Protein - Structure Mapping, Alignments, and Visualization

This notebook gives an example of how to map a single protein sequence to its structure, along with conducting sequence alignments and visualizing the mutations.

Input: Protein ID + amino acid sequence + mutated sequence(s)

Output: Representative protein structure, sequence alignments, and visualization of mutations

Imports

import sys
import logging

# Import the Protein class
from ssbio.core.protein import Protein

# Printing multiple outputs per cell
from IPython.core.interactiveshell import InteractiveShell
InteractiveShell.ast_node_interactivity = "all"

Logging

Set the logging level in logger.setLevel(logging.<LEVEL_HERE>) to specify how verbose you want the pipeline to be. Debug is most verbose.

• CRITICAL
  • Only really important messages shown
• ERROR
  • Major errors
• WARNING
  • Warnings that don’t affect running of the pipeline
• INFO (default)
  • Info such as the number of structures mapped per gene
• DEBUG
  • Really detailed information that will print out a lot of stuff

Warning: DEBUG mode prints out a large amount of information, especially if you have a lot of genes. This may stall your notebook!

# Create logger
logger = logging.getLogger()
logger.setLevel(logging.INFO)  # SET YOUR LOGGING LEVEL HERE #

# Other logger stuff for Jupyter notebooks
handler = logging.StreamHandler(sys.stderr)
formatter = logging.Formatter('[%(asctime)s] [%(name)s] %(levelname)s: %(message)s', datefmt="%Y-%m-%d %H:%M")
handler.setFormatter(formatter)
logger.handlers = [handler]

Initialization of the project

Set these three things:

• ROOT_DIR
  • The directory where a folder named after your PROTEIN_ID will be created
• PROTEIN_ID
  • Your protein ID
• PROTEIN_SEQ
  • Your protein sequence

A directory will be created in ROOT_DIR with your PROTEIN_ID name. The folders are organized like so:

ROOT_DIR
└── PROTEIN_ID
    ├── sequences  # Protein sequence files, alignments, etc.
    └── structures  # Protein structure files, calculations, etc.

# SET FOLDERS AND DATA HERE
import tempfile
ROOT_DIR = tempfile.gettempdir()

PROTEIN_ID = 'SRR1753782_00918'
PROTEIN_SEQ = 'MSKQQIGVVGMAVMGRNLALNIESRGYTVSVFNRSREKTEEVIAENPGKKLVPYYTVKEFVESLETPRRILLMVKAGAGTDAAIDSLKPYLEKGDIIIDGGNTFFQDTIRRNRELSAEGFNFIGTGVSGGEEGALKGPSIMPGGQKDAYELVAPILTKIAAVAEDGEPCVTYIGADGAGHYVKMVHNGIEYGDMQLIAEAYSLLKGGLNLSNEELANTFTEWNNGELSSYLIDITKDIFTKKDEDGNYLVDVILDEAANKGTGKWTSQSALDLGEPLSLITESVFARYISSLKAQRVAASKVLSGPKAQPAGDKAEFIEKVRRALYLGKIVSYAQGFSQLRAASDEYHWDLNYGEIAKIFRAGCIIRAQFLQKITDAYAENADIANLLLAPYFKKIADEYQQALRDVVAYAVQNGIPVPTFSAAVAYYDSYRAAVLPANLIQAQRDYFGAHTYKRTDKEGIFHTEWLE'

class ssbio.core.protein.Protein(ident, description=None, root_dir=None, pdb_file_type=’mmtf’)

Store information about a protein, which represents the monomeric translated unit of a gene.

The main utilities of this class are to:

• Load, parse, and store the same (ie. from different database sources) or similar (ie. from different strains) protein sequences as SeqProp objects in the sequences attribute
• Load, parse, and store multiple experimental or predicted protein structures as StructProp objects in the structures attribute
• Set a single representative_sequence and representative_structure
• Calculate, store, and access pairwise sequence alignments to the representative sequence or structure
• Provide summaries of alignments and mutations seen
• Map between residue numbers of sequences and structures

Parameters:

• ident (str) – Unique identifier for this protein
• description (str) – Optional description for this protein
• root_dir (str) – Path to where the folder named by this protein’s ID will be created. Default is current working directory.
• pdb_file_type (str) – pdb, mmCif, xml, mmtf - file type for files downloaded from the PDB

Todo

• Implement structural alignment objects with FATCAT

# Create the Protein object
my_protein = Protein(ident=PROTEIN_ID, root_dir=ROOT_DIR, pdb_file_type='mmtf')

# Load the protein sequence
# This sets the loaded sequence as the representative one
my_protein.load_manual_sequence(seq=PROTEIN_SEQ, ident='WT', write_fasta_file=True, set_as_representative=True)

<SeqProp WT at 0x7f5f79cbd908>

Mapping sequence –> structure

Since the sequence has been provided, we just need to BLAST it to the PDB.

