Despite access to chemical shift assignments of the isolated transmembrane (solid-state NMR ) and cytosolic (solution NMR ) domains, the assignment of full length GlpG (276 residues) is not trivial. Several peaks are overlapping in 3D spectra and there are many regions where the chemical shifts differ significantly between the full length protein and the isolated domains. The possibility to acquire triple sensitivity-enhanced 4D experiments, enabled by the recent development of TROP and SPEPS pulses, simplified the assignment procedure considerably. Here we used TROP for backbone 13 C-15 N, C- C and N-1 H transfers (see SI Fig. for example strip plots of the backbone spectra). It should be mentioned that the TROP N-1 H transfer is less efficient compared to CP for GlpG (see SI Fig. for a comparison of 1D spectra), but due to the combined sensitivity-enhancement from each indirect dimension it is still possible to record highly sensitive 4D spectra in a reasonable time. Triple sensitivity-enhanced 4D hCACONH and hCOCANH were recorded in ca 48 hours each, hCXCANH in ca 100 hours and hCXCAcoNH in ca 170 hours. All 4D spectra were recorded at 55 kHz MAS on a 600 MHz spectrometer, using 5% NUS. The experiments for chemical shift assignments were performed on a back-exchanged perdeuterated sample. This makes experiments for side-chain assignments challenging since the pool of initial magnetization in the 1 H-13 C CP step is limited to back-exchanged sites, mostly amide protons. With this in mind the main goal was to obtain C assignments. Unambiguous assignments of GlpG would not be possible without access to at least C chemical shifts, additional side-chain information is of course highly beneficial. The initial 1 H- C CP was optimized for highest intensity the region around 10-40 ppm, where most C and other expected side-chains appear. Longer mixing time could potentially improve the side-chain assignments of carbon atoms further down the side-chain, but will lead to an overall loss in sensitivity. Figure shows an example of side-chain assignments for residues W38-Q44 based on the sensitivityenhanced 4D TOCSY experiments, Fig. shows how the spectra are connected in the strip plots. All the C chemical shifts of residues W38-Q44 could be assigned and, additionally, the C chemical shifts of E42 and Q44 were also assigned. But for the leucine residue (L39), with a hydrophobic branched side-chain, only the C was visible in the spectra. Figure shows an AlphaFold2 39,40 model of GlpG with the residues coloured depending on the assignments (see SI tables 4 to 6 for assigned chemical shifts). In total, utilizing sensitivity-enhanced 3D and 4D spectra for backbone and sidechain assignments, the C chemical shifts of 133 residues were successfully assigned (137 residues with any atom assigned). Among these, fifteen residues are glycines and prolines, leaving 118 residues for potential side-chain assignments. Of these, the C chemical shifts were assigned in 91 residues (green in Fig. ), while in 7 residues where the C could not be assigned, another sidechain was assigned (orange in Fig. ). Consequently, only 20 residues remain without any side-chain assignments (dark gray in Fig. ). Figure provides a comprehensive analysis of side-chain assignments for each residue type in GlpG, excluding glycine and proline. Side-chains that were consistently assigned are denoted in green, those occasionally assigned in orange, and those not assigned in red. This pattern aligns relatively well with expected outcomes for side-chain assignments in a perdeuterated sample, where C chemical shifts are typically available to be assigned, and carbons further along the side-chain are less frequently assignable. Notable exceptions where C was not consistently assigned include glutamates (3/8), threonines (2/6), and valines (1/10). For glutamates, the missing assignments appear to be due to the residues' positioning within the protein. For example, two of the glutamates lacking C assignments (E118 and E166) are residues where the amide protons could not be assigned, but the C (and CO) chemical shifts were determined using the amide protons of the preceding residues in the 4D hCACONH spectrum. The reason for the lack of C assignments of several threonines remains unclear, especially since the C were assigned in 5 out of 6 threonines. The sole threonine with a missing C assignment is T97, which is similarly situated as E118 and E166. A plausible explanation for the missing threonine C assignments could be unfavorable dynamics, resulting in inadequate coherence lifetimes for the many magnetization transfer steps in the 4D TOCSY experiments, while the fast-moving methyl groups (C) have longer coherence lifetimes, thus detectable in TOCSY experiments. Another possible reason is the close similarity between C and C chemical shifts in some threonines, hindering their distinction. Valines, expected to behave similarly to other residues with branched hydrophobic side-chains such as leucines and isoleucines, showed an unexpected pattern, with only 1 out of 10 valine C assigned. There is no apparent explanation for this discrepancy. Future structural and dynamic studies of GlpG may elucidate why the valine side-chains are challenging to assign. Intriguingly, among the sidechains within the TOCSY mixing range, the only ones never appearing in the spectra are the C and C of isoleucines and leucines, and the C of lysines (see Fig. ). This implies that nearly complete sidechain assignments are achievable using sensitivity-enhanced TOCSY experiments in solid-state NMR of perdeuterated proteins at 55 kHz. Given the complexity of GlpG as a membrane protein, numerous factors could contribute to the difficulty in assigning certain residues, such as unfavourable dynamics, lipid interactions, incomplete back-exchange of exchangeable protons, and overlapping peaks. Residues with C but no side chain assignments are coloured in dark gray, residues with C assignments are coloured in green and residues with no C assignments but with other carbon side-chains assigned are coloured in orange. (C) Scheme summarizing the side chain assignments for each residue type found in GlpG (except glycines and prolines). Green circles indicate side-chains that were generally assigned, orange circles represent side-chains that were sometimes assigned and red circles represent side-chains that could not be assigned.