Table 1.

Troubleshooting

Step	Problem	Possible Reason	Possible Solution
24	Sequential buffer profiles do not superimpose in a time-independent manner.	Air bubbles in the beam path, sample is moving out of beam (if using flow cell), or problem with intensity normalization.	Repeat steps 22–24 with a clean and dry cell, taking care to avoid air in the beam path. If problem recurs, consult beamline personnel to check intensity normalization.
28	Buffer and protein-solution profiles do not appear to converge at high q.	Buffer mismatch.	Repeat steps 27–28 with freshly made sample. If problem recurs, make up the buffer first, and then exchange into the buffer with a desalting column.
29	Sequential protein-solution profiles do not superimpose in a time-independent manner.	Air bubbles in the beam path, sample is moving out of beam (if using flow cell), or problem with intensity normalization.	Repeat step 27–29 with a clean and dry cell using fresh sample, taking care to avoid air in the beam path. If problem recurs, consult beamline personnel to check intensity normalization.
29	Scattering signal of protein-solution profiles appears to be increasing as a function of time.	Radiation damage.	Repeat step 27–29 with a clean and dry cell using fresh sample. Use faster flow rates (when using a flow cell), shorter exposure times, or 5–10 sec pauses in between exposures to allow radiation-induced aggregates to diffuse away from the beam path. If problem recurs, consider adding or increasing the concentration of additives with protective effects against radiation damage (refer to Buffer Requirements).
31	Buffer profiles before and after protein exposures are not superimposable.	Buildup of protein residue on the walls of the sample cell, contaminants were introduced, or change in the beamline setup (e.g. vacuum leak, beam drift).	Repeat steps 20–31 with more a rigorous cleaning procedure (possibly using harsher cleaning solutions, e.g. detergent). Ask beamline personnel to check the beamline and install a fresh sample cell, if possible.
32	Buffer subtraction yields negative signal or an under-subtracted signal at high q.	Buffer mismatch, poor intensity normalization, poor cleaning of the sample cell, or a change in the beamline setup (e.g. vacuum leak, beam drift).	Perform a buffer exchange with a disposable desalting column. Repeat steps 20–31 with more a rigorous cleaning procedure (possibly using harsher cleaning solutions). Ask beamline personnel to check the beamline setup.
33	The log(I) vs log(q) plot of the background-subtracted profile shows a steep increase at low q.	Aggregation or radiation damage.	Proceed to step 34 for better diagnosis.
34	Guinier curve has an upturn at low q.	Aggregation or radiation damage.	Perform a buffer subtraction of just the first protein exposure and plot as Guinier curve. If there is no upturn, systematically determine the number of exposures that are free of radiation damage and produce a new average (step 28). If upturn is observed in the first exposure, filter or extensively centrifuge fresh sample, and repeat steps 28–29. If the pH of the buffer is close to the isoelectric point (pI) of the protein, reduce the salt concentration or change the pH away from the pI.
34	Guinier curve has a downturn at low q.	Inter-particle repulsion.	Repeat steps 27–29 at a lower protein concentration or higher salt concentration.
35	Not enough data points in the Guinier curve satisfying qRg<1.3 criterion.	Protein is too large for the current beamline setup.	Consult with beamline personnel about changing detector or beamstop position.
40	Negative P(r) at high r.	Inter-particle repulsion.	If severe, repeat steps 27–29 at a lower protein concentration or higher salt concentration.
40	Highly extended P(r) at high r.	Aggregation or oligomerization, though extended P(r) may represent true structural features.	If severe, filter or extensively centrifuge fresh sample and repeat steps 28–29. Alternatively, reduce the protein concentration or change the solution condition. If the pH of the buffer is close to the isoelectric point (pI) of the protein, reduce the salt concentration or change the pH away from the pI.
41	Slightly negative or positive P(r) at high r.	Subtle inter-particle effects or slight aggregation.	Adjust the q-range (particularly qmin).
43	Trends increase significantly or nonlinearly with increasing concentration.	Protein is a mixture of oligomers under this solution condition.	Repeat steps 27–29 at below or above transition if data on monodisperse samples are desired. Alternatively, change the solution conditions.