Rfam
download from Rfam entry RF02519
directory: /rpi_toxIN/sequence_data/
PDB
download from PDB to /rpi_toxIN/sequence_data/toxirna_seq_pdb.fasta
4ATO: Short et al., 2013
2XD0: Blower et al., 2011
2XDB: Blower et al., 2011
2XDD: Fineran et al., 2009
literature 1
/rpi_toxIN/sequence_data/toxirna_seq_literature1.fasta
Blower et al., 2012
include all organisms, which are additional to the ones from Fineran et al. (2009), RNA sequences added from Table S1
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Actinobacillus ureae ATCC 25976
Bacillus cereus Rock 1-15
Bryantella formatexigens DSM 14469
Clostridium botulinum BKT015925
Clostridium cellulovorans 743B
Clostridium nexile DSM 1787
Clostridium phage D-1873
Clostridium sp. HGF2
Coprobacillus sp. 29_1
Coprococcus sp. ART55/1
Eubacterium rectale DSM 17629
Eubacterium rectale M104/1
Eubacterium saburreum DSM 3986
Eubacterium saphenum ATCC 49989
Eubacterium yurii subsp. margaretiae ATCC 43715
Finegoldia magna BVS033A4
Fusobacterium sp. 3_1_5R
Fusobacterium sp. 4_1_13
Fusobacterium sp. D12
Fusobacterium ulcerans ATCC 49185
Haemophilus parasuis
Haemophilus parasuis SH0165
Haemophilus somnus 2336 | no sequence in Tab. S1
Lachnospiraceae bacterium 2_1_46FAA
Lactobacillus gasseri JV-V03
Lactobacillus helveticus DSM 20075 | no sequence in Tab. S1
Lactobacillus helveticus MTCC 5463 | no sequence in Tab. S1
Lactobacillus jensenii 1153 | no sequence in Tab. S1
Lactobacillus jensenii 208-1
Lactobacillus jensenii 269-3
Lactobacillus jensenii 27-2-CHN
Lactobacillus jensenii SJ-7A-US | repeats downstream of ORF Lactobacillus kefiranofaciens ZW3
Leptotrichia goodfellowii F0264
Leptotrichia hofstadii F0254
Pectobacterium atrosepticum SCRI1039
Peptoniphilus duerdenii ATCC BAA-1640
Peptoniphilus harei ACS-146-V-Sch2b | no sequence in Tab. S1
Roseburia intestinalis M50/1 | large gap between end of ToxI and start of ToxN (292bp)
Roseburia intestinalis XB6B4 | large gap between end of ToxI and start of ToxN (232bp)
Ruminococcus sp. 5_1_39B_FAA
Taylorella equigenitalis MCE9
Treponema succinifaciens DSM 2489 | no sequence in Tab. S1
Treponema vincentii ATCC 35580
Veillonella parvula ACS-068-V-Sch12
literature 2
/rpi_toxIN/sequence_data/toxirna_seq_literature2.fasta
Fineran et al., 2009
RNA sequences from Table S3 (putative ToxIN-type loci of multiple bacteria), column "repeat consensus" contains toxI RNA sequences
YP_001513384|Alkaliphilus_oremlandii_OhILAs
CP000047|Bacillus_thuringiensis_serovar_konkukian_97-27_pBT9727
AAZ06636|Bacillus_thuringiensis_serovar_kurstaki_HD73_pAW63 YP_001642731|Bacillus_weihenstephanensis_KBAB4_pBWB402 FJ176937|Erwinia_carotovora_subsp_atroseptica_1039_pECA1039
ZP_02272649|Fusobacterium_nucleatum_subsp_polymorphum_ATCC10953
YP_271719|Haemophilus_influenzae_biotype_aegyptius_pF1947
ZP_00133190|Histophilus_somni_2336
NP_862552|Lactococcus_lactis_subsp_lactis_W-37_pSRQ900
