TABLE 2 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
2	The cysteine mutant is expressed at very low levels	The mutated residue(s) is important for protein expression/stability	Choose another position for SDM. Test different vectors, strains, or mode of expression
4	No or poor cell growth after overnight culture	(1) Only few cells are seeded (2) Some of the minimal media components are missing	(1) Ensure that the pre-culture has sufficient cell density (OD600 of ~0.3–0.5). (2) Check that all the supplements are added to the minimal media (see reagent setup)
		(2) Some of the minimal media components are missing	(2) Check that all the supplements are added to the minimal media (see reagent setup)
8(A)vii and 8(B)xii	Weak signals for the cysteine mutants after spin labeling	(1) Labeling and sample handling under non-optimal conditions, resulting in cell lysis.	(1) For E. coli, process cells quickly after labeling
		(2) Unsuccessful SDM	(2) Verify the cysteine mutation(s) or choose other positions for SDM
		(3) Limited accessibility for the target sites	(3) Optimize the labeling conditions or choose other positions for SDM
	Large amount of free MTSL (as evident from the narrow lines in the RT CW EPR spectrum)	Insufficient washing of the cells or the OM	Increase the number of washing steps after labeling in Step 8(A)iv or 8(B)x
	No difference in signal intensity between WT and the mutant (under identical conditions)	(1) High background labeling	(1) Optimize the labeling and washing steps to reduce background labeling.
		(2) Poor labeling of the target cysteines	(2) Optimise the labeling conditions or change the target sites for labeling.
		(3) For OM preparations, incomplete solubilization of the IM	(3) Use a fresh stock of sarkosyl.
11	When the sample is inserted, there is no shift of the resonator frequency	The sample tube is not inside the cavity	Remove the sample and position it correctly
14	The protection switches (defense pulses) are not visible	Reference arm is off or too low Bias	Switch the reference arm on and open the bias by completely by sliding it to the right-had side. If the problem persists, increase the number of averages and the Video Amplifier Gain.
21	The PELDOR data shows no decay or only an exponential decay	(1) Incorrect ELDOR channel settings (no decay)	(1) Set the mw frequency, which is used for pump pulse optimization as the current ELDOR frequency. Check for the correct ELDOR power
		(2) Low labeling efficiency or very long interspin distances (only an exponential decay)	(2) Check the labeling efficiency and improve it or change the labeling positions as required.
	S/N ratio is not sufficient	(1) Low spin concertation	(1) Try to Increase the amount of sample in the active volume of the resonator
		(2) τ2 is too long	(2) Decrease τ2, but at least one full oscillation should be observed. Add d8-glycerol or deuterate the protein
	The data the WT sample cannot be measured for sufficiently long time window	Small background labeling	Observe the dipolar evolution as long as possible or the measurement may be skipped if the signal is too weak.
22	The WT or the single cysteine mutant shows an intramolecular contribution	(1) Incorrect fitting of the intermolecular background function	(1) Optimize the fitting of B(t). The WT or the Cys-less data often fit into an exponential decay (Figs. 3b and 4b).
		(2) The target protein either aggregates or oligomerizes in-situ	(2) In case of non-specific interaction/aggregation, try to optimize the vector, growth conditions, or the expression strain. For natural oligomers, the oligomeric state could be further characterized with additional single cysteine mutants.
	The doubly labeled protein shows some long-distance contributions	Incorrect fitting of the intermolecular background function	Check the dimensionality of the background function using single cysteine mutants. For the whole cell or the native membrane samples, a value of d between 2.0 to 2.5 fits for the background function.