3.1. Crystallization of SefDdscA
In the chaperone-usher family of pili, fibre polymerization occurs through a process termed donor strand exchange (DSE) [31
], concluding with the N-terminal extension (NTE) of one pilin domain complementing the incomplete immunoglobulin-like fold of an adjacent subunit. To avoid polymerisation we created a donor-strand complemented (DSC) [33
] construct of SefD (SefDdscA
), in which the NTE from the SefA subunit was cloned onto the C-terminus of SefD, separated by a tetrapeptide (DNKQ) linker.
An initial crystallization screen of SefDdscA against more than 1000 standard conditions identified only microcrystals or precipitates which could not be optimized. After several months a solitary crystal developed in one condition (4 M ammonium acetate, 0.1 M sodium acetate pH 4.6), however, despite exhaustive efforts, it was not possible to reproduce this crystal with subsequent optimization in H2O. However, when the protein sample was buffer exchanged into 100% D2O (creating a 50% D2O solution in the final crystallization drop) this led to immediate reproducibility of the initial hit with crystal clusters appearing after a few days (). Complete exchange of both precipitant mixture and protein solution into 100% D2O yielded significant improvements in crystal morphology, with large single crystals developing in the majority of the optimisation drops ().
Representative images of SefDdscA crystals grown in both 50% and 100% D2O.
Diffraction data were collected from the single crystal grown in 0% D2
O and several crystals from 100% D2
O. The structures were solved by molecular replacement and both refined to 3.1 Å ( and Supplementary Fig. 1
). Surprisingly, whilst SefDdscA
in 0% D2
O crystallized in space group P
=218.0), in 100% D2
crystallized in P
=211.8) with additional pseudotranslational non-crystallographic symmetry (Supplementary Fig. 2
3.2. Overall structure of SefDdscA in 0% D2O
There is one molecule of SefDdscA
(154 residues) in the asymmetric unit of the 0% D2
O crystal form, corresponding to 54% solvent content (Vm
=2.67) and all residues could be built except for the flexible N-terminal His6
tag, the flexible C-terminus of SefD (Glu118-Leu119), and the synthetic loop between SefD and the SefA NTE [34
] (G-strand; Asp120-Phe125). Furthermore, the following solvent exposed side chains were not visible in the electron density map: Lys4, lys8, Glu11, Arg17, Lys22, Asn31, Arg32, Lys35, Lys36, Lys45, Lys47, Asn48, Glu72, Asp73, Asp88, Phe89, Glu92, Asn100, Asp102, Glu116, Ile117, Glu118, Lys129, Lys141 and Asn143.
is composed of 9 β-strands (Supplementary Fig. 3
) and its structure is similar to that of other minor pilin domains of the Aaf/Dra family [23
purifies as a stable dimer and the structure shows that the two monomers pack against a hydrophobic face which is stabilized through domain-swapping () via the A2 and G β-strands (which include non-native vector encoded residues: Lys141, Leu142, Asn143).
Figure 2 Global arrangement of SefDdscA molecules within crystals grown in 0% and 100% D2O. (A) Domain-swapped dimer of SefDdscA in 0% D2O. The single molecule of the asymmetric unit is coloured yellow whilst its symmetry mate is coloured olive. The dimer face (more ...)
3.3. Overall structure of SefDdscA in 100% D2O
In the 100% D2O form, there are six molecules of SefDdscA in the asymmetric unit, which corresponds to 49% solvent content (Vm=2.41) and all residues could be built except for the flexible N-terminal His6 tags, the flexible C-terminus of SefD (chains A,D: Glu118; A-D: Leu119) and the synthetic loop (chains A-D: Asp120-Lys122; A-C: Gln123-Phe125; B: Val126). Furthermore, chains E and F show considerable disorder in the loop regions which could not be modelled (chain E: Asn31-Lys36, Ile117-Val126; chain F: Ser20-Lys22, Asn78-Ile81, Phe89-Asn91, Gly99-Asn100, Glu118-Lys129) and it was not possible to build a substantial number of solvent exposed side chains, namely: Ser2 (chain B), Leu3 (chain C), Lys4 (chains A-E), Met6 (chains A,D,F), Lys8 (Chains B-F), Glu11 (chains A-F), Asp16 (chains A,B,D,E), Arg17 (chains A-F), Asn21 (chain E), Lys22 (chains A-E), His28 (chain F), Leu29 (chain F), Phe30 (chain F), Arg32 (chains B,F), Glu33 (chains A,D), Lys35 (chains A-D,F), Lys36 (chains A-D,F), Glu44 (chain E), Lys45 (chains A-F), Lys47 (chains A-C,E,F), Asn48 (chains A-F), Lys64 (chain E,F), Arg66 (chain E), Arg68 (chain F), Glu72 (chains A-F), Asp73 (chains A-D,F), Gln75 (chain E,F), Asn78 (chain E), Asp88 (chains A,B,D-F), Phe89 (chains A,B,C,D,E), Asn91 (chains A,B,C,D,E), Glu92 (chains A,C,E,F), Phe97 (chain F), Asn100 (chains A,B,C,D), Glu116 (chains A-F), Ile117 (chains A,C,D,F), Glu118 (chains B,C), Gln123 (chain D), Phe125 (chain D), Val126 (chains C,D), Asn128 (chains C,D), Lys129 (chains A,E), Lys141 (chains A-F) and Asn143 (chains A-F).
