Example 1: Enzyme active site

In the following example, we will create an active site variant library in the protein with the PBD entry: 5JFM.

The protein was crystallized with multiple chains, but we chose to model the variants on chain A.

The active site residues are 199, 329, 331, 472, 480, 483, 493, 495. Also, it is known that residue 330 is critical for catalysis.

It is highly recommended to work on a PROSS stabilized variant of your protein.

You will enter the parameters of your protein to be modeled, so we can calculate the possible sequence space (i.e., the allowed amino acid identities in each active site position). Make sure you know your protein!!
Email address: <my_email>@<academic domain> <-- email must be from an academic domain and may include up to three digits.
Do you wish to upload your own PDB file? no

PDB ID: 5JFM
Chain identifier to design: A
Amino acid positions to diversify: 199A 329A 331A 472A 480A 483A 493A 495A
Essential amino acid residues: 330A
Ligand designation: leave empty in this case

ΔΔG: 6 (decrease this parameter if the Total number of designs is high)

Minimal PSSM threshold: -2
Minimal number of mutations per design: 2 (all mutants are different from the WT protein by at least 2 active site mutations)
Maximal number of mutations per design: 5 (all mutants are different from the WT protein by at most 5 active site mutations)

Use default parameters for the multiple sequence alignment generation.

A few hours/ days after submission, you will get an email with the results. Please read carefully the attached ReadMe.txt file!!
See the FAQ for further details.

Example 2: Protein-protein interface

In the following example, we will create a designed library of variants of a protein-protein interface (pdb code 3U43).

This structure contains a complex of two proteins, one in chain A and the other in chain B. The proteins interact through an interface formed by two loops: residues 21-36 on chain A, and residues 80-100 on chain B. Also, it is known from previous studies that residues 54A, 55A, 86B are crucial for the interaction (hotspot residues).

Note that FuncLib can also be applied to one chain to increase its affinity for the target chain, which stays fixed.

Here, you will enter the parameters of the complex to be modeled.
Email address: <my_email>@<academic domain> <-- email must be from an academic domain and may include up to three digits.
Do you wish to upload your own PDB file? no
PDB ID: 3U43
Chain identifier to design: A B
Amino acid positions to diversify: 24A 27A 28A 31A 77B 82B 85B 96B 97B
Essential amino acid residues: 54A 55A 86B
ΔΔG: 1 (increase this parameter if the Total number of designs is low)

Minimal PSSM threshold: 0 (this threshold value- zero, is pretty high, so the sequence space will include only amino acids that are slightly favored by the multiple sequence alignment)

Minimal number of mutations per design: 2
Maximal number of mutations per design: 5

Use default parameters for the multiple sequence alignment generation.

Example 3: Light and heavy chain interfaces of an antibody

In the following example, we will create a diversified library of light and heavy chain interfaces of antibody (pdb code 2FJG).

This antibody was crystallized in a complex, and we chose to model the variants on chain H (heavy) and L (light) of the antibody.

Email address: <my_email>@<academic domain> <-- email must be from an academic domain and may include up to three digits.
Do you wish to upload your own PDB file? no
PDB ID: 2FJG
Chain identifier to design: L H W
Amino acid positions to diversify: 43L 49L 55L 89L 94L 95L 97L 100L 59H 95H 107H
ΔΔG: 1 (reduce this parameter if the total number of designs is high)
Minimal PSSM threshold: -1 (this chosen threshold value -1, is a bit permissive, in case you want the sequence space to include only amino acids that are favored by the multiple sequence alignment choose a value above or equal to 0)

Minimal number of mutations per design: 4 (all mutants are different from the WT protein by at least 4 mutations in the light heavy chain interfaces)
Maximal number of mutations per design: 10 (all mutants are different from the WT protein by at most 10 mutations in the light heavy chain interfaces)

Use default parameters for the multiple sequence alignment generation.