Note: These methods do not download any 3D structure files.

Methods

Protein.blast_representative_sequence_to_pdb(seq_ident_cutoff=0, evalue=0.0001, display_link=False, outdir=None, force_rerun=False)

BLAST the representative protein sequence to the PDB. Saves a raw BLAST result file (XML file).

Parameters:

• seq_ident_cutoff (float, optional) – Cutoff results based on percent coverage (in decimal form)
• evalue (float, optional) – Cutoff for the E-value - filters for significant hits. 0.001 is liberal, 0.0001 is stringent (default).
• display_link (bool, optional) – Set to True if links to the HTML results should be displayed
• outdir (str) – Path to output directory of downloaded XML files, must be set if protein directory was not initialized
• force_rerun (bool, optional) – If existing BLAST results should not be used, set to True. Default is False.

Returns:

List of new PDBProp objects added to the structures attribute

Return type:

list

# Mapping using BLAST
my_protein.blast_representative_sequence_to_pdb(seq_ident_cutoff=0.9, evalue=0.00001)
my_protein.df_pdb_blast.head()

['2zyd', '2zya', '3fwn', '2zyg']

	pdb_chain_id	hit_score	hit_evalue	hit_percent_similar	hit_percent_ident	hit_num_ident	hit_num_similar
pdb_id
2zya	A	2319.0	0.0	0.987179	0.963675	451	462
2zya	B	2319.0	0.0	0.987179	0.963675	451	462
2zyd	A	2319.0	0.0	0.987179	0.963675	451	462
2zyd	B	2319.0	0.0	0.987179	0.963675	451	462
2zyg	A	2284.0	0.0	0.982906	0.950855	445	460

Downloading and ranking structures

Methods

Protein.pdb_downloader_and_metadata(outdir=None, pdb_file_type=None, force_rerun=False)

Download ALL mapped experimental structures to the protein structures directory.

Parameters:

• outdir (str) – Path to output directory, if protein structures directory not set or other output directory is desired
• pdb_file_type (str) – Type of PDB file to download, if not already set or other format is desired
• force_rerun (bool) – If files should be re-downloaded if they already exist

Returns:

List of PDB IDs that were downloaded

Return type:

list

Todo

• Parse mmtf or PDB file for header information, rather than always getting the cif file for header info

Warning: Downloading all PDBs takes a while, since they are also parsed for metadata. You can skip this step and just set representative structures below if you want to minimize the number of PDBs downloaded.

# Download all mapped PDBs and gather the metadata
my_protein.pdb_downloader_and_metadata()
my_protein.df_pdb_metadata.head(2)

['2zyd', '2zya', '3fwn', '2zyg']

	pdb_title	description	experimental_method	mapped_chains	resolution	chemicals	taxonomy_name	structure_file
pdb_id
2zya	Dimeric 6-phosphogluconate dehydrogenase compl...	6-phosphogluconate dehydrogenase, decarboxylat...	X-RAY DIFFRACTION	A;B	1.6	6PG	Escherichia coli	2zya.mmtf
2zyd	Dimeric 6-phosphogluconate dehydrogenase compl...	6-phosphogluconate dehydrogenase, decarboxylat...	X-RAY DIFFRACTION	A;B	1.5	GLO	Escherichia coli	2zyd.mmtf

Protein.set_representative_structure(seq_outdir=None, struct_outdir=None, pdb_file_type=None, engine=’needle’, always_use_homology=False, rez_cutoff=0.0, seq_ident_cutoff=0.5, allow_missing_on_termini=0.2, allow_mutants=True, allow_deletions=False, allow_insertions=False, allow_unresolved=True, clean=True, keep_chemicals=None, skip_large_structures=False, force_rerun=False)

Set a representative structure from a structure in the structures attribute.

Each gene can have a combination of the following, which will be analyzed to set a representative structure.

• Homology model(s)
• Ranked PDBs
• BLASTed PDBs

If the always_use_homology flag is true, homology models are always set as representative when they exist. If there are multiple homology models, we rank by the percent sequence coverage.

