Cell Nucleus Structures

All ≈5 500 nucleic acid crystal structures deposited to the Protein Data Bank (PDB; May 2016; resolution ≤ 3.0 Å) were searched for Mg²⁺ binding to purine and pyrimidine imine N1/N3/N7 atoms (or N_b atoms as defined in reference (12 (link))). To determine cut-off distances for the identification of Mg²⁺ bound to imine nitrogens, we relied on a histogram derived from a CSD search (CSD: Cambridge Structural Database, Version 5.37, February 2016) that identified precise Mg²⁺ to water coordination distances as well as ion exclusion zones (Figure 1). Note that the CSD (57 (link)) is a repository for small molecule crystallographic structure that were solved with much better accuracy and, in general, at much higher resolution than those from the PDB. These data parallel those derived from quantum mechanical calculations (58 ), other PDB surveys (23 ,59 (link)) and first principles molecular dynamics simulations of Mg²⁺ in aqueous solution (60 –62 ) that all suggest that: (i) the Mg²⁺…OH₂ coordination distance is slightly below 2.1 Å; (ii) no water oxygens are found within a d(Mg²⁺…Ow) ≈2.2–3.8 Å ‘exclusion zone’; (iii) the second coordination shell starts at a 3.8 Å distance from Mg²⁺ and peaks at 4.2 Å. However, since we mostly deal with medium to low-resolution crystallographic structures (3.0 Å ≥ resolution ≥ 2.0 Å), we used more relaxed criteria to identify solvent species around imine nitrogens. Further, we need to consider that, although the most appropriate Mg²⁺…O coordination distance is in the 2.06–2.08 Å range, the default value in the libraries used by the PHENIX (63 ) and REFMAC (64 ) refinement programs for d(Mg²⁺…Ow) is 2.18 Å. In some instances, this overestimated coordination distance induces serious stereochemical approximations (see below). Bearing in mind that we focus on Mg²⁺ to nitrogen distances, we have also to consider that some authors estimate that the Mg²⁺…N distance is slightly longer (≈2.2 Å) than the Mg²⁺…O distance in agreement with quantum mechanical calculations and PDB/CSD surveys (12 (link),21 25 ). Thus, to a first approximation, our procedures place Mg²⁺ with d(Mg²⁺…N) ≤ 2.4 Å in the pool of possible direct binders, while those with distances in the 2.4–3.8 Å exclusion zone were inspected for misidentification.
Since CSD surveys established that divalent ions directly interacting with a purine or imidazole nitrogen lone pair are located in the C–N=C plane (25 ,65 ), we applied a 1.0 Å cut-off on the distance between the ion and the nucleobase plane. This criterion applies to divalent ions and not to the less strongly bound alkali (Na⁺, K⁺) and the larger alkali earth ions (Ca²⁺, Sr²⁺) that display a greater propensity to lie out-of-plane. The searches included also contacts generated by applying crystallographic symmetry operations.
In the ≤ 3.0 Å resolution range, ions with B-factors ≥ 79 Å² were excluded from our statistics since such high B-factors do not warrant unequivocal binding site characterizations. Further, we excluded ions with B-factors ≤ 1.0 Å² that are definitely not reliable for Mg²⁺ and hint to the presence of a more electron rich atom (see below). Only Mg²⁺ with occupancy of 1.0 were considered unless otherwise specified. Finally, for all Mg²⁺ ions close to imine nitrogens that we identified as suspect, the F_o−F_c and 2F_o−F_c electron density maps deposited to the Uppsala Electron Density Server (EDS) were visualized (66 ). When these maps were not available—typically for large ribosomal structures—we calculated them with phenix.maps by using the structure factors retrieved from the PDB (63 ).
Non-redundant Mg²⁺ binding sites were identified as follows. If two nucleotides from different structures involved in a similar Mg²⁺ binding event shared the same residue numbers, chain codes, trinucleotide sequences, ribose puckers, backbone dihedral angle sequences (we used the g+, g−, t categorization) and syn/anti conformations, they were considered as similar and the one with the best resolution was marked as non-redundant. In case of matching resolutions, the nucleotide with the lowest B-factor was selected. Likewise, if in the same structure two nucleotides involved in a similar Mg²⁺ binding event shared the same residue numbers and trinucleotide sequences (with different chain codes) as well as ribose puckers, backbone dihedral angle sequences and syn/anti conformations, they were considered as similar and the one corresponding to the first biological unit was marked as non-redundant. To further limit redundancy in the largest ribosomal structures, we restricted our analysis to a single biological assembly when more than one was present (see Supplementary Material for selection criteria).
Two non-redundant sets were calculated with a 2.4 and a 3.5 Å d(Mg²⁺…N1/N3/N7) distance cutoff, respectively (Table 1). Note that it is impossible to completely eliminate redundancy from such a complex structural ensemble without eliminating at the same time relevant data. Here, we provide an upper limit for a truly ‘non-redundant’ set. Redundancy issues are further complicated by some systematic assignment errors such as the nucleotide misidentification identified in the first H. marismortui 50S structures that leads to the characterization of two distinct structural ensembles (Supplementary Table S1 and Figure S1).