mM KCl). The resultant thylakoid suspension was adjusted to 10 mg ml −1 chlorophyll (chlorophyll concentrations determined as described previously 33 ). For selective solubilization of cyt b 6 f , the thylakoid suspension (10 mg ml −1 chlorophyll) was diluted with membrane extraction buffer (40 mM Tricine pH 8.0, 10 mM MgCl 2 , 10 mM KCl and 1.25% (w/v) Hecameg) to a final concentration of 2 mg ml −1 chlorophyll, 1% (w/v) Hecameg. The resultant solution was mixed thoroughly then incubated for 2 min at room temperature before dilution to 0.75% (w/v) Hecameg with buffer 4. Unsolubilized material was removed by ultracentrifugation at 244,000 g at 4 °C for 30 min in a Beckman Ti50.2 rotor. The solubilization supernatant was concentrated using a Centriprep 100K centrifugal filter (Merck Millipore) before loading onto a 10–40% (w/v) continuous sucrose gradient containing 40 mM Tricine pH 8, 10 mM MgCl 2 , 10 mM KCl, 0.8% (w/v) Hecameg and 0.1 mg ml −1 egg yolk l-α-phosphatidylcholine (Sigma). This was ultracentrifuged at 174,587 g at 4 °C for 16 h in a Beckman SW32 rotor. A brown-ish band containing cyt b 6 f was collected from a region of the gradient corresponding to ∼16% sucrose. This band was concentrated and loaded onto a ceramic hydroxyapatite column (CHT) (Type I, Bio-Rad) pre-equilibrated in 20 mM Hecameg, 0.1 mg ml −1 phosphatidylcholine and 20 mM Tricine pH 8. The column was washed with 5 column volumes of CHT wash buffer (20 mM Hecameg, 0.1 mg ml −1 phosphatidylcholine and 100 mM ammonium phosphate pH 8) before bound material was eluted with CHT elution buffer (20 mM Hecameg, 0.1 mg ml −1 phosphatidylcholine and 400 mM ammonium phosphate pH 8). Detergent exchange and gel filtration Concentrated CHT eluate was loaded onto a 10–35% (w/v) continuous sucrose gradient containing 50 mM HEPES pH 8, 20 mM NaCl and 0.3 mM 4- trans -(4- trans -propylcyclohexyl)-cyclohexyl α-maltoside (tPCCαM) and ultracentrifuged at 175,117 g at 4 °C for 16 h in a Beck-man SW41 rotor. A single brown band containing cyt b 6 f was collected from a region of the gradient corresponding to ∼22% sucrose. This band was concentrated and loaded onto HiLoad 16/600 Superdex 200 pg gel filtration column (GE Healthcare) connected to an ÄKTA prime plus purification system (GE Healthcare). The column was run at a rate of 0.2 ml min −1 with 145 ml with gel filtration buffer (50 mM HEPES pH 8, 20 mM NaCl, 0.3 mM tPCCαM). Eluted fractions comprising dimeric cyt b 6 f were pooled and concentrated.

SDS–PAGE and BN-PAGE analysis

Samples collected from each purification step were analysed by SDS–PAGE and BN-PAGE. For SDS–PAGE, precast NuPAGE 12% Bis-Tris gels (Invitrogen) were run for 60 min at 180 V before staining with Coomassie blue. For BN-PAGE, precast NativePAGE 3–12% Bis-Tris gels (Invitrogen) were run for 120 min at 160 V before staining with Coomassie blue. Gels were imaged using an Amersham 600 imager (GE Healthcare). Quantification of purified dimeric cyt b 6 f using redox difference spectra Absorbance spectra were recorded at room temperature on a Cary60 spectrophotometer (Agilent). For redox difference spectra cytochromes were first fully oxidized with a few grains of potassium ferricyanide followed by reduction with a few grains of sodium ascorbate (cyt f ) then sodium dithionite (cyt f and cyt b 6 ). At each stage the sample was mixed thoroughly and incubated for ∼1 min before recording spectra. Redox difference spectra (ascorbate-reduced minus ferricyanide-oxidized and dithionite-reduced minus ascorbate-reduced) were calculated and used to determine the concentrations of c -type haem of cyt f and the two b -type haems of cyt b 6 using extinction coefficients of 25 mM cm −1 (haem f ) and 21 mM cm −1 (cyt b 6 haems) 34 . Reduction of decylplastoquinone Approximately 0.1 mg decylplastoquinone (Merck) was dissolved in 100 μl ethanol, mixed with a few grains of sodium dithionite dissolved in 100 μl milliQ H 2 O and vortexed until the solution became colourless. Decylplastoquinol was extracted by mixing with 0.5 ml hexane, vortexing and centrifuging at 16,000 g for 2 min. The hexane layer was carefully removed ensuring none of the aqueous phase was collected. Hexane extraction was repeated on the aqueous phase twice more, then the hexane solutions were pooled and dried in a rotary evaporator at 30 °C for 1 h before re-dissolving in ∼100 μl DMSO. Decylplastoquinol concentration was determined by diluting 10 μl of the DMSO solution into 795 μl ethanol, recording the absorbance spectrum between 250 and 350 nm and using an extinction coefficient 35 of 3,540 M −1 cm −1 at 290 nm.

