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- EMDB-11635: Aquifex aeolicus lumazine synthase-derived nucleocapsid variant NC-4 -

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Basic information

Entry
Database: EMDB / ID: EMD-11635
TitleAquifex aeolicus lumazine synthase-derived nucleocapsid variant NC-4
Map datamap postprocessed in RELION
Sample
  • Complex: Nucleocapsid NC-4
    • Complex: Nucleocapsid NC-4
      • Protein or peptide: Antitermination protein N,6,7-dimethyl-8-ribityllumazine synthase,6,7-dimethyl-8-ribityllumazine synthase
Keywordscapsid / design / virus mimic / VIRUS LIKE PARTICLE
Function / homology
Function and homology information


6,7-dimethyl-8-ribityllumazine synthase / 6,7-dimethyl-8-ribityllumazine synthase activity / riboflavin synthase complex / riboflavin biosynthetic process / RNA polymerase binding / bacterial-type RNA polymerase core enzyme binding / transcription antitermination factor activity, RNA binding / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription termination ...6,7-dimethyl-8-ribityllumazine synthase / 6,7-dimethyl-8-ribityllumazine synthase activity / riboflavin synthase complex / riboflavin biosynthetic process / RNA polymerase binding / bacterial-type RNA polymerase core enzyme binding / transcription antitermination factor activity, RNA binding / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription termination / RNA stem-loop binding / single-stranded RNA binding / regulation of transcription by RNA polymerase II / DNA binding / RNA binding / cytosol
Similarity search - Function
Antitermination protein N, arginine-rich motif / 36-mer N-terminal peptide of the N protein (N36) / Lumazine synthase / Lumazine/riboflavin synthase / Lumazine/riboflavin synthase superfamily / 6,7-dimethyl-8-ribityllumazine synthase
Similarity search - Domain/homology
6,7-dimethyl-8-ribityllumazine synthase / Antitermination protein N
Similarity search - Component
Biological speciesEscherichia virus lambda / Aquifex aeolicus VF5 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsTetter S / Hilvert D
Funding support1 items
OrganizationGrant numberCountry
European Research Council (ERC)ERC-AdG-2012-321295
Citation
Journal: Science / Year: 2021
Title: Evolution of a virus-like architecture and packaging mechanism in a repurposed bacterial protein.
Authors: Stephan Tetter / Naohiro Terasaka / Angela Steinauer / Richard J Bingham / Sam Clark / Andrew J P Scott / Nikesh Patel / Marc Leibundgut / Emma Wroblewski / Nenad Ban / Peter G Stockley / ...Authors: Stephan Tetter / Naohiro Terasaka / Angela Steinauer / Richard J Bingham / Sam Clark / Andrew J P Scott / Nikesh Patel / Marc Leibundgut / Emma Wroblewski / Nenad Ban / Peter G Stockley / Reidun Twarock / Donald Hilvert /
Abstract: Viruses are ubiquitous pathogens of global impact. Prompted by the hypothesis that their earliest progenitors recruited host proteins for virion formation, we have used stringent laboratory evolution ...Viruses are ubiquitous pathogens of global impact. Prompted by the hypothesis that their earliest progenitors recruited host proteins for virion formation, we have used stringent laboratory evolution to convert a bacterial enzyme that lacks affinity for nucleic acids into an artificial nucleocapsid that efficiently packages and protects multiple copies of its own encoding messenger RNA. Revealing remarkable convergence on the molecular hallmarks of natural viruses, the accompanying changes reorganized the protein building blocks into an interlaced 240-subunit icosahedral capsid that is impermeable to nucleases, and emergence of a robust RNA stem-loop packaging cassette ensured high encapsidation yields and specificity. In addition to evincing a plausible evolutionary pathway for primordial viruses, these findings highlight practical strategies for developing nonviral carriers for diverse vaccine and delivery applications.
#1: Journal: Biorxiv / Year: 2021
Title: Evolution of a virus-like architecture and packaging mechanism in a repurposed bacterial protein
Authors: Tetter S / Terasaka N / Steinauer A / Bingham RJ / Clark S / Scott AJP / Patel N / Leibundgut M / Wroblewski E / Ban N / Stockley PG / Twarock R / Hilvert D
History
DepositionAug 19, 2020-
Header (metadata) releaseJun 2, 2021-
Map releaseJun 2, 2021-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7a4j
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7a4j
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11635.png

