Cell tradition and membrane isolation
Copper-replete Methylococcus capsulatus (Bathtub) was cultured in a 12 L reactor with a steady move of 1:3 methane:air at 1 L/min and 45 °C. The tradition was grown as described beforehand in nitrate mineral salts (NMS) buffer containing KNO3, MgSO4·7H2O, CaCl2·2H2O. 3.9 mM phosphate buffer, pH 6.8, hint factor answer, 80 μM NaFeEDTA, and 50 μM CuSO4·5H2O19. Cells had been grown to an optical density at 600 nm (OD600) of seven.0, harvested by centrifugation at 5000 x g for 10 min, flash frozen, and saved at −80 °C.
Copper-starved M. capsulatus (Bathtub) was first grown in 50 ml cultures for two weeks with 10 μM CuSO4 after which passaged into one other 50 ml tradition for two weeks at 0.5 μM CuSO4. This tradition was then used to inoculate a 3 L bioreactor tradition with 0 μM CuSO4 for 1 week till OD600 of 5.0 was reached. Cells had been harvested as described above, flash frozen, and saved at −80 °C.
To isolate membranes, cells had been thawed in buffer containing 25 mM PIPES, pH 7.3, 250 mM NaCl with DNase and protease inhibitor cocktail (Roche). The cells had been sonicated on ice for 10 min at 40 % amplitude with 1 s /1 s on/off pulses (Qsonica 700). The lysate was centrifuged at 20,000 xg for 45 min adopted by assortment of the soluble fraction and centrifugation at 140,000 xg for 1 h at 4 °C to isolate the membranes. The remoted membranes had been washed with 40 ml of the identical buffer, passing by way of a Dounce homogenizer earlier than centrifuging once more. This step was repeated for a complete of three washes. After the ultimate spin, membranes had been resuspended in 8 ml of the identical buffer, aliquoted, and flash frozen. The protein focus within the membranes was measured by the DC Lowry assay (Bio-Rad).
pMMO solubilization, nanodisc reconstitution, and exercise measurements
pMMO was remoted from membranes utilizing n-dodecyl-β-D-maltoside (DDM) blended with the membranes at a 1.2:1 mg:mg ratio at 4 °C for 1 h. The combination was then centrifuged at 140,000 x g to separate out the insoluble portion. Solubilized pMMO was concentrated and exchanged into buffer containing 25 mM PIPES, pH 7.3, 250 mM NaCl, 0.02% DDM. The protein focus was measured utilizing the DC-Lowry assay (Bio-Rad).
To kind nanodiscs of varied sizes, pMMO was blended with lipids remoted from the native organism and membrane scaffold protein (MSP1E3D1), each ready as described beforehand18. A molar ratio of 1:2:120 pMMO:MSP1:lipid was used to generate these nanodiscs; a bigger quantity of lipid than crucial was used to make sure formation of homogeneous nanodiscs. The combination was rotated end-over-end at 4 °C for 15 min together with 20 mM sodium cholate earlier than including 0.8 mg/ml SM-2 Bio-Beads (Bio-Rad). After 2 h, the Bio-Beads had been eliminated utilizing a syringe filter, and the fashioned discs had been purified utilizing a Superose 6 column (Cytiva). Fractions had been collected from the void, early, mid, late, and nanodisc (ND) peaks for additional characterization (Fig. 3). MSP2N2 discs had been generated by the identical technique utilizing a ratio of 1:1:360 pMMO:MSP2:lipid.
Dynamic mild scattering evaluation of fractions was carried out with a Punk instrument (Unchained Labs), analyzing 5 μl of pattern over 10 s with 5 repeats per pattern. Damaging-stain samples had been ready by making use of 3 μl pattern onto a 400-mesh copper grid. Grids had been washed for 2 rounds of 15 s with water and two rounds of 30 s with 1% uranyl formate. Grids had been then blotted and allowed to dry. Pictures had been collected on a JEM-1400 microscope (JEOL) at 30000x magnification at 3.71 Å pixel dimension utilizing Leginon software program.
Exercise assays had been carried out by including 100 μl pattern containing 2 mg/ml pMMO to a screw-top vial, including an extra quantity of duroquinol, eradicating 1 ml headspace, including 1.5 ml 13C-methane and shaking at 45 °C for five min. Damaging management reactions had been carried out omitting enzyme, duroquinol, and methane. Reactions had been stopped by storing at −20 °C. To investigate the methanol produced by the reactions, the samples had been thawed and 500 μl chloroform with 1 mM dicholoromethane was added to every pattern as an inner normal. Samples had been shaken at 2000 rpm at 4 °C for 10 min adopted by centrifugation at 2000 x g for 10 min at 4 °C. Samples had been then analyzed for 13C-methanol focus utilizing an Agilent 7890B/5977 A MSD GC/MS instrument with a PoraBOND Q column (25 m x 250 μm x 3 μm) in response to earlier strategies23,45. The 13C-methanol peak was built-in, in comparison with a regular curve, and normalized to the focus of dichloromethane.
