Fig 1.
Production of the VACV E9-A20-D4 complex.
(A) Chromatogram of the final size exclusion step of the purification. (B) Fractions of panel (A) analyzed by SDS PAGE. The loaded sample is shown between marker proteins and fraction 10. (C) Result of a SEC-MALS run of the complex concentrated from fractions 13 to 17 in panel (B). The red line shows the molecular weight of the complex calculated from the measured scattering signal and the protein concentration determined from the refractive index increment Δn (continuous black line) using dn/dC.
Fig 2.
VACV E9-A20-D4 holoenzyme structure.
(A) The refined 3.8 Å structure of the holoenzyme is displayed in cartoon representation in two views together with the electron density map before sharpening. The disordered thumb domain of E9 (res. 830–1006) is not shown. (B) The D4 binding site of E9 (gray) is shown in a transparent surface representation with the sidechains of the underlying contacting residues coloured in green. Contacting residues of D4 (yellow) are shown as a stick model. (C) Superposition of A20 in the context of the VACV polymerase holoenzyme heterotrimer (violet) with the Alphafold 2 prediction (turquoise for the middle domain, otherwise black). Green circles indicate hinges.
Fig 3.
VACV E9-A20-D4 holoenzyme models and their agreement with SAXS data.
Coloring scheme of the models as in Fig 2A. (A) Refined 3.8 Å cryo-EM structure of the holoenzyme. Green circles indicate the domain boundaries of A20, which are likely flexible hinges for the holoenzyme. (B) Agreement of the experimental SAXS scattering curve and the scattering curve calculated from the cryo-EM model (A) plotted with a red line. (C) Different types of solutions of multiple runs of Alphafold 2 on E9-A20-D4: models similar to the complex observed in cryo-EM shown in panel (A), left; models with an E9-D4 interaction similar to the one of the cryo-EM structure, but with a disrupted A20-D4 interface (middle); models with isolated D4 (right). (D) The middle model of (C) is used but the A20-D4 interface is restored by a repositioning of D4 according to the A20304-426-D4 structure [20]. (E) The calculated scattering curve (blue) of the model shown in (D) is compared to the experimental curve. (F) Model of E9-A20-D4 refined against the experimental scattering curve using Coral using the two hinges indicated in (A) and a flexible connection of the thumb domain. (G) The calculated scattering curve (blue line) of the refined model from Coral shown in (F) is compared to experimental scattering curve.
Table 1.
Parameters derived from SAXS measurements.
Fig 4.
Analysis of the E9-D4 interaction by BLI.
E9 is immobilized on a sensor tip using its 6His-tag. Data have been corrected for the unneglectable dissociation of 6His-tagged E9 from the sensor tip. (A) Interaction with a monomeric mutant of D4 (D4KEK). Controls: (B) Interaction with A20304-426 (A20C); (C) interaction of E9 with a dsDNA composed of a 37 base template strand with a 25 base 5’ overhang and a 12 base primer strand.
Fig 5.
Mutant VACVs generated with the CRISPR/Cas9 system.
Fig 6.
Movements within the holoenzyme complex.
(A) VACV E9 crystal structure (pdb entry 5n2e [21], grey, thumb domain in black) superposed onto the refined structure of the VACV E9-A20-D4 complex (colours as in Fig 2A). (B) Close-up of the fingertip and the insert 2 region of the apo form of VACV holoenzyme (orange) superposed onto the DNA-bound MPXV E9 (gray, finger domain in cyan, residues of the fingertip in stick representation and highlighted in green, insert 2 domain in blue) with an incoming dCTP nucleotide (spheres) [23]. A red arrow shows the direction of the movement of the fingertip upon nucleotide binding. Residues involved in PAA resistance of VACV polymerase [31] are labeled and shown in red. Residues of insert 2 differing between MPXV (GenBank accession ON755039, [2]) and VACV are shown in stick representation and labelled. Residue labels of VACV E9 are printed in black, the corresponding residues from MPXV are printed in blue and additionally in bold if the mutations have been acquired recently. (C) VACV E9-A20-D4 cryo-EM structure where the invisible thumb domain is added according to the crystal structure [21]. For clarity, D4 (yellow) and thumb domain (red) are shown in surface representation. (D) The same view of the MPXV holoenzyme with bound DNA (pdb entry 8hg1 [23]). Domains are colored as in (C); the DNA is shown in green.
Table 2.
Cryo-EM data collection statistics.