It’s been a while since our last “getting
misled by crystal structures” post. That one described unrecognized conformational
heterogeneity of ligands. A more basic issue is ligand occupancy. It’s normally
assumed that every protein in the crystal lattice has a bound ligand. A new
paper in Structure by Timothy Stachowski and Marcus Fischer at St. Jude
Children’s Research Hospital reveals that this is not always the case.
The researchers selected roughly
10,000 protein-ligand structures from the Protein Data Bank (PDB) and did a simple
re-refinement of the ligand occupancies and B-factors (measures of
conformational heterogeneity and modeling errors). 10% of the structures
already presumed ligand occupancies at or below 0.9, but re-refinement saw the
fraction jump to 35%, more than three-fold higher. There were no overall
differences between covalent and non-covalent ligands, but 37% of fragments (defined
as having MW <300 Da) saw decreased occupancy in re-refinement compared to
just 22% of larger ligands. A few structures even saw occupancy drop completely.
The authors wrote that “manual reviewing these revealed that ligands were built
into spurious electron-density.”
Crystallographers use several
metrics to assess the quality of their structures. In addition to B-factors, unique
to each residue or atom, real-space correlation coefficients (RSCC) and real-space
R values are commonly used, and the researchers compared these parameters
before and after re-refinement. In many cases the metrics improved with
decreased occupancy, but not for all metrics, and not always meaningfully. This
means that standard assessments do not always flag partially occupied ligands.
OK, so a ligand binds at only 80%
occupancy rather than the 100% assumed: does this matter? The researchers
describe three categories where the answer may be yes. In the first, correcting
ligand occupancy reveals alternative conformations of protein side chains,
which could be informative for understanding the mechanism of binding. In the
second, correcting ligand occupancy can reveal water molecules that interact
with the ligand and/or protein. As we noted more than a decade ago, water is an
essential player in protein-ligand interactions, and a single water molecule can
make the difference between a binder and a non-binder. Finally, correcting
ligand occupancy can reveal alternative binding modes for the ligand, and even ligands
binding to other sites.
Importantly, this analysis was
done on individually refined structures in the PDB, and it seems likely that the
issues would be even more severe for structures batch-refined in high-throughput
crystallographic fragment screens. As we wrote last year, the community needs
to figure out how to deal with the increasing number of these structures.
The fact that roughly a third of
PDB structures have less than 100% ligand occupancy has implications for training
AI models. It also has implications for individual targets. As the researchers
note, “non-crystallographers who rely on the PDB often assume that deposition
itself is an implicit stamp of approval. Structural biologists, however, know
that this is not always the case.” Before using a structure, it would be wise
to re-refine it yourself.
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