PMID: 22314539 The T788G mutation in the cyp51C gene confers voriconazole resistance in Aspergillus flavus causing aspergillosis.

[CuzickA]

Chemistry paper curating with @martin2urban

curation link
https://canto.phi-base.org/curs/92ed9bf55f48e444

[CuzickA]

Fortunately the Af uniprot ref proteome is the same the control used in this paper. (NRRL3357).

Uniprot help #175342 to add correct gene name to B8NUK6 CYP51c.

[CuzickA]

Also requested uniprot gene names for
B8NFL5 CYP51b
B8N2C8 CYP51a
used email address curation@phi-base.org

[CuzickA]

@ValWood Please could you check this short chemistry paper once you have finished with Tox1/Snn1.
Thanks.

[ValWood]

I checked this one.
This is exactly as I would have curated it. Just the resistance and the normal growth.
These types of papers will be easy.

Only one comment. I think amino acid mutation was selected for the genotytp[es, nut nucleotide mutations were reported ?

I did not fix this because I don't know what you want to do. Even though there is a little more information in the nucleotide description, we always report the amino acids because biologist think in protein sequence alterations (and our users often only report the amino acid change). However, if the nucleotide alteration was reported we add this as a genotype note.

[CuzickA]

Old

New

Well spotted!
Does the above genotype chnage look reasonable?? I have added the amino acid change to the allele description but left the nucleotide change in the allele name as this corresponds more to the name used in Table 3.

[CuzickA]

The paper title also uses 'T788G mutation'

[ValWood]

Yes keep the names but describe as proteins seems good! Best of both worlds.

[CuzickA]

Dear Alayne and Martin,

Moreover, I've completed annotations for cyp51A and cyp51B from /Aspergillus fumigatus/, as well as for cyp51A, cyp51B and cyp51C from /Aspergillus flavus/. The modifications will be publicly available at the UniProt release around the 29-JAN-2019 (Release 2020_01).
If you have any further comments, suggestions or questions regarding the annotation, please do not hesitate to contact me. Once again, we would like to sincerely thank you for having taken the time to help us improve our database.
Best regards,
Marc Feuermann

[CuzickA]

Note- edited these genotypes from expression 'unknown' to 'wild type' as described in text p2602

[CuzickA]

This session is just waiting for

1. UniProt release with updated names DONE
2. key expt for training video Tier1 - plan to make a GO MF annotation and a single pathogen phenotype annotation
   

do we want to include the AE 'has_expressivity' in training video (Tier 1 curation) or does this complicate annotation too much?

also need to add minimal media to the conditions DONE

[ValWood]

I don't think it matters, but if the example you include has it, then include it. Sometimes the "expressivity" is the only difference between similar alleles.

On a related note, we changed "expressivity" to "severity". The community understood the meaning of "severity" better. Odd that this isn't in PHI-Canto. I will open a ticket for this. This has now been DONE and is visble in PHI-Canto

[ValWood]

This one can be approved after the gene name is updated

[CuzickA]

Gene names have been updated. I have approved the session :-)

I will close the ticket for now and it can always be retrieved if required for making the training video.

[CuzickA]

Nichola said there are is only Cyp51A and Cyp51B. Therefore the naming of Cyp51C in this paper is an error. To determine whether Cyp51C should be considered as Cyp51A or Cyp51B for the AE alteration in archetype we decided to use FRAST https://www.frast.com.au/.
Publication provides 'cyp51C (NCBI accession number XM_002383890.1)'
This can be looked up in Genbank but only the nucleotide sequence is downloadable in FASTA. AC: see update below 22_09_2023 https://www.ncbi.nlm.nih.gov/nuccore/XM_002383890.1?report=genbank

For FRAST we need the amino acid sequence in FASTA format.
Therefore search UniProt for 'XM_002383890.1' provides https://www.uniprot.org/uniprotkb/B8NUK6/entry#sequences

The amino acid sequence in FASTA format can be downloaded here

[CuzickA]

Copy and paste into FRAST with the Input 'automatic' selected

Select 'search'

Sequence has highest similarity to Cyp51A

[CuzickA]

Aspergillus flavus
cyp51C-T788G(aaS240A)[WT level]
cyp51C-T161C(aaM54T)[WT level]
cyp51C-C1325A(aaP419T)[WT level]
cyp51C-A1337G(aaN423D)[WT level]

Now, I will look to see if I can add AE alteration in archetype for Cyp51A (assuming this is the cyp51C named by authors)

[CuzickA]

FRAST searches done on 19_09_2023

In FRAST, select 'reference' and 'Cyp51A' than 'next

Enter the AA FASTA file from above and select 'next'

