Schuetz, A. et al.

The structure of the Klf4 DNA-binding domain links to self-renewal and macrophage differentiation

Supplementary Information

Suppl. Fig. 1 Crystal structure of the zinc finger domain of murine Klf4 in complex with a 7-bp DNA duplex. The Klf4 monomer in complex with a heptameric double-stranded DNA molecule representing the consensus binding site is presented as a cartoon model. The right panel corresponds to a 90° rotation of the complex around the x-axis. The zinc-finger motifs are highlighted in blue (motif 1), yellow (motif 2) and green (motif 3), respectively. The N- and C-terminus are marked with N or C, respectively. Zinc ions are shown as gray spheres.The N-terminal zinc-finger motif exhibited poor electron density, but was identified by the examination of the anomalous electron density specific for zinc ions ([F+Zn, peak – F-Zn, peak] – [F+Zn, lrem – F-Zn, lrem]) of the low-remote and peak data set calculated by Sftools and Fft [1].

Suppl. Fig. 2 Superimposition of selected zinc-finger domain structures. The crystal structure of the zinc-finger domain of murine Klf4 (green) bound to its decameric target DNA is superimposed on the crystal structures of a murine Zif268 (blue, 1aay), b human Wilms tumor suppressor protein 1 (orange, 2prt),chuman Ying-Yang 1 protein (brown, 1ubd), andd the transcription factor TFIIIA (purple) from Xenopus laevis (1tf6). The orientation of Klf4 is identical in all four panels.

KLF4:DNA-7 / Zinc MAD of KLF4:DNA-7
peak / edge / hrem / lrem
Beamline / BL 14.2 / BL 14.2
Wavelength [Å] / 0.91841 / 1.28204 / 1.28312 / 1.16967 / 2.00000
Detector / marmosaic 225 / marmosaic 225
Detector distance [mm] / 180.0 / 150.0 / 150.0 / 150.0 / 70.0
Resolution [Å] / 29 - 1.70
(1.75 - 1.70) / 29 – 1.95
(2.00 – 1.95) / 29 - 2.05
(2.05 - 2.00) / 28 – 1.90
(1.95 - 1.90) / 29 – 2.05
(2.10 -2.05)
Space group / P212121 / P212121
Unit cell
parameters [Å] / a=41.0, b=45.97 c=73.91, / a=41.0, b=46.0 c=74.2
No. of reflections
Measured / 68 564 (4 774) / 47 522 (2 466) / 45 789 (2 869) / 60 978 (4 468) / 11 6167 (6 257)
Unique / 15 421 (1 096) / 19 329 (1 228) / 18 174 (1 272) / 21 350 (1 592) / 16 881 (1 202)
Rmeas [%] / 8.3 (68.5) / 6.3 (50.7) / 7.4 (56.6) / 6.1 (45.9) / 6.2 (27.6)
Completeness (%) / 97.5 (98.6) / 97.7 (82.7) / 98.7 (93.0) / 99.3 (99.8) / 99.9 (98.6)
Multiplicity / 4.4 (4.4) / 2.5 (2.0) / 2.5 (2.3) / 2.9 (2.8) / 6.9 (5.2)
<I>/<σ(I)> / 12.2 (2.7) / 13.6 (2.2) / 12.3 (2.2) / 15.7 (3.1) / 22.6 (6.1)
Wilson B-factor [Å2] / 28.9 / 30.5 / 29.1 / 29.1 / 30.2
Crystal mosaicity [°] / 0.20 / 0.21

Values in parentheses are for the highest resolution shell.

Suppl. Table 1Data collection statistics for the Klf4:DNA-7 complex

Numbers in parentheses are for the highest resolution shell.For the Klf4-heptamer DNA complex, data collection was carried out with two crystals. One crystal was used for the multiple-wavelength anomalous diffraction (MAD) experiment at the K edge of zinc. For the native data set another freshly prepared crystal was used to minimize the effect of radiation damage.

KLF4:DNA-7
Resolution range [Å] / 25 - 1.70
Rcryst / 0.200
Rfree (test set of 3%) / 0.232
No. of non-H atoms
protein / 730
DNA / 281
zinc / 3
water / 131
glycerol / 6
Average isotropic B-factor [Å2]
main chain / 35.8
side chain / 37.9
DNA / 24.2
zinc / 28.1
water molecules / 26.8
glycerol / 44.0
Rmsd for bond lengths [Å] / 0.014
Rmsd for bond angles [°] / 1.644
Ramachandran regions
Most favored [%] / 97.6
Allowed [%] / 2.4
Outliers [%] / ---

Suppl. Table 2Data refinement statistics for the Klf4:DNA-7 complex

Numbers in parentheses are for the highest resolution shell.

References

1. Collaborative Computational Project (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr D 50:760-763