Table 1 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
39, 51	A color change and fluorescence is observed in the Fe protein–mPlum fractions during purification, requiring revisions to anaerobic technique starting at Step 1 (Fig. 6a)	Exposure of the sample to oxygen due to:
		insufficient deaeration of buffers	Add redox active dye, such as resazurin (1 mg/mL), to buffers to verify anaerobicity. Perform additional degas cycles as necessary. Running buffers through the columns without sample may be useful to ensure the setup is functioning as intended
		gas permeable points in purification setup	Make sure all FPLC tubing is PEEK tubing. Fully equilibrate the FPLC system with anaerobic buffer. Then, run anaerobic buffer containing redox active dye through FPLC and collect fractions anaerobically (attached to the manifold). If a color change is observed, verify there are no leaks in the system by measuring flow rate
		contaminating oxygen in anaerobic chambers or ‘tents’	Check that the anaerobic monitors read below 10 ppm oxygen. In case of a faulty monitor, bring in a degassed aliquot of redox active dye and check for color change. If high oxygen levels are confirmed, then bring in fresh catalysts and cycle the entire antechamber to lower oxygen levels
89	Too many, too few, or irregularly distributed particles on a grid, requiring re-optimization of grid conditions with the same protein preparation, returning to Step 63 (Fig. 6d)	Suboptimal particle distribution due to:
		too high or too low protein concentration	Verify protein concentration using amino acid analysis, and prepare a set of grids with a range of different protein concentrations using the same blotting parameters
		protein aggregating in solution or on grid, leading to inhomogeneous spreading over grid	Adjust centrifugation settings of protein samples before grid preparation, including longer spin times and higher speeds. If aggregation is still observed on the grid, other surfactants besides CHAPSO, such as n-dodecyl-β-maltoside or fluorinated octyl-maltoside can be explored to minimize particle self-association58
		protein preferring carbon over vitreous ice, or accumulating at the edges of holes	Varying surfactants and blotting conditions, or using graphene-oxide, ultrathin-carbon layered or UltrAUfoil grids may help establish an even distribution of particles across grid holes
	Contamination on surface of cryoEM grids, requiring revision to grid blotting, storage or transfer setup, starting at Step 63	Crystalline ice may accumulate on grids due to:
		high humidity of atmosphere	Cycle the anaerobic tent to lower humidity during grid preparation
		contaminated liquid nitrogen	Always use fresh liquid nitrogen and ethane/propane and use within a short amount of time
90	CryoEM maps appear distorted or highly disordered in particular directions/particle regions, requiring revision of grid freezing conditions at Step 63 (Fig. 6c)	Map distortions due to:
		particles on a grid show a preferred orientation, rather than the desired random distribution of orientations	Use of additives, surfactants or sacrificial proteins may help alter preferred orientation
		adsorption and damage of particles at the AWI	As above, use additives, surfactants, sacrificial proteins or grids layered with graphene oxide, amorphous carbon or other affinity layers to prevent protein interactions with the AWI. Verify random distribution of particles in ice using cryoET
	Damaged or oxidized metallocofactor density, requiring revised anaerobic technique during protein preparation and/or freezing procedures at Step 63 (Fig. 6b)	Metallocofactor density distortion due to:
		exposure of particles to oxygen leading to cofactor damage and/or oxidation	Verify anaerobicity of the solutions, buffers, and Coy tent. If necessary, prepare fresh solutions, cycle the tent and exchange catalysts in the tent
		radiation damage of cofactors or liganding groups during imaging	Alter dose during data collection or analyze dose fractionation of movie frames during processing
		partial occupancy or inhomogeneity of cofactors in protein samples	After purification, verify there is a full complement of metalloclusters using inductively coupled plasma mass spectrometry. Damage to the metallocluster sites may occur downstream due to damage during grid preparation, see solutions above to avoid preferred orientation and interactions with the AWI