We crystallized RMA in complex with the low-complexity domain (LC4) of sorting nexin 9 at room temperature, but the X-ray diffraction was poor as indicated by the large mosaic spread and modest resolution. We were able to improve the quality of our RMA–LC4 crystals by lowering the crystallization temperature to 277 K and reducing the concentration of both protein and precipitant. Although the RMA–LC4 crystals grew larger at room temperature, their diffraction was limited to about 5 Å Bragg spacing. We also attempted to improve these crystals using the Proteros Free Mounting system with various humidity gradients, with no improvement in the observed X-ray diffraction quality (Kiefersauer et al.
Our data set from RMA–LC4 crystals grown at 277 K stands out, with a low mosaic spread (0.1°) compared with all of the liganded RMA crystals that we have collected over the past decades, which had mosaic spreads of 0.5° or greater. Reduced mosaicity in crystals is manifested by an enhanced signal-to-noise ratio of the reflection intensities and is therefore particularly valuable for measuring the weak signal in high-resolution diffraction data (Bellamy et al.
). Even though higher diffraction X-ray data could be obtained for 2ot0
, the quality of the electron-density map of RMA–LC4 was comparable to that of 2ot0
and in certain areas the electron-density map was much clearer.
Both crystal forms A
as well as PDB entry 2ot0
make crystallographic contacts with at least six independent neighboring tetramers and have a total buried interface area ranging from 1173 Å2
(for form A
) to 4290 Å2
). Intuitively, the total surface area involved in crystallographic packing would inversely correlate with mosaicity owing to the stability caused by the interaction surfaces. However, comparison of RMA–LC4 with other RMA crystals shows that the aforementioned notion does not correlate with the reduced mosaicity of RMA–LC4 crystals. Rather, the quality of the contact appears to be more important than the quantity of contacts. Indeed, RMA–LC4 crystals grown at 277 K use the largest number of high-entropy side chains in establishing lattice-packing contacts: of the 18 directional interactions, seven involve Gln residues, six involve Glu residues and another six use Lys residues (Juers & Matthews, 2001
). In comparison, PDB entry 2ot0
, which has the next highest number of interactions (17), has only two interactions involving Gln residues and four involving Glu residues, while only four involve Lys residues. The remaining crystal forms use substantially lower numbers of residues with high-entropy side chains in establishing packing interactions. In all crystal forms, the carboxy-terminal Tyr interacts identically with high-entropy side chains (Lys12, Gln202 and Arg258). In PDB entry 1ado
and RMA–LC4 this same interaction bridges to an adjacent tetramer in the lattice.
The difference in the pH of the crystallization conditions of 7.1 at room temperature versus
7.6 at 277 K is a consequence of the temperature-dependence of the pH of the Tris buffer used in the crystallization solution (Bates & Hetzer, 1961
). Although nucleation and crystal-growth processes are pH-dependent, the temperature-induced change of 0.5 pH units in the experimental conditions does not change the energetics of the crystal-packing interactions, since all of the interacting side chains have solution ionization potentials that differ by at least three pH units from that of the experimental conditions. This large difference in pK
with respect to the pH of the crystallization solutions precludes redistribution of the populations of ionizable species of the interacting side chain owing to the temperature-induced pH change, and thus does not perturb the strength of the side-chain interactions used in lattice packing. For instance, in case of a lysine residue (pK
= 10.5) an increase of 0.5 pH units would merely shift the ratio of neutral to ionized species from 0.04% to 0.16% and would negligibly decrease (by <0.04 kJ mol−1
) the energetic gain of 12.6 kJ mol−1
for a lysine participating in an electrostatic interaction (Fersht et al.
). Similarly, hydrogen-bonding interactions, although twofold to fivefold weaker than electrostatic interactions (Fersht et al.
), would also not be perturbed by the temperature-induced pH change in the experimental conditions. As a result, lattice interactions for all crystal forms are essentially insensitive to pH under the experimental conditions described. Although the temperature-induced pH shift can influence protein solubility and surface charge, because lattice formation is insensitive to pH over the experimental conditions, the temperature-induced pH changes of these parameters would serve to modulate the kinetics of protein crystal growth, namely the rates of nucleation and crystal growth, and not the lattice packing.
The thermodynamic cost of immobilizing high-entropy side chains tends to inhibit their participation in crystal-packing contacts and impedes successful crystal structure determination (Price et al.
). Interactions implicating high-entropy side chains can occur upon cryocooling a protein crystal from room temperature, as the side chains of these residues become thermodynamically easier to order (Juers & Matthews, 2001
). This side-chain ordering accompanying cryocooling is associated with an increase in crystal mosaicity, which is attributed to a change in unit-cell packing that induces strain. Although such an interpretation cannot be excluded in the case of RMA–LC4, the very low mosaicity value observed for the cryocooled crystals would argue against such a possibility and suggest that these side chains were ordered prior to cryocooling.
Surface side-chain ordering in the RMA–LC4 crystals grown at 277 K increases the interaction energy among the aldolase tetramers, thus strengthening lattice packing, which in turn minimizes the lattice strain incurred upon cryocooling and thereby reduces or inhibits changes in mosaicity. It is noteworthy that the solvent content of RMA–LC4 is 53.8%, compared with 50.6, 46.8 and 45.4% for PBD entries 1ado
, respectively, and would allow us to argue that in these RMA–LC4 crystals, the entropic loss owing to side-chain ordering could be offset by an entropy increase resulting from the larger bulk-solvent content of the unit cell. Intriguingly, the crystallization of RMA–LC4 at room temperature requires a nearly twofold increase in precipitant compared with RMA–LC4 crystallized at 277 K (25% PEG MME 550 versus
14% PEG MME 550, respectively). At room temperature, hydration data for PEG–water mixtures indicate that each PEG 600 molecule is hydrated by 31.2 water molecules, compared with 34.2 molecules at 277 K (Branca et al.
), indicating that a significantly smaller total number of water molecules are involved in hydrating PEG in crystallization conditions at 277 K compared with room temperature. Although PEG MME differs from PEG by the presence of a methyl end group instead of a hydroxyl group, it would not significantly affect these conclusions as it merely reduces the number of water molecules that hydrate PEG MME by two. The significantly increased hydration by water molecules by nearly a factor of two at room temperature reduces the bulk-solvent entropy, which would promote side-chain disorder at room temperature as a means of offsetting entropy reduction. This entropy compensation mechanism that increases the likelihood of disordered lattice-packing interactions at room temperature would consequently be incompatible with low mosaicity measurements using cryocooled crystals. Strengthened packing interactions as described would serve to enhance lattice long-range order and mitigate against degradation of resolution limits upon cryocooling.