ZP_01968975|Ruminococcus_torques_ATCC_27756
ZP_01667702|Thermosinus_carboxydivorans_Nor1
commands listed for one specific example; applied the same way for further involved files/data
MSA with mlocarna
mlocarna --stockholm -s 400 --tgtdir /rpi_toxIN/msa_toxirna_pdb/ /rpi_toxIN/sequence_data/toxirna_seq_pdb.fasta > /rpi_toxIN/msa_toxirna_pdb/mlocarna_toxirna_pdb.log
RNAfold for each sequence
RNAfold -p --MEA -o /rpi_toxIN/rnafold_toxirna/pdb/allrna_pdb_rnafold.out -i /rpi_toxIN/sequence_data/toxirna_seq_pdb.fasta
RNAalifold for manual alignment of lit1
RNAalifold --noLP --color --mis -r -p --aln --cfactor 0.6 --nfactor 0.5 /rpi_toxIN/msa_toxirna_literature/lit1_manual/msa_manual_lit1.aln > /rpi_toxIN/msa_toxirna_literature/lit1_manual/RNAalifold/alifold_output.txt
RNAalifold --noLP --color --mis -r -p --aln --cfactor 0.6 --nfactor 0.5 --aln-stk --MEA -C /rpi_toxIN/msa_toxirna_literature/lit1_manual/msa_manual_lit1.aln > /rpi_toxIN/msa_toxirna_literature/lit1_manual/RNAalifold_constraint_hp1/alifold_output.txt
constraint for hairpin loop 1:
.....((((...........))))......................
constraint for hairpin loop 2:
...............||.............................
R-Scape
R-scape -s --fold --outdir /rpi_toxIN/msa_toxirna_literature/lit1_manual/Rscape_hp1/ /rpi_toxIN/msa_toxirna_literature/lit1_manual/RNAalifold/RNAalifold_results.stk
commands listed for one specific example; applied the same way for further involved files/data
download bacterial genomes with esearch v7.40:
esearch -db nucleotide -query "bacteria[filter] AND refseq[filter]" | efetch -format fasta > /rpi_toxIN/all_genomes.fna
Infernal
build and calibrate covariane model
/infernal-1.1.3/src/cmbuild -o /rpi_toxIN/model_toxirna_pdb/toxi_pdb_cmbuild.out /rpi_toxIN/model_toxirna_pdb/toxipdb_model.cm /rpi_toxIN/msa_toxirna_pdb/results/result.stk
/infernal-1.1.3/src/cmcalibrate /rpi_toxIN/model_toxirna_pdb/toxipdb_model.cm <!--optional: --cpu <n> -->
homology search with covariance model
/infernal-1.1.3/src/cmsearch -o /rpi_toxIN/model_toxirna_pdb/toxipdb_cmsearch.out -A /rpi_toxIN/model_toxirna_pdb/toxipdb_cmsearch_msa.fa --tblout /rpi_toxIN/model_toxirna_pdb/toxipdb_cmsearch_hits.txt --cpu 16 --verbose --nohmmonly -E 1000 -Z 549862.597050 /rpi_toxIN/model_toxirna_pdb/toxipdb_model.cm /rpi_toxIN/all_genomes.fna
for more relaxed prefilter add --mid (or --nohmm):
/infernal-1.1.3/src/cmsearch -o /rpi_toxIN/model_toxirna_lit2/toxilit2_cmsearch--mid.out -A /rpi_toxIN/model_toxirna_lit2/toxilit2_cmsearch--mid_msa.fa --tblout /rpi_toxIN/model_toxirna_lit2/toxilit2_cmsearch--mid_hits.txt --mid --cpu 16 --verbose --nohmmonly -E 1000 -Z 549862.597050 /rpi_toxIN/model_toxirna_lit2/toxilit2_model.cm /rpi_toxIN/all_genomes.fna
check covariance model
/infernal-1.1.3/src/cmpress /rpi_toxIN/model_toxirna_lit2/toxilit2_model.cm
