SEC-MALS-SAXS Data Collection and Processing

Purified EtfABCX and EtfAB proteins were thawed in an anaerobic glovebox (Coy Laboratory Products, Grass Lake, MI, USA) maintained in an inert gas atmosphere (95% N₂, 5% H₂) after which they were diluted using standard buffer either with or without 0.01% DDM, respectively, and in the presence or absence of a reductant or coenzyme. All samples in this study have their compositions detailed in Table S1. Buffers were made anaerobic through three cycles each of alternating N₂ purging (15 min) and vacuum pumping (15 min) using a Schlenk line apparatus, after which they were stored either within a glovebox or under positive pressure with N₂ to finish equilibrating toward anaerobicity. Samples were sealed anaerobically in a 96-well plate prior to injection into the anaerobically equilibrated SEC-MALS-SAXS system at the Structurally Integrated BiologY for Life Sciences beamline (SIBYLS, BL 12.3.1) of the Advanced Light Source (ALS) located at Lawrence Berkeley National Laboratory (LBNL).^21,22,26 A 1290 Infinity II series high-performance liquid chromatography (HPLC) system (Agilent, Santa Clara, CA, USA) with an autosampler was used for sample injection and SEC. Autosampler and column temperatures were equilibrated to room temperature before analyses began. The anaerobic running buffer was maintained under positive pressure by using inert gas. The beamline was configured to a 1.24 Å X-ray wavelength and 2075 mm sample-to-detector distance to obtain the relevant wave vector transfer, q = 4πsin(θ)/λ, where 2θ is the scattering angle and λ is the X-ray wavelength, yielding a q-range from 0.01 to 0.4 1/Å.²¹ A KW-803 column (Showa Denko, Tokyo, Japan), selected for its optimal separation of EtfABCX species, was equilibrated with an anaerobic standard buffer containing 1 mM dithiothreitol for at least six h to scrub the silica-based column of residual O₂ before re-equilibration to normal standard buffer. SEC was performed using a flow rate of 0.65 mL/min during 2 s X-ray exposures over the course of 25 min SAXS data collections, wherein a PILATUS3 × 2 M Detector (Dectris, Baden, Switzerland) was used to record images. SAXS images were radially integrated before being background subtracted using BioXTAS RAW (RAW), after which subtracted SAXS profiles were merged and used for analysis in the same software.²⁷ Some SEC-SAXS data sets required the application of a linear baseline correction in RAW to account for shifting baselines in the SAXS signal due to capillary fouling. The performance of SEC-SAXS’s separation of EtfABCX peaks combined with software-assisted singular value decomposition evolving factor analysis (SVD-EFA) ameliorated residual scattering contributions from the supertetramer and allowed obtainment of the NADH-reduced superdimer’s SAXS profile.²⁸

MALS experiments were performed under anaerobic conditions using an 18-angle DAWN HELEOS II light scattering detector connected in tandem to an Optilab T-rEX differential refractive index detector (Wyatt Technology, Goleta, CA, USA), both in-line with the SEC-SAXS system. System normalization and calibration were performed using bovine serum albumin (BSA) with 55 μL injections at 7 mg/mL in buffer matching that of each sample. MALS data analysis was performed using Astra 8 software (Wyatt Technology, Goleta, CA, USA), where dn/dc values were obtained and MALS MW values were determined from the primary SEC peaks of each sample.

In-line UV–visible spectra were measured during each SEC-MALS-SAXS run in order to monitor the cofactor redox state and occupancy through the use of an in-line Diode Array Detector (Agilent, Santa Clara, CA, USA) as part of the 1290 Infinity II series instrument. Absorbance was measured at 280, 374, 390, 454, and 636 nm to monitor the protein, cofactor, and charge-transfer complex behaviors. UV–visible spectral data were exported from Agilent ChemStation software before their normalization to the A₂₈₀ peak maxima of either the EtfABCX superdimer or EtfAB heterodimer reference specimen to correct for increased background from coenzyme presence or to correct for changes in superdimer–supertetramer populations in some conditions. Static, batch-mode UV–visible spectra were measured on exemplar specimens prior to SEC-MALS-SAXS with an N60 Mobile UV/vis spectrophotometer (Implen, Westlake Village, CA, USA).