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Metalation calculators

Revised beta version Metalation Calculator II uses data in Clough et al. (2025) Nature Communications 16, 810.

Conditional calculators for E. coli JM109 (DE3) grown aerobically, exposed to hydrogen peroxide and grown anaerobically were first reported in Foster et al. (2022) Metallomics 14(9), mfac058. This manuscript also contains tutorial information on the uses and constraints of metalation calculators. A spreadsheet version of the original idealised Salmonella calculator was first released in Young et al. (2021) Nature Communications 12, 1195. Iterative refinements, for example to estimates of the intracellular metal availabilities or for additional conditions and cell types, will be catalogued here. The hyper-aerated E. coli calculator was added on 4th August 2022, used vigorously aerated small volume cultures grown in baffled flasks, with notably reduced iron availability, and the underlying data for these hyper-aerated cells has been made available in BioRxiv. Underlying data for M9 calculator is in BioRxiv II.

Notes about kinetics:  (1) Cytosolic available metal is typically bound (and buffered). However, the buffer can become depleted if the rate of demand, for example for an over-expressed recombinant protein, greatly exceeds the rate of metal-import. Metal availability then drops below the steady-state [available metal] (really an activity) value in the calculator. DNA-binding metal sensors, tuned to such changes, have been used to read-out bespoke values enabling speciation of metalation to be predicted (above cited papers and manuscript in preparation); (2) Transfer to/from a nascent protein is likely to follow associative ligand-exchange: This confers rapid on/off rates supporting the accuracy of such calculations; (3) Analogous calculations have been used to estimate the magnitude of ‘kinetic bias’ introduced by metallochaperone-mediated metalation (JACS Au 2023); (4) Relative in vitro binding preferences of proteins that kinetically-trap metals at folding may be used in the calculator in Clough et al. (2025) Nature Communications 16, 810. The calculator is a beta-version with testing ongoing. We are keen to have feedback.

To predict the metalation state of a protein or other biomolecule, fill in values in the table for as many determined metal affinities (and availabilities if known) as possible. Where affinities are unknown, use the toggle buttons to exclude that metal.

Oval Picture of Flasks

Idealised Salmonella

(Default Settings for Metal Availability)

Metal Affinity (M) Metal affinities of molecule (M) (dissociation constants, KD) ∆G (kJ mol-1) ∆G for complexation with the molecule (kJ mol-1) Metal Availability (M) Buffered available metal concentration (M) Available ∆G (kJ mol-1) Buffered available free energy of metal (kJ mol-1) Occupancy Molecule occupancy with metal under these availabilities
Total Metalation

The calculator is based on Nature Communications 12 1195- (2021). Supported by grant BB/V006002/1 from the Biotechnology and Biological Sciences Research Council. Background considerations and underlying assumptions

To predict the metalation state of a protein or other biomolecule, fill in values in the table for as many determined metal affinities (and availabilities if known) as possible. Where affinities are unknown, use the toggle buttons to exclude that metal.

Oval Picture of Flasks

Idealised Salmonella

(Default Settings for Metal Availability)

Metal Affinity (M) Metal affinities of molecule (M) (dissociation constants, KD) ∆G (kJ mol-1) ∆G for complexation with the molecule (kJ mol-1) Metal Availability (M) Buffered available metal concentration (M) Available ∆G (kJ mol-1) Buffered available free energy of metal (kJ mol-1) Occupancy Molecule occupancy with metal under these availabilities
Total Metalation

The calculator is based on Nature Communications 12 1195- (2021). Supported by grant BB/V006002/1 from the Biotechnology and Biological Sciences Research Council. Background considerations and underlying assumptions

To predict the metalation state of a protein or other biomolecule, fill in values in the table for as many determined metal affinities (and availabilities if known) as possible. Where affinities are unknown, use the toggle buttons to exclude that metal.

Oval Picture of Flasks

Idealised Salmonella

(Default Settings for Metal Availability)

Metal Affinity (M) Metal affinities of molecule (M) (dissociation constants, KD) ∆G (kJ mol-1) ∆G for complexation with the molecule (kJ mol-1) Metal Availability (M) Buffered available metal concentration (M) Available ∆G (kJ mol-1) Buffered available free energy of metal (kJ mol-1) Occupancy Molecule occupancy with metal under these availabilities
Total Metalation

The calculator is based on Nature Communications 12 1195- (2021). Supported by grant BB/V006002/1 from the Biotechnology and Biological Sciences Research Council. Background considerations and underlying assumptions

To predict the metalation state of a protein or other biomolecule, fill in values in the table for as many determined metal affinities (and availabilities if known) as possible. Where affinities are unknown, use the toggle buttons to exclude that metal.

