3.3. Model accuracy
We assessed the CASP14 predictions of TSP4-N by individual domains
because the flexible interdomain linkers may adopt different
conformations than those seen in the crystal structures. Several groups
predicted the correct folds of domains XD1-XD3, with different level of
accuracy. Overall, the AlphaFold2 predictions (DeepMind team, group 427)
were the most accurate with respect to the XD domains. Superposition of
the crystal structure and the AlphaFold2 model 1 using PyMOL43 yielded root mean
squared deviation (RMSD) values for aligned Cα atoms of 0.38 Å for XD1
(90 of 100 superposed amino acid residues), 0.36 Å for XD2 (52 of 60
superposed amino acid residues) and 0.63 Å for XD3 (63 of 65 superposed
amino acid residues). Several other CASP14 participants predicted the
structures of these domains successfully, typically with twice or more
the RMSD values. Fig. 5B shows the superposition of the XD2 domain,
illustrating the remarkable similarity between the experimental and
Alphafold2 structures, and also an excellent structure similarity
produced by another group, even though not as good as the AlphaFold2
structure (ZhangTBM server, group 226, RMSD = 0.7-0.8 Å).
None of the structure predictions of the AD domain resembled the entire
triple β-helix region. Nevertheless, the AlphaFold2 model of the AD
subunit contains a meandered polypeptide chain covering residues 20-50
that resembles the β-helix trace seen in the crystal structure, with
RMSD value for 28 of the 31 aligned Cα atoms of 1.7 Å. Moreover, the
predicted ensuing 3-stranded antiparallel β-sheet that forms the
trimeric antiparallel β-prism II (residues 51-75) is quite accurate,
with RMSD value for all 25 aligned Cα atoms of 0.65 Å. In contrast, the
AlphaFold2 residues 1-19 diverge from the experimental structure and the
five deposited models exhibit a wide range of extended polypeptide chain
orientations. Fig. 5C illustrates this by superposing only the closely
related 3-stranded antiparallel β-sheet regions of the X-ray structure
and the AlphaFold2 model 1. Considering that the AD triple β-helix
polypeptide lacks significant amino acid sequence homology to those of
known protein structures, and that there are no intra subunit
interactions in triple β-helices, it is surprising that the fold
calculated by the AI methods resembles at all the actual fold.
AlphaFold2 models enable solving crystal structure of Af1503
transmembrane receptor (CASP: T1100) that withstood experimental
approaches for years – by MDH, RA and ANL.From email to the CASP Prediction Center: I cannot overstate my
excitement at the fact that Marcus Hartmann solved the structure of
Af1503 by molecular replacement with the models of group g427. Andrei
Lupas
Brief description of the target
Our department has a long-standing interest in coiled coils and their
role in transmembrane signal transduction. Coiled coils are bundles of
α-helices with a specific regular and repetitive packing44; they are found in
innumerable structural contexts in essentially all aspects of cell
biology 45. While their
structural and functional roles are well understood in many contexts,
their role in transmembrane signal transduction is still debated. Many
transmembrane receptors are homo-dimeric proteins in which a
membrane-spanning coiled coil connects extracellular sensor domains to
intracellular effector domains, such that signals have to be passed
along the coiled-coil segment. To study this process, we have been
working on the minimalistic putative receptor Af1503 fromArchaeoglobus fulgidus - fortuitously, we had already entered its
genomic neighbor, Af1502, into the CASP 11 experiment46,47.
Sequence analysis suggested that Af1503 forms a homo-dimer merely
consisting of an extracellular PAS domain connected to an intracellular
HAMP domain via an antiparallel tetrameric coiled coil. While we
conducted several structural studies on the isolated HAMP domain48,49and on chimeric fusion proteins in which we fused the Af1503 HAMP domain
to other coiled coil-based signaling domains50,51,
we were so far unable to determine the structure of the full receptor52.
How AlphaFold2 models helped to solve the structure
Our problems in obtaining the structure of the full receptor did not lie
in the behavior of the protein. The protein was very well behaved,
stable, and readily crystallized in a range of conditions. However,
crystal quality was very erratic, could not be improved systematically,
and diffraction was generally strongly anisotropic and not to high
resolution. This led to the failure of experimental phasing approaches,
despite several different strategies employed. On the other hand,
molecular replacement (MR) was not successful, as we only had the
structure of the HAMP domain as an available search model, and as the
approach was further complicated by the presence of translational
non-crystallographic symmetry. To aid MR, we decided to tackle a
truncated construct covering the extracellular PAS domain, but this
construct failed to crystallize. In contrast, we succeeded with an NMR
analysis of this construct, revealing the fold of the PAS domain, but
the structural models derived from the NMR data were too far from the
actual crystal structure to succeed in MR attempts.
Finally, years later, we easily managed to solve the crystal structure
using the AlphaFold2 models. As the predictions were modeled as
monomers, without constraints for the homo-dimeric state, they were not
fully compatible with the dimeric state along the whole chain, and a
very first, naive MR attempt employing a full model did not succeed.
However, in the second attempt, only employing a single PAS domain with
a short coiled-coil segment as a search model, the structure was
essentially solved using MOLREP with standard parameters53; after the correct
placement and initial refinement of the PAS domains, the electron
density for the rest of the protein was clearly traceable.