Parameters:

• seq_outdir (str) – Path to output directory of sequence alignment files, must be set if Protein directory was not created initially
• struct_outdir (str) – Path to output directory of structure files, must be set if Protein directory was not created initially
• pdb_file_type (str) – pdb, mmCif, xml, mmtf - file type for files downloaded from the PDB
• engine (str) – biopython or needle - which pairwise alignment program to use. needle is the standard EMBOSS tool to run pairwise alignments. biopython is Biopython’s implementation of needle. Results can differ!
• always_use_homology (bool) – If homology models should always be set as the representative structure
• rez_cutoff (float) – Resolution cutoff, in Angstroms (only if experimental structure)
• seq_ident_cutoff (float) – Percent sequence identity cutoff, in decimal form
• allow_missing_on_termini (float) – Percentage of the total length of the reference sequence which will be ignored when checking for modifications. Example: if 0.1, and reference sequence is 100 AA, then only residues 5 to 95 will be checked for modifications.
• allow_mutants (bool) – If mutations should be allowed or checked for
• allow_deletions (bool) – If deletions should be allowed or checked for
• allow_insertions (bool) – If insertions should be allowed or checked for
• allow_unresolved (bool) – If unresolved residues should be allowed or checked for
• clean (bool) – If structure should be cleaned
• keep_chemicals (str, list) – Keep specified chemical names if structure is to be cleaned
• skip_large_structures (bool) – Default False – currently, large structures can’t be saved as a PDB file even if you just want to save a single chain, so Biopython will throw an error when trying to do so. As an alternative, if a large structure is selected as representative, the pipeline will currently point to it and not clean it. If you don’t want this to happen, set this to true.
• force_rerun (bool) – If sequence to structure alignment should be rerun

Returns:

Representative structure from the list of structures. This is a not a map to the original structure, it is copied and optionally cleaned from the original one.

Return type:

StructProp

Todo

• Remedy large structure representative setting

# Set representative structures
my_protein.set_representative_structure()

<StructProp REP-2zyd at 0x7f5f79bea1d0>

Loading and aligning new sequences

You can load additional sequences into this protein object and align them to the representative sequence.

my_protein.__dict__

{'_root_dir': '/tmp',
 'description': None,
 'id': 'SRR1753782_00918',
 'notes': {},
 'pdb_file_type': 'mmtf',
 'representative_chain': 'A',
 'representative_chain_seq_coverage': 95.7,
 'representative_sequence': <SeqProp WT at 0x7f5f79cbd908>,
 'representative_structure': <StructProp REP-2zyd at 0x7f5f79bea1d0>,
 'sequence_alignments': [<<class 'Bio.Align.MultipleSeqAlignment'> instance (2 records of length 468, SingleLetterAlphabet()) at 7f5f79be7a90>,
  <<class 'Bio.Align.MultipleSeqAlignment'> instance (2 records of length 468, SingleLetterAlphabet()) at 7f5f79299080>],
 'sequences': [<SeqProp WT at 0x7f5f79cbd908>],
 'structure_alignments': [],
 'structures': [<PDBProp 2zyd at 0x7f5fdb48e080>,
  <PDBProp 2zya at 0x7f5fd538a550>,
  <PDBProp 3fwn at 0x7f5f79cbde48>,
  <PDBProp 2zyg at 0x7f5f79cbdda0>,
  <StructProp REP-2zyd at 0x7f5f79bea1d0>]}

Methods

Protein.load_manual_sequence(seq, ident=None, write_fasta_file=False, outdir=None, set_as_representative=False, force_rewrite=False)

Load a manual sequence given as a string and optionally set it as the representative sequence. Also store it in the sequences attribute.

Parameters:

• seq (str, Seq, SeqRecord) – Sequence string, Biopython Seq or SeqRecord object
• ident (str) – Optional identifier for the sequence, required if seq is a string. Also will override existing IDs in Seq or SeqRecord objects if set.
• write_fasta_file (bool) – If this sequence should be written out to a FASTA file
• outdir (str) – Path to output directory
• set_as_representative (bool) – If this sequence should be set as the representative one
• force_rewrite (bool) – If the FASTA file should be overwritten if it already exists

Returns:

Sequence that was loaded into the sequences attribute

Return type:

SeqProp

# Input your mutated sequence and load it
mutated_protein1_id = 'N17P_SNP'
mutated_protein1_seq = 'MSKQQIGVVGMAVMGRPLALNIESRGYTVSVFNRSREKTEEVIAENPGKKLVPYYTVKEFVESLETPRRILLMVKAGAGTDAAIDSLKPYLEKGDIIIDGGNTFFQDTIRRNRELSAEGFNFIGTGVSGGEEGALKGPSIMPGGQKDAYELVAPILTKIAAVAEDGEPCVTYIGADGAGHYVKMVHNGIEYGDMQLIAEAYSLLKGGLNLSNEELANTFTEWNNGELSSYLIDITKDIFTKKDEDGNYLVDVILDEAANKGTGKWTSQSALDLGEPLSLITESVFARYISSLKAQRVAASKVLSGPKAQPAGDKAEFIEKVRRALYLGKIVSYAQGFSQLRAASDEYHWDLNYGEIAKIFRAGCIIRAQFLQKITDAYAENADIANLLLAPYFKKIADEYQQALRDVVAYAVQNGIPVPTFSAAVAYYDSYRAAVLPANLIQAQRDYFGAHTYKRTDKEGIFHTEWLE'

my_protein.load_manual_sequence(ident=mutated_protein1_id, seq=mutated_protein1_seq)

