step	problem	Possible reason	solution
4	Poor sequence coverage for the detergent-solubilized sample	The rate of deuterium exchange is slow. The protein has aggregated	Increase deuteration time, number of deuteration time points and /or pH. Screen for other buffers which sustain protein’ stability
9	Poor sequence coverage in nanodiscs. Good digestion on the detergent-solubilized protein.	The nanodisc disassembly didn’t work. Alternately, the protein concentration is too low.	Increase the concentration of DDM in Quench buffer. The use of a harsher detergent such as foscholine can help for dissociation. However, a harsher detergent can lead to aggregation. Different concentrations of both detergents should be tested Alternately, concentrate the sample more on mini-spin concentrators.
9	There is little or no deuterium uptake.	The protein is not solvent accessible or the kinetics of exchange are too slow.	Use shorter chain lipids or increase the pD of HDX labelling buffer.
18	Protein is unfolding during the simulation and structure is not stable.	System is not neutralized Wrong simulation condition i.e, temperature, pressure and ensemble Small simulation box with inappropriate space between periodic images.	Neutralize the system by adding appropriate counter ions using “autoionize” plugin on VMD. Simulation should be performed in NPT ensemble at 310 K and 1 atm pressure. Generate a larger simulation box, ensuring that the minimum distance between the periodic images is at least half of the cut-off distance.
4 and 9	Poor digestion	Enzyme pepsin not active anymore.	Test the efficiency of the pepsin column by digesting the standard sample phosphorylase B. Alternately, place the column upside-down or change column