Purification of PC

PC was purified in its oxidized form from market spinach. In brief, spinach leaves were homogenized in buffer containing 50 mM sodium phosphate pH 7.4, 5 mM MgCl 2 and 300 mM sucrose. Homogenate was then filtered and centrifuged for 15 min at 4,000 g . Following centrifugation, the supernatant containing cell debris was discarded and the pellet was resuspended in buffer containing 10 mM Tricine pH 7.4 and 5 mM MgCl 2 . The solution was incubated on ice for 1 min before diluting twofold with buffer containing 10 mM Tricine pH 7.4, 5 mM MgCl 2 , 400 mM sucrose and centrifuging for 15 min at 4,000 g . Following centrifugation, the pellet was resuspended to a chlorophyll concentration of 2 mg ml −1 in buffer containing 10 mM HEPES pH 7.6, 5 mM NaCl and 5 mM EDTA, and sonicated for 10 min, at 30 s intervals. The solution was centrifuged at 200,000 g for 1 h to pellet any large unbroken material. The supernatant was applied to four 5-ml GE Healthcare Hi-TRAP Q FF anion-exchange columns in series, equilibrated in HEPES pH 8, 5 mM NaCl. A gradient of 0.005–1 M NaCl was used for elution, with PC eluting at around 200 mM. PC-containing fractions were identified by the blue colour on addition of potassium ferricyanide. These fractions were pooled, concentrated in a Vivaspin 3-kDa molecular-weight cut-off spin concentrator and loaded onto a Superdex 200 16/600 FPLC column, equilibrated with 20 mM HEPES pH 8 and 20 mM NaCl. The resulting PC fractions were pooled, concentrated and frozen at −80 °C until use.

Activity assays

Reduction of PC by cyt b 6 f was monitored by stopped-flow absorbance spectroscopy using an Olis RSM 1000 rapid-scanning spectrophotometer equipped with a USA-SF stopped flow cell at 20 °C. Solution A (231.25 nM cyt b 6 f and 62.5 μM PC in 50 mM HEPES pH 8, 20 mM NaCl and 0.3 mM tPCCαM) and solution B (1.25 mM decylplasto-quinol in the same buffer) were prepared and the reaction was initiated by mixing the solutions in a 4:1 volumetric ratio (final concentrations: 185 nM cyt b 6 f , 50 μM PC and 250 μM decylplastoquinol). PC reduction was monitored by recording absorbance spectra between 420 and 750 nm at a rate of 62 scans s −1 and plotting the change in absorbance 36 at 597 nm. In a control reaction, cyt b 6 f was omitted to record the uncatalysed reduction of PC by decylplastoquinol. Fitting of the initial reaction rates was performed in Origin. All measurements were carried out in triplicate.

