Supercharge Server Documentation

Supercharge illustration


Supercharging refers to increasing the net charge of a protein by mutating surface residues. Increasing net charge can improve reversibility of unfolding by preventing aggregation of partially unfolded states. Aggregation is a common obstacle for use of proteins in biotechnology and medicine. Net charge, rather than number of charged residues, is often an indicator of aggregation propensity. Additionally, highly cationic proteins and peptides are capable of nonviral cell entry, and highly anionic proteins are filtered by kidneys more slowly than neutral or cationic proteins.

Supercharge application

This is a surface redesign protocol. Optimal positions for incorporation of charged side chains should be determined, as numerous mutations and accumulation of like-charges can also destabilize the native state. A previously demonstrated approach (AvNAPSA) deterministically mutates flexible polar residues (amino acids DERKNQ) with the fewest average neighboring atoms per side chain atom. Our approach uses Rosetta-based energy calculations to choose the surface mutations. Both automated approaches for supercharging are implemented in this protocol.

The AvNAPSA approach varies net charge by adjusting the surface cutoff. The Rosetta approach varies net charge by adjusting reference energies of the positive or negatively charged residues.

Supercharging does not predict the optimal net charge for a given application, it is intended to generate a series of sequences with different mutations and net charges for experimental testing.


AvNAPSA supercharge philosophy: mutate the most exposed polar residues to minimize structural change or destabilization. Only DE-RK-NQ residues can be mutated. This final sequence is deterministic.

Drawbacks: mutating surface polar residues can eliminate hydrogen bonds. Helix capping, edge-strand interaction, and loop stabilization all result from surface hydrogen bonds. Furthermore, this automated protocol mutates N to D and Q to E, but N and Q sometimes act simultaneously as a donor and acceptor for hydrogen bonds.

Reference: Lawrence MS, Phillips KJ, Liu DR. Supercharging proteins can impart unusual resilience. J Am Chem Soc. 2007 Aug 22;129(33):10110-2.


Rosetta supercharge philosophy (Rsc): mutate residue positions that preserve and/or add favorable surface interactions. Hydrophobic and small polar surface residues can also be mutated.

Rosetta drawbacks: mutating less-exposed positions can lead to better computed energies, but mistakes at these positions can be destabilizing. AvNAPSA favors charge swaps, so Rosetta requires more mutations to accomplish the same net charge.

Reference: Miklos AE, Kluwe C, Der BS, Pai S, Sircar A, Hughes RA, Berrondo M, Xu J, Codrea V, Buckley PE, Calm AM, Welsh HS, Warner CR, Zacharko MA, Carney JP, Gray JJ, Georgiou G, Kuhlman B, Ellington AD. Structure-based design of supercharged, highly thermoresistant antibodies. Chem Biol. 2012 Apr 20;19(4):449-55.

Supercharge flowchart

Supercharge options

The only required input file is the PDB to be redesigned. If homology models, NMR ensembles, or relaxed crystal structures are the starting structure, -l can be used.
Optionally, the user can use a resfile to specify residue positions to NOT mutate (NATAA or NATRO). This would be useful to preserve a known binding surface, for example. The default for the input resfile should be ALLAA and the supercharge protocol restricts the allowed residues at designable positions. For example, Gly, Pro, Cys residues and hbonded sidechains are not allowed to mutate by default.


For homology model ensembles or NMR ensembles, we recommend supercharging all the input structures and choosing consensus mutations. For supercharging a single crystal structure, or ensembles, we recommend generating designs with a spectrum of net charges. Since repacking the surface converges on similar sequences, we do not recommend using nstruct more than 10 (actually, nstruct is not used for AvNAPSA-mode because the sequence is deterministic). When positive-supercharging, you can bias the choice of Arg vs. Lys by giving different reference energies for the two residues. Likewise for negative-supercharging. The protocol dumps output PDBs with customized self-documenting names.



Please email Bryan Der with questions or suggestions.