Write here any specific comment you might have about experimental approaches and methods used in the study.
HTT is a very difficult molecule to work with and its large size precludes use of traditional methods for conformational and structural analysis i.e. NMR and X-ray crystallography. The methods used to assess huntingtin structure and conformational flexibility in this study are similar to those previously described inVijayvargia et al 2016 eLIFE with marginal gains in resolution i.e. EM maps at 30 A compared 9 A give only marginal gains with respect to revealing structural details of this molecule.
It is not clear how GFP linked to the mobile exon1 region of the huntingtin protein would be located in a single position in relation to the globular structure of HTT, nor how this could be determined to be GFP in a low resolution (15 A) volume. Therefore, reliance on this method for volume orientation prior to modelling is risky in our opinion and the authors should justify this methodology in more detail. Without access to the data in the EMdb and PDB it is hard to comment further.
The SAXS analysis is not a novel approach and was recentlypublished by our group. Although the in-line size-exclusion chromatography would help to separate different oligomer species, the monomer and dimer forms of HTT form overlapping peaks on a Superose6 column which would still render the sample a mixture of oligomeric states. This could account for the high Dmax values compared to Rg. NB: Dmax values are similar to our own experimentally determined values for HTT and HTT-HAP40 where samples were analysed without cross-linking. The inclusion of HTT-HAP40 complex samples in the work by Jung et al would be a good control.
The phosphosite specific antibodies are a fantastic resource and we hope they will be shared widely with the HD community, in addition to details of their validation. A missed opportunity is mapping of the PTMs onto the models generated to see if this would give any insight into potential mechanisms of conformational changes in the protein molecule. For example, S2116 resides close to the interface of C-HEAT and BRIDGE domains, so phosphorylation could lead to a change in electrostatic interaction of the domains and conformational change. Again, without access to the data in the EMdb and PDB it is hard to comment further.
Phosphorylation mimic mutations for S2116 could also be investigated to determine the effects of phosphorylation.
For the other phosphorylation sites (S434, S1181, S1201, S2114) that were mutated to A, the authors should ensure that there are no structural or stability changes in HTT as a result of the amino acid changes. These studies would ensure that they are probing only changes related to phosphorylation.