/infernal-1.1.3/src/cmscan -o /rpi_toxIN/cmscan_checkup/lit2_cmscan.out --tblout /rpi_toxIN/cmscan_checkup/lit2_cmscan_hits.txt /rpi_toxIN/model_toxirna_lit2/toxilit2_model.cm /rpi_toxIN/sequence_data/toxirna_seq_literature2.fasta
RNAlien
webserver used
download to /rpi_toxIN/rnaalien/
download bacterial proteomes from NCBI FTP server
hmmsearch
hmmsearch -o /rpi_toxIN/pfam_hmm/toxnpfam_hmmsearch.out -A /rpi_toxIN/pfam_hmm/toxnpfam_hmmsearch_msa.fa --tblout /rpi_toxIN/pfam_hmm/toxnpfam_hmmsearch_hits.txt -Z 47079205 -E 1000 --cpu 10 /rpi_toxIN/pfam_hmm/PF13958_ToxN.hmm /rpi_toxIN/all_proteomes.fa
clustering
cd-hit -i /rpi_toxIN/sequence_data/PF13958_full_sequences.fasta -o /rpi_toxIN/pfam_clusters/pfam100 -c 1.0 -n 5 -d 0
-n 5 for -c 1.0 to 0.7
-n 4 for -c 0.65 to 0.6
results processing
extract database IDs and organism name from entries in all sets:
/rpi_toxIN/all_toxin_hits/extract_id-species.py
> input file for each set:
rfam: /rpi_toxIN/sequence_data/RF02519.fa
pfam: /rpi_toxIN/sequence_data/PF13958_msafull.fasta
/rpi_toxIN/sequence_data/PF13958_msaseed.fasta
pdb: /rpi_toxIN/sequence_data/toxirna_seq_pdb.fasta
literature1: /rpi_toxIN/sequence_data/toxirna_seq_literature1+idgi.fasta
literature2: /rpi_toxIN/sequence_data/toxirna_seq_literature2.fasta
cmsearch: rfam: /rpi_toxIN/model_rfam/toxi_cmsearch_hits.txt
pdb: /rpi_toxIN/model_toxirna_pdb/toxipdb_cmsearch_hits.txt
lit1: /rpi_toxIN/model_toxirna_lit1manual/toxilit1man_cmsearch_hits.txt (deactivate filter for inclusion threshold)
/rpi_toxIN/model_toxirna_lit1manual/toxilit1man_cmsearch--mid_hits.txt (deactivate filter for inclusion threshold)
rfam+lit1: /rpi_toxIN/model_toxirna_rfam+lit1/version_2.0.0RC8/toxirfamlit1_cmsearch--mid_hits.txt (deactivate filter for inclusion threshold)
hmmsearch: /rpi_toxIN/pfam_hmm/toxnpfam_hmmsearch_hits.txt
> write output to /rpi_toxIN/all_toxin_hits/
retrieve taxonomy ID for entries of sets:
/rpi_toxIN/all_toxin_hits/retrieve_genus.py
> options:
t 1: rfam, cmsearch, lit1
t 2: hmm, lit2
t 3: pfam
t 4: pdb
> input files are output files from extract_id-species.py
> write output to /rpi_toxIN/all_toxin_hits/
sort each file according to taxonomy ID and reduce multiple matches for the same taxonomy ID to one entry:
sort -k1 -n -u /rpi_toxIN/all_toxin_hits/taxidlist_rfam.txt > /rpi_toxIN/all_toxin_hits/taxidlist_rfam_unique.txt
append all files into one for R script, adapt the line start (sed) for each set:
for f in /rpi_toxIN/all_toxin_hits/taxidlist_rfam_unique.txt; do sed "s/^/rfam\t/" $f; done | cut -d ',' -f1 >> /rpi_toxIN/species_comparison/inputfile_taxids.txt
for hmmsearch (/rpi_toxIN/all_toxin_hits/taxidlist_hmm_unique.txt) use:
- get all matches annotated as taIII:
for f in /rpi_toxIN/all_toxin_hits/taxidlist_hmm_unique.txt; do sed "/MULTISPECIES/d; /hypothetical/d; s/^/hmmsearch_taIII\t/" $f; done | cut -d ',' -f1 >> /rpi_toxIN/species_comparison/inputfile_taxids.txt
- get all matches annotated as hypothetical protein:
for f in /rpi_toxIN/all_toxin_hits/taxidlist_hmm_unique.txt; do sed "/MULTISPECIES/d; /type_III/d; s/^/hmmsearch_hyppr\t/" $f; done | cut -d ',' -f1 >> /rpi_toxIN/species_comparison/inputfile_taxids.txt
overlap analysis with UpSet plot
generate plot with R script: /rpi_toxIN/species_comparison/species_visualization.R
RPI tools
web services with default parameters
sequences from PDB:2XD0, PDB:4ATO for RPISeq and PRIdictor
PDB:2XD0 and PDB:4ATO for PRI Hotscore and PLIP
download results to /rpi_toxIN/rpi_tools/
clustering
protein
cd-hit -i /rpi_toxIN/alignments_toxIN/protein/seq_co.fasta -o /rpi_toxIN/alignments_toxIN/protein/cdhit_cluster/seqco95 -c 0.95 -n 5 -d 0
-n 5 for -c 0.95 to 0.7
RNA
cd-hit-est -i /rpi_toxIN/alignments_toxIN/rna/seq_co.fasta -o /rpi_toxIN/alignments_toxIN/rna/cdhit_cluster/seqco95 -c 0.95 -n 10 -d 0
-n 10 for -c 0.95
-n 8 for -c 0.90
-n 6 for -c 0.85
-n 5 for -c 0.80
co-alignments
protein
MSA with mafft
mafft /rpi_toxIN/alignments_toxIN/protein/seq_co.fasta > /rpi_toxIN/alignments_toxIN/protein/msa-co_toxnproteins.fasta
convert fasta to stockholm files (command line)
from Bio import AlignIO
msa = AlignIO.read("msa-co_toxnproteins.fasta","fasta")
AlignIO.write(msa,"msa-co_toxnproteins.stk","stockholm")
RNA
shift starting positions of species sequences manually
MSA with mlocarna
mlocarna --stockholm --tgtdir /rpi_toxIN/alignments_toxIN/rna/mlocarna_co /rpi_toxIN/alignments_toxIN/rna/seq_co.fasta > /rpi_toxIN/alignments_toxIN/rna/mlocarna_co/mlocarna_cotoxi.log
annotation of interactions
-
literature-based interactions for PDB:2XD0 (Blower et al., 2011) and PDB:4ATO (Short et al., 2013)
-
annotation of both reference sequences into the MSA stk files (RNA and protein) with stk annotation lines:
#=GS DE
#=GR AS -
pseudoknot secondary structure annotation for RNA MSA stk files
5'HP (co-MSA) and 3'HP (all-MSA) merged in consensus:
#=GC SS_cons ....AAAAA....<<<<aaaaa......>>>>........
(3'HP adjusted according to annotation in Blower et al., 2012, Fig.4) -
visualization with Jalview (as feature annotations)
-
annotations from literature and CoDNaS-RNA database (cluster 99): see /rpi_toxIN/alignments_toxIN/analysis_protein.txt + _rna.txt and /rpi_toxIN/alignments_toxIN/coevolution.md
Weblogos
web service of WebLogo 3
PyMOL
annotation selection for PDB:2XD0
select protein, chain A
select aminoacids_blower, chain A & resi 2+3+19+21+23+29+33+52+53+55+58+73+74+79+88+99+100+102+110+112+114+115+116+117+118+119+122
select aminoacids_codnas, chain A & resi 20+143
select rna, chain H+G
select bases_blower, chain H+G & resi 1+6+17+31+32
select bases_codnas, chain H+G & resi -3+-2+5+7+8+21+22+23+26+27+28+30
annotation selection for PDB:4ATO
select protein, chain A
select aminoacids, chain A & resi 25+26+27+29+31+37+56+57+58+86+110+117+127+130+131+134+148
select rna, chain G
select bases, chain G & resi 1+2+3+4+5+10+16+17+18+19+20+29+30+31+32+33+34