Renumbering

Overview

To highlight the Complimentary Determining Regions (CDRs) and remove the constant domain of the molecule, EMLy can renumber the antibody.

Inputs

• PDB filename

• Filename to save renumebered output to

• Renumbering scheme to follow (Kabat, Chothia, or IGMT, IGMT is default)

Outputs

• Renumbered PDB for the user to download

• 3D Viewer output shows the constant domain removed and the CDRs highlighted

Description

EMLy renumbers a molecule by reading the ATOM data type from the PDB file using panda's readpdb, the 3 letter amino acid sequences are converted into 1 letter with the seq1 function from the Bio.SeqUtils library. This data is stores in a pandas data frame. ANARCI is used to detect an immune receptor - if alignment is successful, an immune receptor has been found. The start and end of the variable regions are found so the constant regions can be removed. The residues are then added to their corresponding amino acid from the residue list and set to light grey while the CDRs are coloured according to the Etcembly colour scheme.

ANARCI

(https://academic.oup.com/bioinformatics/article/32/2/298/1743894)

ANARCI is a tool used to classify and number antibody and T-cell receptor amino-acid variable domain sequences. It aligns TCR variable sequences to a numbering scheme which allows the equivalent residue positions to be annotated and for different molecules to be compared. It works by aligning a single or multiple amino acid protein sequence input to a set of Hidden Markov Models (HMMs). HMMs describe the putative germ-line sequence for a domain type of a particular species. The most significant of these alignments is then used to apply the chosen numbering scheme. HMMs are built by downloading pre-aligned sequences for v and j gene segments of each available species and domain type and assigning this data to a single reference sequence using Muscle, with a large gap-open penalty. The HMMs are built by using all possible pairwise combinations of relevant v and j gene segments of each available species. All possible pairwise combinations are then taken to form a set of putative germ-line domain sequences. Each position represents one of the 128 positions in the IGMT numbering scheme. The hmmbuild tool is then used to build an alignment from the HMM. The hmmscan tool is then used to align an input sequence to each HMM. A position in each HMM alignment is annotated with the equivalent position in the numbering scheme - the most significant alignment classifies its domain type and the alignment is translated into a chosen numbering scheme. For each numbered domain, a header is written that describes the most significant alignment including the species, chain type, and alignment range, then the numbering follows in a column delimited format. ANARCI can also be imported as a Python module and used as an API within seperate scripts.

Difference Between Numbering Schemes (https://pmc.ncbi.nlm.nih.gov/articles/PMC6198058/)

There are three different renumbering schemes EMLy can use - IGMT, Kabat, and Chothia.

Kabat

The Kabat scheme works by defining a variability parameter, which is the number of different amino acids at a given position divided by the frequency of the most occurring amino acid at that position. This parameter helped to identify hypervariable regions in the variable regions of light and heavy chains, which are now known as CDRs. After aligning and numbering the variable region of heavy and light amino acids, and the variable regions of TCRs, it was discovered that the analysed sequences exhibited variable lengths and gaps and insertions could only be inserted at certain positions - these positions were located inside CDRs. In numbering schemes, these insertion points are found and identified to allow the CDRs to be found. This method ignores antibody chains of unconventional lengths and it doesn’t match well with 3D structure of antibodies.

Chothia

 Structure based, align crystal structures of antibody variable regions, define the loop structures that form the CDRs, and correct the position numbers of the insertion points so they better fit their topological positions. The CDRs defined on hypervariable amino acids according to Kabat are in a slightly different location to those defined by Chothia loop topology.

IGMT

Used for all protein sequences of immunoglobulin superfamily, including antibody light and heavy chains and TCRs. Based on amino acid alignment of germ-line V genes, which meant numbering stopped where CDR3 should be. To fix this, the numbering scheme was extended to the entire variable domains and various tools were developed to analyse the full length sequences. IGMT works by counting residues continuously from 1 to 128 based on germ-line V sequence alignment. This means it avoids the use of insertion codes. IGMT is default for EMLy as it is the primary reference in immunogenetics and immuno-informatics - it is used by WHO. The numbering method is based on alignments of sequences from a complete reference gene database, including the whole immunoglobulin family. The numbering scheme is less flexible as it does not allow visualisation of insertion positions. It is more difficult to adapt the IGMT scheme for potential sequences with new amino acid insertions.