Downloads & links

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Map

FileDownload / File: emd_11635.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap postprocessed in RELION
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 600 pix.
= 660. Å		1.1 Å/pix.
x 600 pix.
= 660. Å		1.1 Å/pix.
x 600 pix.
= 660. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.10272622 - 0.18146865
Average (Standard dev.)-0.00015571428 (±0.006383867)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 660.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z600600600
origin x/y/z0.0000.0000.000
length x/y/z660.000660.000660.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS600600600
D min/max/mean-0.1030.181-0.000

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Supplemental data

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Mask #1

Fileemd_11635_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: map from 3D autorefinement in RELION

Fileemd_11635_additional_1.map
Annotationmap from 3D autorefinement in RELION
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_11635_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11635_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Nucleocapsid NC-4

EntireName: Nucleocapsid NC-4
Components
  • Complex: Nucleocapsid NC-4
    • Complex: Nucleocapsid NC-4
      • Protein or peptide: Antitermination protein N,6,7-dimethyl-8-ribityllumazine synthase,6,7-dimethyl-8-ribityllumazine synthase

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Supramolecule #1: Nucleocapsid NC-4

SupramoleculeName: Nucleocapsid NC-4 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Evolved variant of a virus-inspired nucleocapsid design based on the Aquifex aeolicus riboflavin synthase

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Supramolecule #2: Nucleocapsid NC-4

SupramoleculeName: Nucleocapsid NC-4 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all
Source (natural)Organism: Escherichia virus lambda

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Macromolecule #1: Antitermination protein N,6,7-dimethyl-8-ribityllumazine synthase...

MacromoleculeName: Antitermination protein N,6,7-dimethyl-8-ribityllumazine synthase,6,7-dimethyl-8-ribityllumazine synthase
type: protein_or_peptide / ID: 1 / Number of copies: 240 / Enantiomer: LEVO / EC number: 6,7-dimethyl-8-ribityllumazine synthase
Source (natural)Organism: Aquifex aeolicus VF5 (bacteria)
Molecular weightTheoretical: 21.475307 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGNARTRRRE RRAEKQAQWK AANAGAGAGA MATPHFDYNA SVVSKGLANL SLELRKPVSF DIITADTLEQ AIERAGTKHG NKGWEAALS AIEMANLYKS LRGTEHHHHL HGSSIEIYEG KLTAEGLRFG IVASRFNHTL VDRLVEGAID CIVRHGGRGE D ITLVRVPG ...String:
MGNARTRRRE RRAEKQAQWK AANAGAGAGA MATPHFDYNA SVVSKGLANL SLELRKPVSF DIITADTLEQ AIERAGTKHG NKGWEAALS AIEMANLYKS LRGTEHHHHL HGSSIEIYEG KLTAEGLRFG IVASRFNHTL VDRLVEGAID CIVRHGGRGE D ITLVRVPG AWEIPVAADE LARKEDIDAV IAFGDLIRG

UniProtKB: Antitermination protein N, 6,7-dimethyl-8-ribityllumazine synthase, 6,7-dimethyl-8-ribityllumazine synthase

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 12 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 22 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 69723
Startup modelType of model: INSILICO MODEL
In silico model: generated in RELION from the data themselves
Details: with icosahedral symmetry imposed (I1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 15392
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1hqk


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 83
Output model

PDB-7a4j:
Aquifex aeolicus lumazine synthase-derived nucleocapsid variant NC-4

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