Exercise:AntibodyStructurePrediction ans
Antibody Structure Modelling — Answers
SI135 CLL antibody modelling exercise.
Part 1 — Antibody modelling with LYRA
Q1. Framework template selection
Answer:
LYRA selected the following framework templates:
| Chain | Selected template | Sequence identity | BLOSUM score |
|---|---|---|---|
| Heavy (VH) | 3H42 | 80.5% | 514 |
| Lambda light (VL) | 1W72 | 85.7% | 518 |
Interestingly, 2UZI has a slightly higher sequence identity to the heavy chain (81.7%), but LYRA selects 3H42, which has a higher BLOSUM score and compatible canonical structures.
This illustrates that template selection depends on more than sequence identity alone.
Q2. Light-chain type
Answer:
The SI135 antibody has a lambda (λ) light chain.
Lambda is less common than kappa (κ) in human antibodies. Approximately 60% of human antibodies have kappa light chains and 40% have lambda light chains.
Q3. Sequence differences between target and templates
Answer:
The target antibody is relatively similar to the selected framework templates, but several substitutions are observed in both framework and CDR regions.
Some examples from the LYRA alignments include:
| Region | Template → Target | Interpretation |
|---|---|---|
| Heavy-chain framework | K → R | Conservative substitution between positively charged residues |
| CDR-H1 | S → T | Conservative polar substitution |
| CDR-H2 | S → N | Changes side-chain size and hydrogen-bonding possibilities |
| CDR-H3 | R → N | Removes a positive charge |
| CDR-H3 | Q → T | Changes side-chain size and hydrogen-bonding properties |
| CDR-L2 | D → Y | Removes a negative charge and introduces a bulky aromatic residue |
| CDR-L3 | L → S | Changes from hydrophobic to polar |
| CDR-L3 | S → D | Introduces a negative charge |
| CDR-L3 | A → H | Introduces a larger, potentially ionizable side chain |
| CDR-L3 | D → P | Removes a negative charge and introduces a residue that restricts backbone conformation |
The CDR-L2 substitution D → Y and the CDR-L3 substitution D → P are particularly interesting because they involve substantial changes in physicochemical properties.
In the light-chain framework alignment, a D → K substitution near the C-terminus also reverses the side-chain charge.
Observation: The impact of a substitution depends not only on its BLOSUM score but also on its structural location and whether the residue is buried or exposed.
Q4. CDR loop lengths and expected accuracy
Answer:
All six selected CDR templates have the same loop length as the corresponding SI135 target loops.
| Loop | Template | Template sequence | Target sequence | Length |
|---|---|---|---|---|
| H1 | 3H42 | GFTFSSY | GFTFSTY | 7 |
| H2 | 3H42 | SSSS | SSSN | 4 |
| H3 | 2NR6 | VRQMD | VNTMD | 5 |
| L1 | 2G75 | GNNIGSKS | GNNIGSKS | 8 |
| L2 | 1W72 | DDS | YDN | 3 |
| L3 | 3GHB | WDSGLSADW | WDSGSDHPW | 9 |
These are the loop-core lengths reported by LYRA. Other CDR definitions may include additional residues.
Matching loop lengths generally make template-based modelling easier because no insertions or deletions are required in the loop core.
However, there is one interesting exception in the canonical structure classification.
For CDR-L1:
- Template canonical structure: 7
- Predicted canonical structure: 8
Thus, although L1 has the same sequence length and an identical loop-core sequence in the selected template, the predicted canonical structure class is different.
This introduces additional uncertainty in the loop conformation.
Note: Matching loop length does not guarantee that the target and template have the same three-dimensional conformation.
Q5. Number of templates and most dissimilar region
Answer:
LYRA uses five distinct PDB templates to construct the SI135 antibody model:
| PDB template | Regions modelled |
|---|---|
| 3H42 | Heavy-chain framework, H1, H2 and heavy/light chain packing |
| 2NR6 | H3 |
| 1W72 | Light-chain framework and L2 |
| 2G75 | L1 |
| 3GHB | L3 |
A total of eight framework/loop regions are modelled, but several use the same structural template.
The sequence identity within each selected loop core is:
| Loop | Identical residues | Identity |
|---|---|---|
| H1 | 6/7 | 85.7% |
| H2 | 3/4 | 75.0% |
| H3 | 3/5 | 60.0% |
| L1 | 8/8 | 100% |
| L2 | 1/3 | 33.3% |
| L3 | 5/9 | 55.6% |
CDR-L2 has the lowest direct sequence identity between its selected loop template and the target.
CDR-L3 also contains several substitutions, including the potentially important D → P change.
The loop identities above are calculated directly from the sequences reported in the PDB annotations, not from the overall framework-template scores.
Part 2 — Exploring the antibody structure in PyMOL
Q6. Shape of the antigen-binding site
Expected answer:
The antigen-binding site is formed by the six CDR loops.
Its overall shape should be classified by displaying the predicted antibody as a molecular surface and examining the arrangement of these loops.
SI135 classification: To be confirmed from the PyMOL surface representation.
The LYRA sequence alignments do not establish whether the binding site is flat, pocket-like, groove-like or mountain-like.
The classification is qualitative, and students should justify their answer based on the model.
