Next generation sequence counts for different E. coli Cra mutant libraries after cell survival selection
Data files
Apr 21, 2026 version files 63.14 KB
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Cra_Deep_Mutational_Scanning_Counts.xlsx
24.67 KB
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Cra_Individual_Selections.xlsx
9.91 KB
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Cra_Positional_Scanning_Amino_Acid_Counts.xlsx
10.86 KB
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Cra_Positional_Scanning_Codon_Counts.xlsx
13.28 KB
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README.md
4.42 KB
Abstract
Understanding the relationship between protein sequence changes and their resulting impacts on structural stability and biological function is leading to advances in protein engineering and design. The N-terminal DNA binding domain of the bacterial catabolite repressor/activator protein (Cra) contains a non-parallel three-helix bundle with the helix-turn-helix major structural motif. While past efforts have comprehensively uncovered the sequence-fitness relationship of alpha helical proteins with parallel and anti-parallel packing arrangements, considerably less is known about non-parallel alpha helical proteins. We have developed a cell-based selection technology that leverages bacterial growth differences on pyruvate to rapidly identify stable and functional variants of E. coli Cra. The selection was applied to 19 individual single point mutants, five positional scanning NNN codon libraries, and a deep mutational scanning library of the N-terminal DNA binding domain containing 1,027 variants. We collected Illumina next generation sequencing counts for each variant before and after selection. The calculated sequence enrichment scores were used to establish trends in the sequence-fitness relationship for non-parallel helical bundle proteins.
Dataset DOI: 10.5061/dryad.41ns1rnw3
Description of the data and file structure
These excel sheets contain the next generation sequencing counts for all Cra individual mutant libraries, positional scanning libraries, and deep mutational scanning libraries that were experimented on. Sequence counts are included for the constructed naive libraries and for the mutant pools after rounds of growing cells in pyruvate minimal media.
Files and variables
File: Cra_Individual_Selections.xlsx
- This file contains next generation sequencing counts for the Cra individual mutant libraries containing only wildtype and one mutant at a time. Data is included for the naive and round 1 plasmid pools. The variable descriptions are below. All values have units of counts.
- Mutant: Name of the single point mutant being tested. First letter is the wildtype amino acid. Number is the position of the amino acid in the protein sequence. Second letter is the mutant amino acid.
- Counts: Number of next generation sequencing counts.
- Naive: Sequence counts in the original plasmid pool before selection.
- Round 1: Sequence counts in the plasmid pool after one round of selection.
- Wildtype: Sequence counts in the naive and round 1 plasmid pools for the wildtype plasmid.
- Mutant: Sequence counts in the naive and round 1 plasmid pools for the mutant plasmid.
File: Cra_Deep_Mutational_Scanning_Counts.xlsx
- This file contains next generation sequencing counts for the Cra mutational scanning library. Data is included for the naive, round 1, and round 2 plasmid pools. The variable descriptions are below. All values have units of counts.
- Amino Acid: The amino acid present at the randomized position.
- Counts: Number of next generation sequencing counts.
- K2, L3, D4, E5, etc.: The randomized positions. Letter is the wildtype amino acid. Number is the position of the amino acid in the protein sequence.
- Naive: Sequence counts for each amino acid at each randomized position in the original plasmid pool before selection.
- Round 1 and Round 2: Sequence counts for each amino acid at each randomized position in the plasmid pool after one or two rounds of selection.
File: Cra_Positional_Scanning_Amino_Acid_Counts.xlsx
- This file contains next generation sequencing counts for the Cra positional scanning libraries, given by amino acid counts. Data is included for the naive and round 3 plasmid pools for the I6, T15, and K33 positions. Data is included for the naive and round 1 plasmid pools for the V20 and K24 positions. The variable descriptions are below. All values have units of counts.
- Amino Acid: The amino acid present at the randomized position.
- Counts: Number of next generation sequencing counts.
- I6, T15, K33, V20, and K24: The randomized position. Letter is the wildtype amino acid. Number is the position of the amino acid in the protein sequence.
- Naive: Sequence counts for each amino acid at the randomized position in the original plasmid pool before selection.
- Round 1 and Round 3: Sequence counts for each amino acid at the randomized position in the plasmid pool after one or three rounds of selection.
File: Cra_Positional_Scanning_Codon_Counts.xlsx
- This file contains next generation sequencing counts for the Cra positional scanning libraries, given by codon counts. Data is included for the naive and round 3 plasmid pools for the I6, T15, and K33 positions. Data is included for the naive and round 1 plasmid pools for the V20 and K24 positions. The variable descriptions are below. All values have units of counts.
- Codon: The codon present at the randomized position.
- Counts: Number of next generation sequencing counts.
- I6, T15, K33, V20, and K24: The randomized position. Letter is the wildtype amino acid. Number is the position of the amino acid in the protein sequence.
- Naive: Sequence counts for each codon at the randomized position in the original plasmid pool before selection.
- Round 1 and Round 3: Sequence counts for each codon at the randomized position in the plasmid pool after one or three rounds of selection.
Code/software
Excel is needed to view the data.