CryoET pattern preparation
For M. capsulatus (Bathtub) micro organism, the cells had been diluted to an OD600 of two with NMS buffer. Quantifoil grids (300 mesh, Cu R2/1) had been glow-discharged for 45 s. 4 μl of the pattern had been added onto the carbon facet of the grid, which had been blotted from the mesh facet for 10 s earlier than plunge freezing into liquid ethane with a Leica GP2 (Leica Microsystems). The humidity was set to 100% and the temperature was set to fifteen °C throughout blotting.
For the remoted M. capsulatus (Bathtub) membranes, a 5 mg/ml inventory membrane answer was diluted to 1.25 mg/ml with buffer containing 25 mM PIPES, pH 7.3 and 250 mM NaCl, and blended with 6 nm gold beads earlier than plunge-freezing. Lacey carbon-coated copper grid (300 mesh, Agar Scientific) was glow-discharged for 45 s. 3 μl of the pattern had been added onto the carbon facet of the grid, and the grid was blotted for 3.5 s with a blotting power of −15 earlier than plunge freezing into liquid ethane utilizing a Vitrobot (Thermo Fisher Scientific). The humidity was set to 100% and the temperature was set to 4 °C throughout blotting.
Serial CryoFIB/SEM
Samples had been imaged on a ZeissCrossbeam 550XL (Carl Zeiss Microscopy, LLC), fitted with a Quorum switch station and cryo-stage (Quorum Applied sciences). They had been mounted on a Quorum-compatible customized pattern holder and coated with Pt for 60 s at 10 mA on the Quorum switch stage, earlier than loading on the cryo-stage. The stage temperature was set at −165 °C, whereas the anticontaminator was held at −185 °C. Samples had been imaged at 45° tilt after being coated once more with Pt for 2x 30 s utilizing the FIB-SEM’s inner GIS system, with the Pt reservoir held at 25 °C.
Preliminary trapezoid trenches had been milled at 30 kV 700 pA over 8.5 μm to achieve a closing depth of three.5 μm, with no additional sharpening step. Serial sectioning and imaging acquisition was carried out as follows: for top Cu samples, FIB milling was carried out utilizing the 30 kV 50 pA probe, whereas for low Cu samples, it was accomplished at 30 kV 20pA, and each had been acquired with a z-slice step of 10 nm and a depth of three.5 μm over all the milling field of 8 × 8 μm; SEM imaging was carried out at a pixel depth of 2048 ×1 536 pixels, which resulted in a pixel dimension of 5 nm, with the beam set at 1.5 kV 20pA, dwell time 50 nsec and scan velocity 0, averaging the sign over 100 line scans as a noise-reduction technique.
CryoFIB milling for lamella preparation
Lamella preparation of M. capsulatus (Bathtub) was carried out utilizing a Scios DualBeam cryoFIB/SEM (Thermo Fisher Scientific) outfitted with a PP3010T switch system and stage (Quorum Applied sciences) utilizing the xT v7.6 software program (Thermo Fisher Scientific). Grids had been sputter coated with Pt throughout the PP3010T switch chamber maintained at −175 °C. After loading onto the Scios stage at −168 °C, the grids had been inspected utilizing the SEM (operated at 5 kV and 13 pA). The grid floor was coated utilizing the fuel injection system trimethyl (methylcyclopentadienyl) platinum (IV) (Thermo Fisher Scientific) for 4 s, to offer a thickness of ~3 µm. Milling was carried out utilizing the ion beam operated at 30 kV and currents lowering from 300 to 30 pA. At 30 pA, lamella thickness was thinned to < 200 nm. Throughout the closing stage of milling, SEM lamella inspection was performed at 2 kV and 13 pA.
CryoET information assortment
For remoted membranes, cryoET tilt sequence had been acquired utilizing a Thermo Fisher Titan Krios operated at 300 keV outfitted with a Gatan Quantum post-column power filter (Gatan Inc) operated in zero-loss mode with 20 eV slit width, and Gatan K3 direct electron detector in eBIC (electron BioImaging Centre, Diamond). Tilt sequence had been collected with SerialEM48 with a nominal magnification of 64k and a bodily pixel dimension of 1.34 Å per pixel. They had been acquired utilizing dose-symmetric tilt-scheme49 ranging from 0° with a 3° tilt increment by a bunch of three and an angular vary of ± 63°. The collected dose of every tilt sequence was round 129 e–/Å2 with a defocus vary between −2.5 and −6 µm. Ten frames had been saved in every uncooked tilt picture. Particulars of information assortment parameters are listed in Supplementary Desk 1.
For cell lamellae, cryoET tilt sequence had been collected with SerialEM48 with a nominal magnification of 42k and bodily pixel dimension of two.13 Å/pix. They had been acquired usng dose-symmetric tilt-scheme49 ranging from 0° with a 3° tilt increment by a bunch of three and an angular vary of ± 54°. The collected dose of every tilt sequence was round 111 e–/Å2 with a defocus vary between −3.5 and −6 µm. Ten frames had been saved in every uncooked tilt picture.
