Methods
The calculation methods, reference data, and sequence assumptions used by ResidueBench.
Amino acid composition
Count each of the 20 supported amino acids. Divide each count by the sequence length and multiply by 100 to get its percentage. The table includes absent residues and shows the total. Rounded percentages can differ slightly from 100 when added by hand.
Molecular weight
Mass in daltons equals the sum of amino acid residue masses plus the mass of one water molecule. The terminal water accounts for the free ends of a single chain. One neutral, linear, unmodified chain with free N- and C-termini. Cysteines are modeled without disulfide bonds.
Average mass uses isotope weighted masses and adds 18.01524 Da for water. Monoisotopic mass uses the principal isotope of each element and adds 18.01056 Da for water. Each calculation uses the residue masses and water mass from the same mode.
Results are theoretical neutral masses. They do not model modifications, cyclic or disulfide bonded chains, adducts, or mass-to-charge ratios.
Theoretical isoelectric point
Each basic group contributes 1 / (1 + 10pH − pKa). Each acidic group contributes −1 / (1 + 10pKa − pH). Net charge is the sum across the two free termini and all K, R, H, D, E, C, and Y side chains. The first and last residues can change the pKa of their respective termini.
The solver bisects pH 0–17 to find zero net charge. It uses an interval tolerance of 0.000001 pH units with a cap of 32 steps. The calculator displays two decimal places. The interval extends beyond pH 14 to capture mathematical roots for extremely basic sequences.
- One linear, unmodified chain with free N and C termini and reduced cysteines.
- Fixed pKa values approximate independent ionization. Structure, solvent conditions, and neighboring charges can shift experimental pI.
- Treat short-peptide estimates with care because sequence context can have a large effect on ionization.
- Bjellqvist (Biopython 1.86) parameter table
- pKa values used by the calculator
- What is the isoelectric point?
- Isoelectric Point Calculator
Theoretical net charge across pH
The charge curve uses the same terminal groups, side chains, and pKa values as the theoretical isoelectric point calculation. Each point adds the modeled fractional charges at one pH. Values are expressed in elementary charge units (e).
The curve evaluates 141 points at 0.1 pH intervals from pH 0 to 14. The table displays the 15 whole-number pH values, while the TSV export includes every point. Display and export values use four decimal places.
Molar extinction coefficient at 280 nm
For a protein in water, add 5,500 per tryptophan, 1,490 per tyrosine, and 125 per cystine pair. The result is a theoretical molar coefficient in M⁻¹·cm⁻¹.
The default estimate assumes all cysteines are reduced. The comparison uses the largest whole number of disjoint cysteine pairs within one chain. Use structural or experimental evidence to determine how many pairs form in the sample.
Kyte–Doolittle hydropathy
GRAVY is the arithmetic mean of the residue scores across the sequence. The local profile uses the same scores with equal weight for all residues in each full window. An input of length N and window size w produces N − w + 1 values.
Plot each value at the center of its one-based residue interval. Even window sizes place their centers between residues. The tool uses no partial windows at the termini and reports four decimal places.
Peptide sequence report
The analyzer validates a single sequence, separates any FASTA header, and normalizes residue letters to uppercase. It then reports the normalized sequence, counts and percentages, and both neutral mass estimates. The copied TSV includes the sequence.
Multi-record protein FASTA validation
Each record begins with a header that starts with > in column 1 and contains an identifier. The following lines contain the sequence until the next header. The validator counts non-whitespace symbols and flags anything outside the 20 standard residue codes.
Lines and columns start at 1. Columns count UTF-16 code units, with an optional initial byte order mark skipped. The validator accepts up to 1,000 records and 260,000 total input code units. Headers and sequence symbols may use up to 250,000 code units, excluding line breaks and whitespace in sequences. It reports the first 50 issues and the total found in the scanned input.
Format a valid batch into a separate copy with sequence lines up to 60 residues and LF line endings. Headers, residue order, and letter case are preserved by default. The other sequence calculators continue to accept one record per calculation.
One-letter and three-letter amino acid codes
Conversion uses the same 20 standard residues in each direction. ASCII letter case does not affect matching. Three-letter input requires whitespace between codes. Positions are one-based UTF-16 offsets into the original text.
B, J, and Z and their three-letter counterparts represent ambiguous residues. X and Xaa represent an unknown residue. The converter identifies those codes, as well as Sec and Pyl, and waits for corrected input before returning a sequence.
Input requirements
Paste a plain one-letter sequence or one FASTA record. For FASTA, start with a header beginning with > and put the sequence on the following lines. The calculators accept up to 10,000 standard amino acid residues. Ambiguous residue codes and explicit modification annotations produce input errors.
Verification
The molecular weight calculator was checked against Biopython 1.86 using its free amino acid masses and terminal water correction. Small differences reflect the reference tables. The values shown are theoretical estimates.
The isoelectric point calculator was checked against Biopython 1.86 using the same pKa set and matching search bounds. Separate tests verify terminal-only cases analytically.
Sequence handling
The calculators run in the browser. Sequence input stays on the page during calculation. Copying results places the selected output on your device clipboard.
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