Select 'alignment'

For Aspergillus flavus cyp51C-T788G(aaS240A)[WT level]

Codon 240 in the sequence index is 'S' which aligns with codon 241 'S' in the Cyp51A archetype sequence.
Maybe we could use 'S241A; Cyp51C (Cyp51A); ASPEFU
(Note Cyp51A archetype species is Aspergillus fumigatus)

[CuzickA]

For Aspergillus flavus cyp51C-T161C(aaM54T)[WT level]

Codon 54 in the sequence index is 'M' which aligns with with archetype index codon 55 but a different wildtype AA 'I' in the Cyp51A archetype sequence.
Maybe we could use '55T; Cyp51C (Cyp51A); ASPEFU', no first AA listed as Wts different.

[CuzickA]

For Aspergillus flavus cyp51C-C1325A(aaP419T)[WT level]

Codon 419 in the sequence index is 'P' which aligns with with archetype index codon 420 but a different wildtype AA 'T' in the Cyp51A archetype sequence.
Maybe we could use '420T; Cyp51C (Cyp51A); ASPEFU', no first AA listed as Wts different. The second AA (mutation) is the same in the Af primary genotype as the archetype - does it make sense to have this AE?

[CuzickA]

For Aspergillus flavus cyp51C-A1337G(aaN423D)[WT level]

Codon 423 in the sequence index is 'N' which aligns with archetype index codon 424 but a different wildtype AA 'E' in the Cyp51A archetype sequence.
Maybe we could use '424D; Cyp51C (Cyp51A); ASPEFU', no first AA listed as Wts different.

[CuzickA]

logged in with Google
Saved session

[CuzickA]

Nichola says these AEs' look ok.

[CuzickA]

After discussion with Nichola, she recommended not using the UniProt download option to obtain an AA FASTA file. this is because there may be cases where the appropriate UniProt Id is not available. Instead use the following method

1. Look up author provided gene id in Genbank
   Publication provides 'cyp51C (NCBI accession number XM_002383890.1)'
   https://www.ncbi.nlm.nih.gov/nuccore/XM_002383890.1?report=genbank
2. Copy and paste the AA sequence into 'notepad'. Delete unwanted tabs. Insert a FASTA header eg '>XM_002383890.1 Aspergillus flavus NRRL3357 cytochrome P450|Cyp51C (Cyp51A)|PMID 22314539'
3. Save a copy of this AA FASTA file
   
4. Now enter this AA sequence into FRAST in the same way as above.

[CuzickA]

22_09_2023

[CuzickA]

'Reference'
'Archetype input Cyp51A'
Copy and paste

View output provides info in images above. (Also see below for double check)

Download MAFFT file into Notepad and save a copy. To view alignments this file would need to be opened using appropriate sequence alignment software (not sure which).

Session saved in FRAST

[CuzickA]

Double check (uniprot vs genbank generated AA FASTA file)

1. Looks the same

For Aspergillus flavus cyp51C-T788G(aaS240A)[WT level]

Codon 240 in the sequence index is 'S' which aligns with codon residue 241 'S' in the Cyp51A archetype sequence.
Maybe we could use 'S241A; Cyp51C (Cyp51A); ASPEFU
(Note Cyp51A archetype species is Aspergillus fumigatus)

2. Looks the same
   For Aspergillus flavus cyp51C-T161C(aaM54T)[WT level]

Codon 54 in the sequence index is 'M' which aligns with archetype index codon residue 55 but a different wildtype AA 'I' in the Cyp51A archetype sequence.
Maybe we could use '55T; Cyp51C (Cyp51A); ASPEFU', no first AA listed as Wts different.

3. Looks the same
   For Aspergillus flavus cyp51C-C1325A(aaP419T)[WT level]

Codon 419 in the sequence index is 'P' which aligns with archetype index codon residue 420 but a different wildtype AA 'T' in the Cyp51A archetype sequence.
Maybe we could use '420T; Cyp51C (Cyp51A); ASPEFU', no first AA listed as Wts different. The second AA (mutation) is the same in the Af primary genotype as the archetype - does it make sense to have this AE?

4. Looks the same
   For Aspergillus flavus cyp51C-A1337G(aaN423D)[WT level]

Codon 423 in the sequence index is 'N' which aligns with archetype index codon residue 424 but a different wildtype AA 'E' in the Cyp51A archetype sequence.
Maybe we could use '424D; Cyp51C (Cyp51A); ASPEFU', no first AA listed as Wts different.

[CuzickA]

AEs now added

Session approved, closing ticket.

[CuzickA]

Just to note: I can open the MAFFT output from FRAST using the Geneious software.