CryoET information processing and subtomogram averaging
The automated cryoET pipeline developed in-house was used for preliminary tomograms (https://github.com/ffyr2w/cet_toolbox) by way of performing movement correction50 of the uncooked frames, tilt-series alignment and closing reconstruction with IMOD51. Appropriate tilt sequence had been chosen. For remoted membranes, the fiducial markers had been manually inspected to make sure the centering of predicted markers for every tilt sequence in eTOMO. For cell lamellae, a fiducial-less tilt-series alignment was carried out through the use of AreTomo52.
Subtomogram averaging from the remoted intracytoplasmic membranes was carried out following the workflow of emClarity36,37. The pMMO trimer crystal construction (PDB 3RGB)21 was low-pass filtered to 30 Å and used because the preliminary template for template search. A small dataset of 10 tilt sequence was used for template search with 4x binned tomograms with a pixel dimension of 5.36 Å. About 2,000 subtomograms had been chosen based mostly on their preliminary positions and orientations within the membrane. Subtomogram averaging and alignment had been carried out with a quantity together with 7-pMMO particles. After a number of refinement cycles, the density map with 7-pMMO was obtained.
An extra 127,417 subtomograms had been chosen from all the dataset of 187 tilt sequence. The subtomogram averaging and alignment had been carried out iteratively utilizing 3x and 2x binned tomograms. A cylindrical alignment masks together with one pMMO particle and a 3-fold symmetry had been used all through the alignment process. We transformed the coordinates from emClarity to Relion-4.053 for Relion 3D refinement in bin3 (4.02 Å pixel dimension) and bin2 (2.68 Å pixel dimension). The ultimate density map was reconstructed at bin 1 with Relion-4.053 and sharpened with a b-factor of −50.
For the three-trimer subtomogram averaging, the orientations for every particle from the above step was saved and the place shifted to the centre of three trimers. A cylindrical alignment masks together with the central 3 trimers and a 3-fold symmetry had been used for extra averaging and alignment in bin 2 (2.68 Å pixel dimension) in Relion-4.053.
Subtomogram averaging of pMMO from lamellae was processed equally because the remoted intracytoplasmic membranes utilizing emClarity36,37. A template search utilizing the 7-particle reference was carried out in 4x binned tomograms with a pixel dimension of 8.52 Å for the skinny lamellae. A complete of 500 subtomograms had been chosen based mostly on their preliminary positions and orientations within the intracellular membranes. The subtomogram averaging and alignment had been carried out iteratively utilizing 3x and 2x binned tomograms, with pixel sizes of 6.39 Å and 4.26 Å. respectively. A cylindrical alignment masks and a 3-fold symmetry had been utilized all through the alignment process. The ultimate density map was reconstructed at bin 2 with emClarity.
Mannequin constructing and refinement and evaluation
A beginning mannequin of the pMMO trimer was constructed by rigid-body docking the cryoEM SPA buildings of PmoA, PmoB and PmoC from pMMO nanodiscs (PDB 7S4H)18 into our cryoET STA map utilizing Chimera54. A mannequin of the prolonged pMMO array was constructed by hexagonally tiling the pMMO trimer utilizing a lattice fixed of 9.4 nm as decided from the subtomogram common of three pMMO trimers (Fig. 4b, c). A four-trimer area was then manually embedded in a lipid bilayer constructed and equilibrated utilizing CHARMM-GUI55 and containing 57% PVPE, 18% PVPG, 16% POPC, and 9% cardiolipin56 and subsequently solvated with TIP3P water and 150 mM NaCl utilizing VMD v1.9457. The ensuing system contained a complete of ~420,000 atoms, together with ~52,000 water molecules and ~730 lipid molecules, and was positioned in a simulation field with dimensions x = y = 187 Å, z = 123 Å and axial angles α = β = 90°, ɣ = 60°.
Molecular dynamics simulations
All-atom molecular dynamics simulations had been carried out utilizing NAMD v2.1458 and the CHARMM36 power area59.The four-trimer hexagonal patch was first subjected to a sequence of conjugant gradient power minimizations adopted by a 10-ns equilibration simulation with spine restraints and at last a 100-ns manufacturing simulation with out restraints. Simulations had been performed within the NPT ensemble with situations maintained at 1 atm and 310 Ok utilizing the Nosé–Hoover Langevin piston and Langevin thermostat respectively. The r-RESPA integrator scheme was employed with an integration time step of two fs and SHAKE constraints utilized to all hydrogen atoms. Brief-range, nonbonded interactions had been calculated each 2 fs with a cut-off of 12 Å; long-range electrostatics had been evaluated each 6 fs utilizing the particle-mesh-Ewald technique. Periodic boundary situations had been appropriately chosen to allow the protein to work together with its periodic picture within the x-y airplane, mimicking the molecular interactions throughout the prolonged hexagonal array.
Reporting abstract
Additional info on analysis design is obtainable within the Nature Analysis Reporting Abstract linked to this text.
