Increasing the bulk of the 1TEL–target linker and retaining the 10×His tag in a 1TEL–CMG2-vWa construct improves crystal order and diffraction limits

Gajjar, P.L.; Pedroza Romo, M.J.; Litchfield, C.M.; Callahan, M.; Redd, N.; Nawarathnage, S.; Soleimani, S.; Averett, J.; Wilson, E.; Lewis, A.; Stewart, C.; Tseng, Y.-J.; Doukov, T.; Lebedev, A.; Moody, J.D.

doi:10.1107/S2059798323007246

Figure 3
Possible models of crystal disorder. (a) Lattice contacts in the 1TEL-TV-vWa (SUMO) unit cell, with proteins shown in cartoon representation. Chain A (3₂ axis) is coloured cyan (1TEL) and yellow (vWa). Chain B (6₅ axis) is coloured green (1TEL) and brown (vWa). Chain C (3₂ axis) is coloured magenta (1TEL) and dark blue (vWa). The unit cell is shown as a thin black line. (b) As in (a) but showing the chain A, B and C 1TEL-TV-vWa domains superimposed via their 1TEL domains. (c) Representative diffraction pattern of the 1TEL-TV-vWa (SUMO) crystal, showing diffuse scattering between the strong reflections. (d) Projection of reciprocal space viewed perpendicular to c. (e) As in (d) but viewed along c. (f) Packing ambiguity of adjacent helical polymers. (g) Possible model of the crystal architecture. The bright and dark hexagons represent 3₂-axes (chains A and C) and 6₅-axes (B chain) polymers, respectively. On the large scale, the difference between 3₂-axes and 6₅-axes polymers is that they are related by twofold rotation around an axis in the plane of the figure as shown in (f), and on a smaller scale the relative orientation of the 1TEL and vWa protein domains differs by 6.8° (chains A and B), 5.8° (chains A and C) and 11.7° (chains C and B). (h, i) Comparison of intensity statistics of reflection subsets. (h) Reflections h − k = 3n. (i) Reflections h − k = 3n ± 1. Left column: CC_1/2 shows considerable differences in the useful resolution range of the two subsets. Centre column: the results of the L-test for twinning strongly depend on data processing. Right column: the results of H-tests are consistent between the two subsets of reflections and indicate no twinning relative to the twofold axis perpendicular to c. Thin solid black lines in the L-test plots represent theoretical curves for twinned (top) and nontwinned data (bottom). Thin black lines in the H-test plots represent the theoretical curve for the twinning operation or crystallographic symmetry operation (top line, step function) and the lattice symmetry operation unrelated to crystal symmetry or twinning (bottom line).

STRUCTURAL
BIOLOGY

ISSN: 2059-7983

Volume 79| Part 10| October 2023| Pages 925-943

https://doi.org/10.1107/S2059798323007246

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