Table 1 |.

Troubleshooting table

Step	Problem	Possible reason	Solutions
20	No PCR product, low intensity band, or multiple bands are observed	Primers have low specificity PCR parameters are not optimal	Increase the length of primers (can be > 25 bases) to increase specificity. Adjust primer or template concentration, change annealing or extension temperature.
26	No PCR band or wrong size of band is observed	Original template is incompletely digested Overlap sequence (between the primers for vector and coupler) has low Tm	Lower template concentration, or PCR verify more colonies Redesign primers with increased length, introduce silent mutations (when possible) on the vector primer to make it GC rich.
35 38	Restrained and unrestrained membrane proteins both show abnormal subcellular location	Membrane protein is heterologously expressed in Pichia cells Protein overexpression in mammalian cells interferes with protein sorting or results in protein degradation	For mammalian proteins, analyze subcellular location in HEK393 cells, but purification and crystallization of the restrained membrane proteins expressed in Pichia are usually unaffected.
74	Restrained protein remains unstable in detergent	Membrane protein is highly unstable	Add high affinity ligand to stabilize protein conformation, add cholesterol derivative (e.g., cholesteryl hemisuccinate)
97	Density map at the membrane protein region is of poor quality	The dataset is of low resolution (worse than 3.5 Å) or poor quality (e.g., severe anisotropy)	Use multi-crystal averaging (Step 102), perform anisotropy correction