How it works

To get from CSMs to residue links or PPIs xiFDR needs to aggregate CSMs. This is done in multiple stages/levels where on each level an FDR cutoff may be applied. Below you find a summary for each aggregation level.

Aggregation levels

CSM level

The CSM level is the plain input data to the FDR calculation. A lower FDR cutoff here can greatly reduce computation time.

Peptide level

To aggregate from the CSM level to the peptide level xiFDR combines all CSMs with the same peptide sequences (including modifications), crosslink positions inside the sequence, positions of the sequences in the according proteins diregarding the scores and charges of the individual matches.

Protein level

This is special in the sense, that it does not operate on crosslinks but on the proteins involved in the peptide level crosslinks. This is done by splitting each peptide level match into the linked proteins and distributing the peptide level score based on the fragment coverage for the according peptide level link. If no fragment coverage is supplied in the input DataFrame, the score is divided 50/50 for the proteins. After this, same proteins get aggregated and a linear FDR calculation and filtering is applied. Finally the peptide level matches gets filtered to only include matches where each interacting protein also passes the protein level FDR cutoff.

Note

For each protein passing the protein FDR filter, the complementing decoy protein must pass as well. If not, the decoy proteins would no longer represent the false positives within the targets.

Link level

This level aggregated the protein FDR filtered matches to links of specific position of the complete protein sequence. For this, aggregation is performed for matches with the same interacting proteins and the same link position in the protein sequence (i.e. the same residue). This level is relevant if you are not only interested in which proteins interact with each other but also how this interaction looks in 3D.

PPI level

The final aggregation is the protein-protein interaction level. Here, all link level matches with the same interacting proteins are aggregated.

Aggregation functions

By default the scores in each aggregation are performed using the geometric norm:

\[score_{agg} = \sqrt{\sum (score_i)^2}\]

However, you can define custom aggregation functions. Please refer to the API documentation.

FDR groups

Due to the different nature of self-links (i.e. homomeric [1]) and between-links (i.e. heterometric [1]) the FDR is calculated separately for these groups.

For similar reasons we also split the protein level FDR in groups of proteins with only between-link, proteins with only linear or self-link matches and proteins with self-link or linear matches additional to between-links.

[1] (1,2)

Homomeric links sometimes refer to links explicitly within a single molecule of a protein. As we usually don’t know whether the link is within one molecule or between multiple molecule of the same protein, we rather use the nomenclature of self- and between-links.

Validity checks

For some datasets, very low FDR cutoffs may result in too little data coming through. This may make confidnent FDR estimation impossible. To avoid this, we check if the number of target-target matches results in a minimum number of decoys td_prob to be observed (statistically).