# Input another mutated sequence and load it
mutated_protein2_id = 'Q4S_N17P_SNP'
mutated_protein2_seq = 'MSKSQIGVVGMAVMGRPLALNIESRGYTVSVFNRSREKTEEVIAENPGKKLVPYYTVKEFVESLETPRRILLMVKAGAGTDAAIDSLKPYLEKGDIIIDGGNTFFQDTIRRNRELSAEGFNFIGTGVSGGEEGALKGPSIMPGGQKDAYELVAPILTKIAAVAEDGEPCVTYIGADGAGHYVKMVHNGIEYGDMQLIAEAYSLLKGGLNLSNEELANTFTEWNNGELSSYLIDITKDIFTKKDEDGNYLVDVILDEAANKGTGKWTSQSALDLGEPLSLITESVFARYISSLKAQRVAASKVLSGPKAQPAGDKAEFIEKVRRALYLGKIVSYAQGFSQLRAASDEYHWDLNYGEIAKIFRAGCIIRAQFLQKITDAYAENADIANLLLAPYFKKIADEYQQALRDVVAYAVQNGIPVPTFSAAVAYYDSYRAAVLPANLIQAQRDYFGAHTYKRTDKEGIFHTEWLE'

my_protein.load_manual_sequence(ident=mutated_protein2_id, seq=mutated_protein2_seq)

Protein.pairwise_align_sequences_to_representative(gapopen=10, gapextend=0.5, outdir=None, engine=’needle’, parse=True, force_rerun=False)

Pairwise all sequences in the sequences attribute to the representative sequence. Stores the alignments in the sequence_alignments DictList attribute.

Parameters:

• gapopen (int) – Only for engine='needle' - Gap open penalty is the score taken away when a gap is created
• gapextend (float) – Only for engine='needle' - Gap extension penalty is added to the standard gap penalty for each base or residue in the gap
• outdir (str) – Only for engine='needle' - Path to output directory. Default is the protein sequence directory.
• engine (str) – biopython or needle - which pairwise alignment program to use. needle is the standard EMBOSS tool to run pairwise alignments. biopython is Biopython’s implementation of needle. Results can differ!
• parse (bool) – Store locations of mutations, insertions, and deletions in the alignment object (as an annotation)
• force_rerun (bool) – Only for engine='needle' - Default False, set to True if you want to rerun the alignment if outfile exists.

# Conduct pairwise sequence alignments
my_protein.pairwise_align_sequences_to_representative()

# View IDs of all sequence alignments
[x.id for x in my_protein.sequence_alignments]

# View the stored information for one of the alignments
my_alignment = my_protein.sequence_alignments.get_by_id('SRR1753782_00918_N17P_SNP')
my_alignment.annotations
str(my_alignment[0].seq)
str(my_alignment[1].seq)

Protein.sequence_mutation_summary(alignment_ids=None, alignment_type=None)

Summarize all mutations found in the sequence_alignments attribute.

Returns 2 dictionaries, single_counter and fingerprint_counter.

single_counter:

Dictionary of {point mutation: list of genes/strains} Example:

{
    ('A', 24, 'V'): ['Strain1', 'Strain2', 'Strain4'],
    ('R', 33, 'T'): ['Strain2']
}

Here, we report which genes/strains have the single point mutation.

fingerprint_counter:

Dictionary of {mutation group: list of genes/strains} Example:

{
    (('A', 24, 'V'), ('R', 33, 'T')): ['Strain2'],
    (('A', 24, 'V')): ['Strain1', 'Strain4']
}

Here, we report which genes/strains have the specific combinations (or “fingerprints”) of point mutations

Parameters:

• alignment_ids (str, list) – Specified alignment ID or IDs to use
• alignment_type (str) – Specified alignment type contained in the annotation field of an alignment object, seqalign or structalign are the current types.