Oval Picture of Flasks

Idealised Salmonella

(Default Settings for Metal Availability)

Metal Affinity (M) Metal affinities of molecule (M) (dissociation constants, KD) ∆G (kJ mol-1) ∆G for complexation with the molecule (kJ mol-1) Metal Availability (M) Buffered available metal concentration (M) Available ∆G (kJ mol-1) Buffered available free energy of metal (kJ mol-1) Occupancy Molecule occupancy with metal under these availabilities
Total Metalation

The calculator is based on Nature Communications 12 1195- (2021). Supported by grant BB/V006002/1 from the Biotechnology and Biological Sciences Research Council. Background considerations and underlying assumptions

To predict the metalation state of a protein or other biomolecule, fill in values in the table for as many determined metal affinities (and availabilities if known) as possible. Where affinities are unknown, use the toggle buttons to exclude that metal.

Oval Picture of Flasks

Idealised Salmonella

(Default Settings for Metal Availability)

Metal Affinity (M) Metal affinities of molecule (M) (dissociation constants, KD) ∆G (kJ mol-1) ∆G for complexation with the molecule (kJ mol-1) Metal Availability (M) Buffered available metal concentration (M) Available ∆G (kJ mol-1) Buffered available free energy of metal (kJ mol-1) Occupancy Molecule occupancy with metal under these availabilities
Total Metalation

The calculator is based on Nature Communications 12 1195- (2021). Supported by grant BB/V006002/1 from the Biotechnology and Biological Sciences Research Council. Background considerations and underlying assumptions

To predict the metalation state of a protein or other biomolecule, fill in values in the table for as many determined metal affinities (and availabilities if known) as possible. Where affinities are unknown, use the toggle buttons to exclude that metal.

Oval Picture of Flasks

Idealised Salmonella

(Default Settings for Metal Availability)

Metal Affinity (M) Metal affinities of molecule (M) (dissociation constants, KD) ∆G (kJ mol-1) ∆G for complexation with the molecule (kJ mol-1) Metal Availability (M) Buffered available metal concentration (M) Available ∆G (kJ mol-1) Buffered available free energy of metal (kJ mol-1) Occupancy Molecule occupancy with metal under these availabilities
Total Metalation

The calculator is based on Nature Communications 12 1195- (2021). Supported by grant BB/V006002/1 from the Biotechnology and Biological Sciences Research Council. Background considerations and underlying assumptions

To predict the metalation state of a protein or other biomolecule, fill in values in the table for as many determined metal affinities (and availabilities if known) as possible. Where affinities are unknown, use the toggle buttons to exclude that metal.

Oval Picture of Flasks

Idealised Salmonella

(Default Settings for Metal Availability)

Metal Affinity (M) Metal affinities of molecule (M) (dissociation constants, KD) ∆G (kJ mol-1) ∆G for complexation with the molecule (kJ mol-1) Metal Availability (M) Buffered available metal concentration (M) Available ∆G (kJ mol-1) Buffered available free energy of metal (kJ mol-1) Occupancy Molecule occupancy with metal under these availabilities
Total Metalation

The calculator is based on Nature Communications 12 1195- (2021). Supported by grant BB/V006002/1 from the Biotechnology and Biological Sciences Research Council. Background considerations and underlying assumptions

To predict the metalation state of a protein or other biomolecule, fill in values in the table for as many determined metal affinities (and availabilities if known) as possible. Where affinities are unknown, use the toggle buttons to exclude that metal.

Oval Picture of Flasks

Idealised Salmonella

(Default Settings for Metal Availability)

Metal Affinity (M) Metal affinities of molecule (M) (dissociation constants, KD) ∆G (kJ mol-1) ∆G for complexation with the molecule (kJ mol-1) Metal Availability (M) Buffered available metal concentration (M) Available ∆G (kJ mol-1) Buffered available free energy of metal (kJ mol-1) Occupancy Molecule occupancy with metal under these availabilities
Total Metalation

The calculator is based on Nature Communications 12 1195- (2021). Supported by grant BB/V006002/1 from the Biotechnology and Biological Sciences Research Council. Background considerations and underlying assumptions

To predict the metalation state of a protein or other biomolecule, fill in values in the table for as many determined metal affinities (and availabilities if known) as possible. Where affinities are unknown, use the toggle buttons to exclude that metal.