CryoEM specimen preparation and data acquisition

In brief, 3 μl of purified cyt b 6 f (∼17 μM) was applied to freshly glow-discharged holey carbon grids (Quantifoil R1.2/1.3, 400 mesh Cu). The grids were blotted for 2 s at 8 °C then plunge frozen into liquid ethane using a Leica EM GP at 90% relative humidity. Data acquisition was carried out on a Titan Krios microscope operated at 300 kV (Thermo Fisher) equipped with an energy filtered (slit width 20 eV) K2 summit direct electron detector. A total of 6,035 movies were collected in counting mode at a nominal magnification of 130,000× (pixel size of 1.065 Å) and a dose of 4.6 e − Å −2 s −1 (see Extended Data Table 1 ). An exposure time of 12 s was used and the resulting movies were dose-fractionated into 48 fractions. A defocus range of −1.5 to −2.5 μm was used.

Image processing and 3D reconstruction

Beam-induced motion correction and dose-fractionation were carried out using MotionCor2. Contrast transfer function (CTF) parameters of the dose-weighted motion-corrected images were then estimated using GCTF 37 . All subsequent processing steps were performed using RELION 2.1 38 or 3.0 39 unless otherwise stated. In total, 422,660 particles were manually picked from 6,035 micrographs. These particles were extracted using a box size of 220 × 220 pixels and subjected to reference-free 2D classification. A typical micro-graph showing picked particles is shown in Extended Data Fig. 2a, b . Particles that categorized into poorly defined classes were rejected, while the remaining 292,242 (69.2%) particles were used for further processing. A subset of 30,000 particles was used to generate a de novo initial model using the ‘3D initial model’ subroutine. The initial model low-pass filtered to 20 Å was used as a reference map for subsequent 3D classification into 10 3D classes. One stable 3D class at a resolution of 5.38 Å was selected for high-resolution 3D auto-refinement; this class accounted for a subset of 108,560 particles (25.6%). This subset of refined particles was then re-extracted and re-centred before another round of 3D auto-refinement was carried out. The resultant 4.85 Å density map was corrected for the modulation transfer function (MTF) of the Gatan K2 summit camera then further sharpened using the post-processing procedure to 4.02 Å. Per-particle CTF-refinement was carried out and a soft mask was created which included the detergent shell. The final global resolution estimate of 3.58 Å was based on the gold-standard Fourier shell correlation (FSC) cut-off of 0.143. Local resolution was determined using one of two unfiltered half-maps as an input, a calibrated pixel size of 1.065 and a B-factor of −103. The output local resolution map is shown in Extended Data Fig. 2d, e . Model building Initially, a homology-based approach was performed using the crystal-lographic structure of Nostoc sp. PCC 7120 cyt b 6 f (PDB: 4OGQ) 40 as a template. Sequence alignments of the eight polypeptide subunits of cyt b 6 f were carried out using Clustal Omega ( Extended Data Figs. 7 , 8 ). The model was rigid-body docked into the density using the ‘fit in map’ tool in Chimera 41 . This was then followed by manual adjustment and real-space refinement using COOT 42 . Sequence assignment and fitting was guided by bulky residues such as Arg, Trp, Tyr and Phe. After fitting of the polypeptide chains and cofactors in one half of the dimeric complex, the other half of the complex was then independently fitted into the C1 density map. Once both halves of the complex were fitted, cofactors, lipids and plastoquinone-9 molecules were fitted into regions of unassigned density. The final model underwent global refinement and minimization using the real space refinement tool in PHENIX 43 . The final refinement statistics are summarized in Extended Data Table 1 . Pigment analysis by reversed-phase HPLC Pigments were extracted from purified cyt b 6 f with 7:2 acetone:methanol (v/v) and clarified extracts were separated by reversed-phase HPLC at a flow rate of 1 ml min −1 at 40 °C using a Supelco Discovery HS C18 column (5 μm particle size, 120 Å pore size, 25 cm × 4.6 mm) on an Agilent 1200 HPLC system. The column was equilibrated in acetonitrile: water:trimethylamine (9:1:0.01 v/v/v) and pigments were eluted by applying a linear gradient of 0–100% ethyl acetate over 15 min followed by isocratic elution with 100% ethyl acetate for a further 5 min. Elution of carotenoid and chlorophyll species was monitored by absorbance at 400, 450, 490 and 665 nm. Chlorophyll a was identified by its absorption spectra and known retention time 44 . The major carotenoid species was confirmed as 9- cis β-carotene using a standard obtained from Sigma-Aldrich (product no. 52824).