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- EMDB-40224: Cryo-ET reconstruction of HSV-1 DN/SUP mutant NEC coat -

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Basic information

Entry
Database: EMDB / ID: EMD-40224
TitleCryo-ET reconstruction of HSV-1 DN/SUP mutant NEC coat
Map dataCryo-ET reconstruction of HSV-1 DN/SUP mutant NEC coat
Sample
  • Complex: Hexameric coat of the HSV-1 nuclear egress complex
    • Protein or peptide: UL31 SUP
    • Protein or peptide: UL34 DN
KeywordsHSV-1 / Nuclear egress / NEC lattice / UL31 / UL34 / suppressor mutation / cryoET / VIRAL PROTEIN
Biological speciesHuman alphaherpesvirus 1 strain 17
Methodsubtomogram averaging / cryo EM / Resolution: 5.4 Å
AuthorsWang H / Draganova EB
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P30 GM124165 United States
Department of Energy (DOE, United States)DE-AC02-06CH11357 United States
CitationJournal: PLoS Pathog / Year: 2024
Title: The universal suppressor mutation restores membrane budding defects in the HSV-1 nuclear egress complex by stabilizing the oligomeric lattice.
Authors: Elizabeth B Draganova / Hui Wang / Melanie Wu / Shiqing Liao / Amber Vu / Gonzalo L Gonzalez-Del Pino / Z Hong Zhou / Richard J Roller / Ekaterina E Heldwein /
Abstract: Nuclear egress is an essential process in herpesvirus replication whereby nascent capsids translocate from the nucleus to the cytoplasm. This initial step of nuclear egress-budding at the inner ...Nuclear egress is an essential process in herpesvirus replication whereby nascent capsids translocate from the nucleus to the cytoplasm. This initial step of nuclear egress-budding at the inner nuclear membrane-is coordinated by the nuclear egress complex (NEC). Composed of the viral proteins UL31 and UL34, NEC deforms the membrane around the capsid as the latter buds into the perinuclear space. NEC oligomerization into a hexagonal membrane-bound lattice is essential for budding because NEC mutants designed to perturb lattice interfaces reduce its budding ability. Previously, we identified an NEC suppressor mutation capable of restoring budding to a mutant with a weakened hexagonal lattice. Using an established in-vitro budding assay and HSV-1 infected cell experiments, we show that the suppressor mutation can restore budding to a broad range of budding-deficient NEC mutants thereby acting as a universal suppressor. Cryogenic electron tomography of the suppressor NEC mutant lattice revealed a hexagonal lattice reminiscent of wild-type NEC lattice instead of an alternative lattice. Further investigation using x-ray crystallography showed that the suppressor mutation promoted the formation of new contacts between the NEC hexamers that, ostensibly, stabilized the hexagonal lattice. This stabilization strategy is powerful enough to override the otherwise deleterious effects of mutations that destabilize the NEC lattice by different mechanisms, resulting in a functional NEC hexagonal lattice and restoration of membrane budding.
History
DepositionMar 24, 2023-
Header (metadata) releaseJan 10, 2024-
Map releaseJan 10, 2024-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40224.png

Downloads & links

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Map

FileDownload / File: emd_40224.map.gz / Format: CCP4 / Size: 28.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-ET reconstruction of HSV-1 DN/SUP mutant NEC coat
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.69 Å/pix.
x 196 pix.
= 331.24 Å		1.69 Å/pix.
x 196 pix.
= 331.24 Å		1.69 Å/pix.
x 196 pix.
= 331.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.69 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.005
Minimum - Maximum-0.008064327 - 0.020691603
Average (Standard dev.)0.000000000000006 (±0.0015758745)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions196196196
Spacing196196196
CellA=B=C: 331.24002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40224_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-ET reconstruction of HSV-1 DN/SUP mutant NEC coat (half map 2)

Fileemd_40224_half_map_1.map
AnnotationCryo-ET reconstruction of HSV-1 DN/SUP mutant NEC coat (half map 2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-ET reconstruction of HSV-1 DN/SUP mutant NEC coat (half map 1)

Fileemd_40224_half_map_2.map
AnnotationCryo-ET reconstruction of HSV-1 DN/SUP mutant NEC coat (half map 1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hexameric coat of the HSV-1 nuclear egress complex

EntireName: Hexameric coat of the HSV-1 nuclear egress complex
Components
  • Complex: Hexameric coat of the HSV-1 nuclear egress complex
    • Protein or peptide: UL31 SUP
    • Protein or peptide: UL34 DN

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Supramolecule #1: Hexameric coat of the HSV-1 nuclear egress complex

SupramoleculeName: Hexameric coat of the HSV-1 nuclear egress complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Human alphaherpesvirus 1 strain 17

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Macromolecule #1: UL31 SUP

MacromoleculeName: UL31 SUP / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Human alphaherpesvirus 1 strain 17
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPGSYDTDPH RRGSRPGPYH GKERRRSRSS AAGGTLGVVR RASRKSLPPH ARKQELCLHE RQRYRGLFAA LAQTPSEEIA IVRSLSVPLV KTTPVSLPFC LDQTVADNCL TLSGMGYYLG IGGCCPACNA GDGRFAATSR EALILAFVQQ INTIFEHRAF LASLVVLADR ...String:
GPGSYDTDPH RRGSRPGPYH GKERRRSRSS AAGGTLGVVR RASRKSLPPH ARKQELCLHE RQRYRGLFAA LAQTPSEEIA IVRSLSVPLV KTTPVSLPFC LDQTVADNCL TLSGMGYYLG IGGCCPACNA GDGRFAATSR EALILAFVQQ INTIFEHRAF LASLVVLADR HNAPLQDLLA GILGQPELFF VHTILRGGGA CDPRLLFYPD PTYGGHMLYV IFPGTSAHLH YLLIDRMLTA CPGYRFVAHV WQSTFVLVVR RNAEKPTDAE IPTVSAADIY CKMRDISFDG GLMLEYQRLY ATFDEFPPP

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Macromolecule #2: UL34 DN

MacromoleculeName: UL34 DN / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Human alphaherpesvirus 1 strain 17
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPLGSPEFPG RPMAGLGKPY TGHPGDAFEG LVQRIRLIVP STLRGGAGAA GPYSPSSLPS RCAFQFHGHD GSDESFPIEY VLRLMNDWAE VPCNPYLRIQ NTGVSVLFQG FFHRPHNAPG GAITPERTNV ILGSTETTGL SLGDLDTIKG RLGLDARPMM ASMWISCFVR ...String:
GPLGSPEFPG RPMAGLGKPY TGHPGDAFEG LVQRIRLIVP STLRGGAGAA GPYSPSSLPS RCAFQFHGHD GSDESFPIEY VLRLMNDWAE VPCNPYLRIQ NTGVSVLFQG FFHRPHNAPG GAITPERTNV ILGSTETTGL SLGDLDTIKG RLGLDARPMM ASMWISCFVR MPRVQLAFRF MGPEDAGRTR RILCRAAEQA ITRRRRTRRS REAYGAEAGL GVAGTGFRAR GD

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation state2D array

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Sample preparation

BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMHEPESN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
100.0 mMNaClsodium chloride
1.0 mMTCEPtris(2-carboxyethyl)phosphine
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 2.34 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.5 µm / Nominal defocus min: 2.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 5.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 35039
ExtractionNumber tomograms: 35 / Number images used: 48481
Final angle assignmentType: MAXIMUM LIKELIHOOD

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