Oval Picture of Flasks

Idealised Salmonella

(Default Settings for Metal Availability)

Metal Affinity (M) Metal affinities of molecule (M) (dissociation constants, KD) ∆G (kJ mol-1) ∆G for complexation with the molecule (kJ mol-1) Metal Availability (M) Buffered available metal concentration (M) Available ∆G (kJ mol-1) Buffered available free energy of metal (kJ mol-1) Occupancy Molecule occupancy with metal under these availabilities
Total Metalation

The calculator is based on Nature Communications 12 1195- (2021). Supported by grant BB/V006002/1 from the Biotechnology and Biological Sciences Research Council. Background considerations and underlying assumptions

To predict the metalation state of a protein or other biomolecule, fill in values in the table for as many determined metal affinities (and availabilities if known) as possible. Where affinities are unknown, use the toggle buttons to exclude that metal.

Oval Picture of Flasks

Idealised Salmonella

(Default Settings for Metal Availability)

Metal Affinity (M) Metal affinities of molecule (M) (dissociation constants, KD) ∆G (kJ mol-1) ∆G for complexation with the molecule (kJ mol-1) Metal Availability (M) Buffered available metal concentration (M) Available ∆G (kJ mol-1) Buffered available free energy of metal (kJ mol-1) Occupancy Molecule occupancy with metal under these availabilities
Total Metalation

The calculator is based on Nature Communications 12 1195- (2021). Supported by grant BB/V006002/1 from the Biotechnology and Biological Sciences Research Council. Background considerations and underlying assumptions

To predict the metalation state of a protein or other biomolecule, fill in values in the table for as many determined metal affinities (and availabilities if known) as possible. Where affinities are unknown, use the toggle buttons to exclude that metal.

Oval Picture of Flasks

Idealised Salmonella

(Default Settings for Metal Availability)

Metal Affinity (M) Metal affinities of molecule (M) (dissociation constants, KD) ∆G (kJ mol-1) ∆G for complexation with the molecule (kJ mol-1) Metal Availability (M) Buffered available metal concentration (M) Available ∆G (kJ mol-1) Buffered available free energy of metal (kJ mol-1) Occupancy Molecule occupancy with metal under these availabilities
Total Metalation

The calculator is based on Nature Communications 12 1195- (2021). Supported by grant BB/V006002/1 from the Biotechnology and Biological Sciences Research Council. Background considerations and underlying assumptions

*Uses availabilities within E. coli grown in LB media except Rhizobium availabilities for Co and Zn calculated using KCo-DmeR = 3.5 x 10-8 M (note Cys-Pro substitution relative to E.coli RcnR) plus qPCR values for dmeF, zntA and znuA, noting Rhizobium MgIIGTPCobW KCo = 2.5 x 10-11 M-1 rounded to 3 x 10-11 M-1

To predict the metalation state of a protein or other biomolecule, fill in values in the table for as many determined metal affinities (and availabilities if known) as possible. Where affinities are unknown, use the toggle buttons to exclude that metal.

Oval Picture of Flasks

Idealised Salmonella

(Default Settings for Metal Availability)

Metal Affinity (M) Metal affinities of molecule (M) (dissociation constants, KD) ∆G (kJ mol-1) ∆G for complexation with the molecule (kJ mol-1) Metal Availability (M) Buffered available metal concentration (M) Available ∆G (kJ mol-1) Buffered available free energy of metal (kJ mol-1) Occupancy Molecule occupancy with metal under these availabilities
Total Metalation

The calculator is based on Nature Communications 12 1195- (2021). Supported by grant BB/V006002/1 from the Biotechnology and Biological Sciences Research Council. Background considerations and underlying assumptions

*Uses availabilities within E. coli grown in LB media except Rhizobium availabilities for Co and Zn calculated using KCo-DmeR = 3.5 x 10-8 M (note Cys-Pro substitution relative to E.coli RcnR) plus qPCR values for dmeF, zntA and znuA, noting Rhizobium MgIIGTPCobW KCo = 2.5 x 10-11 M-1 rounded to 3 x 10-11 M-1

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