Frequently Asked Questions

• What are the main advantages of AbLIFT?
• What type of proteins can be submitted to AbLIFT?
• Can AbLIFT be used without a structure?
• Can I submit a structure with missing density (missing residues/loops)?
• Can I submit an NMR structure?
• How long does an AbLIFT calculation take?
• What do the result files include?
• How to proceed towards experimental validation?
• How to proceed when receiving an error email?

• The novelty in AbLIFT is that it does not change the antigen-binding surface, thereby reducing the odds of changing specificity.
• Since AbLIFT does not target antigen-binding residues, mutations introduced through AbLIFT can collaborate with previously introduced mutations from natural or laboratory affinity maturation processes.
• Experience in the field shows that the vL-vH interface, which AbLIFT targets for design, can be modeled quite accurately. This suggests that antibodies may be subjected to AbLIFT without an experimental structure.
  We recommend to use AbPredict webserver for antibody modeling(AbPredict)
• AbLIFT does not require high-throughput screens - we recommend to test the top 20 ranked designs.
• AbLIFT can be used to improve antibody that is not in complex (we did not test the results experimentally).

• An excel file with the clustered designs sorted by Rosetta score(Rosetta Energy Units).
  • Design clustering- we select the best designs (in terms of predicted Rosetta score) that are different from one another by N mutations (by default, N=2).
• A zip file with the structures of the top 50 ranked designs, and their protein sequences in fasta format (please read carefully also How to proceed towards experimental validation?).
• A text file with the sequence space, e.g. the allowed residues in each position

• We strongly recommend to order the full genes rather than inserting mutations one by one.
• Select the designs for experimental testing- we recommend to order the top 20 designs, but you can order more/less variants according to your screening capacity. For each selected design, align its amino acid sequence (provided to you in the results email) with your WT sequence, and verify that there are no gaps.
• Structures with missing densities: the sequences in the attached Top50.fa file Do Not include the missing densities in the structure. If you have missing densities in your structure, you should align each mutant sequence to the full sequence of your protein, and fill in the missing residues.
• Back-translate the amino acid sequences to DNA sequences with dnaworks or EMBOSS websites.

• PDB id
  • The desired PDB id contains the letter 'o' but you typed the digit 0 or vice versa.
  • Wrong id - copy the id reported in the email and paste it in the rcsb web. Make sure it is your protein of interest.
  • The id is of a NMR structure - AbLIFT is not compatible with NMR structures.
  • The PDB files contain negative residue numbers. AbLIFT is incompatible with negative numbers.
  • One of the residues have more than one conformation- edit the file manually and keep only one conformation.
  • (rare) The PDB file contains a residue for which one or more of the backbone atoms is missing. Note that if an entire residue is missing this is not a problem. However, if some atoms exist, then all backbone atoms should also exist. To solve this problem remove all other atom lines related to the specific residue (i.e., turn it into a missing density).
• Chain identifier to design
  • Wrong identifier - not of a protein chain but of DNA or RNA chains.
  • Wrong identifier - the identifier is of a protein chain but the wrong one and is incompatible with the rest of the parameters.
  • Numerical identifier - AbLIFT is incompatible with numerical identifiers (1, 2...). You can change the identifiers to letters and resubmit the query using the upload files option.
• Essential amino acid residues
  • Non protein residue cannot be specified in this option(e.g. ligands, sugars). If needed, specify the protein residues in contact with these other atoms
• Amino acid positions to diversify and Essential amino acid residues
  • One or more of the positions (a pair of residue number and chain, e.g. 106A) does not exist in the pdb file
  • Typos
  • A missing comma leading to the merge of 2 numbers.
  • Using a numbering system from a paper or from a database that does not match the numbering system in the pdb file.
  • Trying to specify a position for which there is missing X-ray density.
  • Specifying a number of a nonnative amino acid that is
  • Some residues positions are both in Amino acid positions to diversify and Essential amino acid residues
  We suggest to open the pdb file on PyMOL; then go over all the position numbers you entered here and make sure you can select them on the designed chain in PyMOL.
• Self uploaded structure and sequence files
  • You uploaded an NMR solved PDB file or a PSE file (generated by PyMOL). These formats are not supported.
  • There is more than one sequence in the sequence file.
  • One of the residues have more than one conformation- edit the file manually and keep only one conformation.
  • The PDB file has amino acids that do not appear in the sequence file. For instance, the PDB file has a HIS-tag at the end that the FASTA sequence file does not show. Remember, the sequence file should be equal or longer (in the case of missing densities in the structure) than the sequence in the PDB file.
  • The PDB sequence and the one from the sequence file have point mutations compared to one another. This is not allowed.
  • One of the uploaded files has a character/sign that are not allowed. For instance, a whitespace before an ATOM line in the PDB file.
  • One of the uploaded files has a hidden character that is not allowed. This can happen if you prepared the file in a rich text editor and is even more likely if you used a PC (windows) to prepare the files. Try to open your files in other editors or other operating systems (mac) to check if this is the case.