Nucleocapsid assembly in pneumoviruses is regulated by conformational switching of the N protein
- University of Oxford, United Kingdom
- Diamond Light Source, United Kingdom
Figures
Figure 1 with 1 supplement
Structure of the HMPV N0-P complex.
(A) Crystal structure of RNA-free HMPV N0 bound to P1-28. The C-terminal domain (CTD) of N is colored in light blue and the N-terminal domain (NTD) in dark blue. Secondary structure elements …
Figure 1—figure supplement 1
Construct design and purification of the HMPV N0-P hybrid.
(A) Schematic of the N0-P hybrid construct. The N-terminal (NTD) and C-terminal (CTD) domains of N are coloured in dark and light blue, respectively. The N-terminal and C-terminal arms are …
Figure 2 with 4 supplements
Comparison of N in assembled RNA-bound and monomeric RNA-free states.
(A) top- and side-views of RNA-bound HMPV subnucleocapsid rings. N protomers and RNA are shown as surfaces with RNA rendered in brown. The diameter and height of the ring are indicated. (B) Three …
Figure 2—figure supplement 1
Purification of HMPV N-RNA and characterisation of oligomeric state.
(A) Size exclusion chromatogram (Superose 6) of N-RNA after purification from E. coli. The fractions containing N-RNA are indicated by a red bar. The broad peak centred around 10 mL (indicated with …
Figure 2—figure supplement 2
Electron density maps of N-RNA.
(A–C) Samples of electron density of the N-RNA crystal at 4.2 Å. A 2Fo-Fc map contoured at 1.0 σ after density modification with Parrot and B-factor map sharpening is shown. (A) zoomed-out overview …
Figure 2—figure supplement 3
RNA-binding cleft of HMPV N.
(A) External side view showing RNA inserted into three neighbouring protomers of assembled N. The two outer protomers are shown as surface representation coloured by electrostatics, highlighting the …
Figure 2—figure supplement 4
Role of a conserved aromatic residue in N hinge motion.
(A) Monomeric N0 (blue) is superposed onto RNA-bound N (grey). The dotted arrow indicates the tilting of α-helix αC3 during the transition from N0 to N-RNA. Tyr252 is thereby pushed upwards, …
Figure 3 with 1 supplement
Role of the CTD-arm in inhibiting premature RNA uptake.
(A) Conformational switch of the CTD-arm. The CTD-arm (red) is shown in a upward conformation assumed in the N-RNA state and downward conformation of the N0 state (indicated). (B) Polar interactions …
Figure 3—figure supplement 1
CTD-arms in other Mononegavirales family members.
(A) Conformational change of the CTD-arm in HMPV as in Figure 3A. (B and C) negative charges within the CTD-arm are topologically conserved in Borna virus (Bornaviridae) Rudolph et al., 2003 and …
Tables
Table 1
Data collection and refinement statistics.
| N0-P | N-RNA | |
|---|---|---|
| Data collection | ||
| Space group | P 1 | C 2 2 21 |
| Cell dimensions | ||
| a, b, c (Å) | 40.9, 62.8, 86.7 | 202.0, 233.2, 203.6 |
| α, β, γ (°) | 91.0, 96.4, 109.0 | 90, 90, 90 |
| Wavelength (Å) | 0.979 | 0.917 |
| Resolution (Å) | 28.42-1.86 (1.91-1.86) | 101.19-4.17 (4.28-4.17) |
| CC (1/2) | 1.00 (0.47) | 1.00 (0.38) |
| Rmerge | 0.055 (0.590) | 0.220 (2.924) |
| I / σI | 9.2 (1.1) | 9.2 (1.0) |
| Completeness (%) | 94.8 (75.0) | 99.9 (100) |
| Redundancy | 1.7 (1.6) | 13.5 (13.8) |
| Refinement | ||
| Resolution (Å) | 28.42-1.86 | 101.19-4.17 |
| No. reflections | 64451 (3743) | 36125 (2617) |
| Rwork / Rfree | 17.1/20.53 | 19.1/23.0 |
| No. atoms | ||
| Protein | 5707 | 27957 |
| Non-protein | 588 | 1400 |
| B-factors | ||
| Protein | 34.54 | 216.06 |
| Non-protein | 42.56 | 215.08 |
| R.m.s. deviations | ||
| Bond lengths (Å) | 0.007 | 0.010 |
| Bond angles (°) | 1.000 | 1.120 |
| Ramachandran plot quality | ||
| Favoured (%) | 99.72 | 95.01 |
| Allowed (%) | 0.28 | 4.96 |
| Outliers (%) | 0.00 | 0.03 |
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Numbers in parentheses refer to the highest resolution shell.
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Rfree was calculated as per Rwork for a 5% subset of reflections that was not used in the crystallographic refinement.
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Molprobity scores are included in the Methods section.