Returns:

single_counter, fingerprint_counter

Return type:

dict, dict

# Summarize all the mutations in all sequence alignments
s,f = my_protein.sequence_mutation_summary(alignment_type='seqalign')
print('Single mutations:')
s
print('---------------------')
print('Mutation fingerprints')
f

Some additional methods

Getting binding site/other information from UniProt

import ssbio.databases.uniprot

this_examples_uniprot = 'P14062'
sites = ssbio.databases.uniprot.uniprot_sites(this_examples_uniprot)
my_protein.representative_sequence.features = sites
my_protein.representative_sequence.features

Mapping sequence residue numbers to structure residue numbers

Methods

Protein.map_seqprop_resnums_to_structprop_resnums(resnums, seqprop=None, structprop=None, chain_id=None, use_representatives=False)

Map a residue number in any SeqProp to the structure’s residue number for a specified chain.

Parameters:

• resnums (int, list) – Residue numbers in the sequence
• seqprop (SeqProp) – SeqProp object
• structprop (StructProp) – StructProp object
• chain_id (str) – Chain ID to map to
• use_representatives (bool) – If the representative sequence and structure should be used. If True, seqprop, structprop, and chain_id do not need to be defined.

Returns:

Mapping of sequence residue numbers to structure residue numbers

Return type:

dict

# Returns a dictionary mapping sequence residue numbers to structure residue identifiers
# Will warn you if residues are not present in the structure
structure_sites = my_protein.map_seqprop_resnums_to_structprop_resnums(resnums=[1,3,45],
                                                                       use_representatives=True)
structure_sites

Viewing structures

The awesome package nglview is utilized as a backend for viewing structures within a Jupyter notebook. ssbio view functions will either return a NGLWidget object, which is the same as using nglview like the below example, or act upon the widget object itself.

# This is how NGLview usually works - it will load a structure file and return a NGLWidget "view" object.
import nglview
view = nglview.show_structure_file(my_protein.representative_structure.structure_path)
view

Methods

StructProp.view_structure(only_chains=None, opacity=1.0, recolor=False, gui=False)

Use NGLviewer to display a structure in a Jupyter notebook

Parameters:

• only_chains (str, list) – Chain ID or IDs to display
• opacity (float) – Opacity of the structure
• recolor (bool) – If structure should be cleaned and recolored to silver
• gui (bool) – If the NGLview GUI should show up

Returns:

NGLviewer object

# View just the structure
view = my_protein.representative_structure.view_structure()
view

Protein.add_mutations_to_nglview(view, alignment_type=’seqalign’, alignment_ids=None, seqprop=None, structprop=None, chain_id=None, use_representatives=False, grouped=False, color=’red’, unique_colors=True, opacity_range=(0.8, 1), scale_range=(1, 5))

Add representations to an NGLWidget view object for residues that are mutated in the sequence_alignments attribute.

Parameters:

• view (NGLWidget) – NGLWidget view object
• alignment_type (str) – Specified alignment type contained in the annotation field of an alignment object, seqalign or structalign are the current types.
• alignment_ids (str, list) – Specified alignment ID or IDs to use
• seqprop (SeqProp) – SeqProp object
• structprop (StructProp) – StructProp object
• chain_id (str) – ID of the structure’s chain to get annotation from
• use_representatives (bool) – If the representative sequence/structure/chain IDs should be used
• grouped (bool) – If groups of mutations should be colored and sized together
• color (str) – Color of the mutations (overridden if unique_colors=True)
• unique_colors (bool) – If each mutation/mutation group should be colored uniquely
• opacity_range (tuple) – Min/max opacity values (mutations that show up more will be opaque)
• scale_range (tuple) – Min/max size values (mutations that show up more will be bigger)

# Map the mutations on the visualization (scale increased) - will show up on the above view
my_protein.add_mutations_to_nglview(view=view, alignment_type='seqalign', scale_range=(4,7),
                                    use_representatives=True)

Protein.add_features_to_nglview(view, seqprop=None, structprop=None, chain_id=None, use_representatives=False)

Add select features from the selected SeqProp object to an NGLWidget view object.

Currently parsing for:

• Single residue features (ie. metal binding sites)
• Disulfide bonds

Parameters:

• view (NGLWidget) – NGLWidget view object
• seqprop (SeqProp) – SeqProp object
• structprop (StructProp) – StructProp object
• chain_id (str) – ID of the structure’s chain to get annotation from
• use_representatives (bool) – If the representative sequence/structure/chain IDs should be used

# Add sites as shown above in the table to the view
my_protein.add_features_to_nglview(view=view, use_representatives=True)

Saving

Protein.save_json(outfile, compression=False)

Save the object as a JSON file using json_tricks

import os.path as op
my_protein.save_json(op.join(my_protein.protein_dir, '{}.json'.format(my_protein.id)))