EMDB-11849: Asymmetric reconstruction of a native Bunyamwera virus ribonucleo...

Basic information

Entry

Database: EMDB / ID: EMD-11849

• Title: Asymmetric reconstruction of a native Bunyamwera virus ribonucleoprotein

Sample

• Complex: Native ribonucleoprotein extracted from Bunyamwera virus

• Biological species: Bunyamwera virus
• Method: single particle reconstruction / cryo EM / Resolution: 15.0 Å
• Authors: Hopkins FR / Fontana J / Barr JN

Funding support

United Kingdom, 1 items

Organization	Grant number	Country
Biotechnology and Biological Sciences Research Council (BBSRC)		United Kingdom

• Citation: Journal: mBio / Year: 2022; Title: The Native Orthobunyavirus Ribonucleoprotein Possesses a Helical Architecture.; Authors: Francis R Hopkins / Beatriz Álvarez-Rodríguez / George R Heath / Kyriakoulla Panayi / Samantha Hover / Thomas A Edwards / John N Barr / Juan Fontana / ; Abstract: The order is the largest group of negative-sense RNA viruses, containing many lethal human pathogens for which approved anti-infective measures are not available. The bunyavirus genome consists of multiple negative-sense RNA segments enwrapped by the virus-encoded nucleocapsid protein (NP), which together with the viral polymerase form ribonucleoproteins (RNPs). RNPs represent substrates for RNA synthesis and virion assembly, which require inherent flexibility, consistent with the appearance of RNPs spilled from virions. These observations have resulted in conflicting models describing the overall RNP architecture. Here, we purified RNPs from Bunyamwera virus (BUNV), the prototypical orthobunyavirus. The lengths of purified RNPs imaged by negative staining resulted in 3 populations of RNPs, suggesting that RNPs possess a consistent method of condensation. Employing microscopy approaches, we conclusively show that the NP portion of BUNV RNPs is helical. Furthermore, we present a pseudo-atomic model for this portion based on a cryo-electron microscopy average at 13 Å resolution, which allowed us to fit the BUNV NP crystal structure by molecular dynamics. This model was confirmed by NP mutagenesis using a mini-genome system. The model shows that adjacent NP monomers in the RNP chain interact laterally through flexible N- and C-terminal arms only, with no longitudinal helix-stabilizing interactions, thus providing a potential model for the molecular basis for RNP flexibility. Excessive RNase treatment disrupts native RNPs, suggesting that RNA was key in maintaining the RNP structure. Overall, this work will inform studies on bunyaviral RNP assembly, packaging, and RNA replication, and aid in future antiviral strategies. Bunyaviruses are emerging RNA viruses that cause significant disease and economic burden and for which vaccines or therapies approved for humans are not available. The bunyavirus genome is wrapped up by the nucleoprotein (NP) and interacts with the viral polymerase, forming a ribonucleoprotein (RNP). This is the only form of the genome active for viral replication and assembly. However, until now how NPs are organized within an RNP was not known for any orthobunyavirus. Here, we purified RNPs from the prototypical orthobunyavirus, Bunyamwera virus, and employed microscopy approaches to show that the NP portion of the RNP was helical. We then combined our helical average with the known structure of an NP monomer, generating a pseudo-atomic model of this region. This arrangement allowed the RNPs to be highly flexible, which was critical for several stages of the viral replication cycle, such as segment circularization.

History

Deposition	Oct 15, 2020	-
Header (metadata) release	May 4, 2022	-
Map release	May 4, 2022	-
Update	May 17, 2023	-
Current status	May 17, 2023	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_11849.map.gz / Format: CCP4 / Size: 489.3 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 4.28 Å

Density

Contour Level	By AUTHOR: 0.0709
Minimum - Maximum	-0.075650975 - 0.17875257
Average (Standard dev.)	0.004170107 (±0.029601403)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 -49 Dimensions 50 50 50 Spacing 50 50 50
Cell	A=B=C: 214.00002 Å α=β=γ: 90.0 °

Supplemental data

Sample components

Entire : Native ribonucleoprotein extracted from Bunyamwera virus

• Entire: Name: Native ribonucleoprotein extracted from Bunyamwera virus

Components

• Complex: Native ribonucleoprotein extracted from Bunyamwera virus

Supramolecule #1: Native ribonucleoprotein extracted from Bunyamwera virus

• Supramolecule: Name: Native ribonucleoprotein extracted from Bunyamwera virus; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
• Source (natural): Organism: Bunyamwera virus

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: filament

Sample preparation

• Buffer: pH: 7.4
• Vitrification: Cryogen name: NITROGEN / Instrument: FEI VITROBOT MARK II

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000
• Specialist optics: Phase plate: VOLTA PHASE PLATE
• Image recording: Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 9.0 sec. / Average electron dose: 51.3 e/Ų
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Startup model: Type of model: OTHER; Details: Previously generated sub-tomogram average of the same sample
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD
• Final angle assignment: Type: MAXIMUM LIKELIHOOD
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 5077