Q7. Longest and most protruding CDR
Answer:
According to LYRA, the six loop-core lengths are:
| Loop | Length |
|---|---|
| H1 | 7 |
| H2 | 4 |
| H3 | 5 |
| L1 | 8 |
| L2 | 3 |
| L3 | 9 |
The longest loop is CDR-L3.
This is notable because CDR-H3 is often the longest and most structurally variable CDR in human antibodies.
The loop that protrudes furthest should be determined from the three-dimensional structure, since length alone does not determine protrusion.
Most protruding loop: To be confirmed by visual inspection in PyMOL.
Part 3 — Somatic mutations
Q8. Somatic mutations near the CDRs
Expected answer:
Somatic mutations must be identified by comparing the SI135 sequence with the closest germline immunoglobulin genes using IMGT/DomainGapAlign.
The target-template substitutions identified by LYRA are not necessarily somatic mutations, because the structural templates are unrelated antibody sequences rather than the germline ancestors of SI135.
Mutations located inside the CDRs or affecting the residues that support them are particularly interesting.
The most likely candidates to affect binding are substitutions that:
- Change the charge of an exposed residue.
- Introduce or remove aromatic residues.
- Affect hydrogen bonding.
- Change loop geometry or flexibility.
SI135-specific mutation list: Requires the IMGT germline alignment.
Part 4 — Electrostatic properties
Q9. Charged CDR residues
Answer:
The LYRA loop definitions allow us to identify the following charged or potentially charged residues.
| Loop | Sequence | Relevant residues |
|---|---|---|
| H1 | GFTFSTY | None of K, R, D or E |
| H2 | SSSN | None of K, R, D or E |
| H3 | VNTMD | D (negative) |
| L1 | GNNIGSKS | K (positive) |
| L2 | YDN | D (negative) |
| L3 | WDSGSDHPW | Two D residues (negative); H may be protonated |
The principal charged residues within these CDR definitions are therefore:
- One aspartate in H3.
- One lysine in L1.
- One aspartate in L2.
- Two aspartates in L3.
The light-chain loops contain several negatively charged residues, which may contribute to negatively charged patches on the antigen-binding surface.
However, the actual electrostatic distribution depends on the spatial arrangement of these residues.
Residue numbering in the provided LYRA PDB:
| Loop | Residue | PDB position |
|---|---|---|
| H3 | Aspartate | H101 |
| L1 | Lysine | L31 |
| L2 | Aspartate | L51 |
| L3 | Aspartate | L92 |
| L3 | Aspartate | L95A |
| L3 | Histidine | L95B |
Students can highlight the main charged CDR residues using the following PyMOL commands:
select charged_cdrs, (chain H and resi 101) or (chain L and resi 31+51+92+95A) show sticks, charged_cdrs color yellow, charged_cdrs
These residue numbers refer specifically to the supplied LYRA PDB file.
Q10. Introducing a positive charge (optional)
Expected answer:
Replacing an exposed negatively charged residue with lysine or arginine should make its local electrostatic environment more positive.
For example, a D → K mutation at a solvent-exposed CDR position would reverse the formal side-chain charge.
After recalculating the electrostatic potential, the surface coloring near the mutation should change.
Actual changes: Require performing the mutation and recalculating the potential in PyMOL.
A change in electrostatic potential does not necessarily improve antigen binding.
Part 5 — Comparing antibody modelling approaches
Q11. LYRA versus ABodyBuilder2
Expected answer:
LYRA and ABodyBuilder2 use different approaches to structure prediction.
| LYRA | ABodyBuilder2 |
|---|---|
| Template-based homology modelling | Deep-learning-based prediction |
| Uses experimentally determined structural templates | Uses a trained neural network |
| Models framework and loops using selected templates | Predicts the antibody structure from sequence |
| Depends on availability and suitability of structural templates | Provides prediction-error estimates |
Both methods should produce a similar overall immunoglobulin fold.
Differences are expected to be more pronounced in variable loops, particularly where the sequence similarity to the LYRA template is low or the canonical structures differ.
For SI135, potentially interesting regions include:
- CDR-L1, because of the canonical structure mismatch.
- CDR-L2, because of its low sequence identity with the selected loop template.
- CDR-L3, because it contains several non-conservative substitutions.
- CDR-H3, because antibody H3 loops can have diverse conformations.
Actual differences between the two SI135 predictions: Require the ABodyBuilder2 model for structural superposition.
In PyMOL, the models can be superimposed using:
align ABodyBuilder2, LYRA
The object names should be adapted to the names used in the PyMOL session.
Take-home messages
- The SI135 antibody has a lambda light chain.
- The selected framework templates show relatively high sequence identity: 80.5% for VH and 85.7% for VL.
- Five distinct structural templates were used to build the antibody model.
- All six loop templates have the same lengths as their corresponding target loops.
- CDR-L1 has a mismatch in canonical structure classification despite matching loop length.
- CDR-L2 has the lowest sequence identity to its selected loop template.
- CDR-L3 is the longest loop in the LYRA model.
- Several negatively charged residues are present in the CDRs, especially in the light chain.
- Template similarity does not guarantee structural accuracy, particularly in flexible loops.