The structure of SefDdscA
in 100% D2
O is in essence identical to the 0% D2
O form (rmsd between 0.37-0.56 Å) (Supplementary Fig. 4
). The only deviations are seen in the A2 and G strands which are involved in domain-swapping within the dimer.
3.4. Comparisons between SefDdscA in 0% and 100% D2O
Crystals of SefDdscA are formed from rod-like structures packing side-by-side that are themselves composed of domain-swapped dimers, which rotate about a central crystallographic c-axis with either 65 (0% D2O) or 21 (100% D2O) screw-symmetry (). The spacegroup C2221 (a=52.5, b=90.7, c=218.0) is a subgroup of P6522 and the two crystal forms are clearly similar, where the centred orthorombic has become a primitive orthorhombic system related by non-crystallographic translational symmetry in the 100% D2O form.
Whilst the overall structures of SefDdscA
from both crystal forms are highly similar, substantial variations are evident in the orientation of the monomers within the chain A-E and B-D domain-swapped dimers where one subunit is twisted and translated in relation to the other (; Supplementary Fig. 5
). This inter-dimer interface is formed in part by a hydrophobic surface and also significant backbone hydrogen-bonding within the A2 and G β-strands. In addition to this domain-swapped dimer, two other lattice dimers are also present. The ‘back-to-back’ dimer is identical in both crystal forms (Supplementary Fig. 6
), whilst the ‘face-to-face’ dimer exhibits some deviations (Supplementary Fig. 7
). This latter dimer is responsible for the non-crystallographic pseudo-translational symmetry, where chains A and D, and chains B and C are related by the vector (0.5,0.5,0.031), and superimposes well with the 0% D2
O structure (Supplementary Fig. 2
; Supplementary Fig. 7
). However, chains E and F do not form a dimer and as such they show substantial disorder in these ‘face-to-face’ loop regions.
Figure 3 (A) Effects of deuterium on the inter-domain domain-swapped dimer interface. Top panel: A SefDdscA dimer from a 100% D2O crystal (blue) is shown superimposed onto a single chain of a 0% D2O dimer (yellow) and clearly demonstrates a twisting and displacement (more ...)
3.5. Interpreting the increased propensity of SefDdscA crystallization in D2O
Whilst studies have been published describing only subtle effects of D2
O on crystal formation [36
], these have only been conducted on highly compact, single domain proteins that readily crystalize in many conditions and generally contain low solvent levels. It is therefore not possible to extrapolate these conclusions to biological macromolecules that do not readily crystallise or of those crystallization conditions which cannot be reproduced. In the case of SefDdscA
, the improvements in crystal reproducibility are attributed to the altered interactions in the 100% D2
O crystal form. These changes are largely mediated by the interface within the domain-swapped dimer which does not contribute to the structured core. It is clear that this region is more malleable and that deuterium has altered the lengths and geometry of the hydrogen bond networks. The overall effects of these changes are propagated down the c
-axis of the 100% D2
O crystal form. In these crystals, the chain E-F dimer does not form, increasing the entropy of SefDdscA
, whilst the chain C-F domain-swapped dimer is identical to the 0% D2
O crystal form which allows it to stay in register and maintain lattice formation (; Supplementary Fig. 5
; Supplementary Fig. 7
3. 4 Conclusions
Panaceas for crystallization problems do not exist and whilst replacing H2O with D2O in the crystallization medium is unlikely to be a miracle additive, our study reveals that this approach can offer genuine promise in lost causes. The ease at which any benefits of D2O can be tested, by merely buffer exchanging the protein sample into 100% D2O prior to crystallization, should enable it to become routine for sparse-matrix screening in macromolecular crystallography. Furthermore, the D/H isotope effect may have more pronounced effects with targets such as membrane proteins, large RNAs or macromolecules with substantially dynamic properties.