I have a sequence of a GPCR (PDB code 3NY8) which I am using to create a dataset of homologs (via a homology modelling pipeline) so I can perform electrostatic calculations on them. In order to create this GPCR dataset I need an alignment which I will feed to the pipeline so that it will create one homolog per alignment member.
My problem is sequence-based: I'm doing PSI/DELTA-BLASTs on the query and I am getting some odd results. When I do the 1st iteration, I get 196 hits, but on the second I get 155. It either stays the same or keeps decreasing on each successive iteration, and I have no idea how that's happening. Does anyone have any idea how that can happen? My understanding was that PSI-BLAST searches are supposed to increase the number of hits on each successive iteration since the algorithm is using a PSSM as opposed to a sequence to detect distant members.
The parameters used to construct the alignment were as follows:
Database: Non-redundant protein sequence databases (includes GenBank CDS translations, PDB, SwissProt, PIR, PRF)
Organism: Homo sapiens
Exclude: Models/uncultured sample sequences (both excluded)
Maximum target sequences: 1000
Expect threshold: 10
Word size: 3
Maximum matches in a query range: 0
Gap Costs: Existence: 12, Extension: 1
Compositional adjustments: Composition-based statistics
Filter: Low complexity regions
PSI-BLAST Threshold: 0.005
Any ideas would be appreciated!