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- EMDB-26319: Influenza Neuraminidase N1-MI15-sNAp-174 -

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Basic information

Entry
Database: EMDB / ID: EMD-26319
TitleInfluenza Neuraminidase N1-MI15-sNAp-174
Map data
Sample
  • Complex: Structure-based design of an engineered Influenza Neuraminidase tetramer
    • Protein or peptide: Influenza N1-MI15-sNAp-174
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsInfluenza / Antigen / Engineered Protein / Structural Genomics / Seattle Structural Genomics Center for Infectious Disease / SSGCID / VIRAL PROTEIN
Biological speciesInfluenza A virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsActon OJ / Veesler D / Seattle Structural Genomics Center for Infectious Disease (SSGCID)
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nat Commun / Year: 2022
Title: Structure-based design of stabilized recombinant influenza neuraminidase tetramers.
Authors: Daniel Ellis / Julia Lederhofer / Oliver J Acton / Yaroslav Tsybovsky / Sally Kephart / Christina Yap / Rebecca A Gillespie / Adrian Creanga / Audrey Olshefsky / Tyler Stephens / Deleah ...Authors: Daniel Ellis / Julia Lederhofer / Oliver J Acton / Yaroslav Tsybovsky / Sally Kephart / Christina Yap / Rebecca A Gillespie / Adrian Creanga / Audrey Olshefsky / Tyler Stephens / Deleah Pettie / Michael Murphy / Claire Sydeman / Maggie Ahlrichs / Sidney Chan / Andrew J Borst / Young-Jun Park / Kelly K Lee / Barney S Graham / David Veesler / Neil P King / Masaru Kanekiyo /
Abstract: Influenza virus neuraminidase (NA) is a major antiviral drug target and has recently reemerged as a key target of antibody-mediated protective immunity. Here we show that recombinant NAs across non- ...Influenza virus neuraminidase (NA) is a major antiviral drug target and has recently reemerged as a key target of antibody-mediated protective immunity. Here we show that recombinant NAs across non-bat subtypes adopt various tetrameric conformations, including an "open" state that may help explain poorly understood variations in NA stability across viral strains and subtypes. We use homology-directed protein design to uncover the structural principles underlying these distinct tetrameric conformations and stabilize multiple recombinant NAs in the "closed" state, yielding two near-atomic resolution structures of NA by cryo-EM. In addition to enhancing thermal stability, conformational stabilization improves affinity to protective antibodies elicited by viral infection, including antibodies targeting a quaternary epitope and the broadly conserved catalytic site. Stabilized NAs can also be integrated into viruses without affecting fitness. Our findings provide a deeper understanding of NA structure, stability, and antigenicity, and establish design strategies for reinforcing the conformational integrity of recombinant NA proteins.
History
DepositionFeb 24, 2022-
Header (metadata) releaseApr 20, 2022-
Map releaseApr 20, 2022-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26319.png

Downloads & links

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Map

FileDownload / File: emd_26319.map.gz / Format: CCP4 / Size: 32.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.16 Å/pix.
x 204 pix.
= 236.64 Å		1.16 Å/pix.
x 204 pix.
= 236.64 Å		1.16 Å/pix.
x 204 pix.
= 236.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.16 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.84
Minimum - Maximum-4.1257744 - 5.9580526
Average (Standard dev.)0.006789739 (±0.15822068)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions204204204
Spacing204204204
CellA=B=C: 236.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_26319_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Structure-based design of an engineered Influenza Neuraminidase t...

EntireName: Structure-based design of an engineered Influenza Neuraminidase tetramer
Components
  • Complex: Structure-based design of an engineered Influenza Neuraminidase tetramer
    • Protein or peptide: Influenza N1-MI15-sNAp-174
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Structure-based design of an engineered Influenza Neuraminidase t...

SupramoleculeName: Structure-based design of an engineered Influenza Neuraminidase tetramer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Influenza A virus
Molecular weightTheoretical: 210 kDa/nm

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Macromolecule #1: Influenza N1-MI15-sNAp-174

MacromoleculeName: Influenza N1-MI15-sNAp-174 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus
Molecular weightTheoretical: 42.577742 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: VKLAGNSSLC PVSGWAPLSK DNSVRIGSKG DVFVIREPFI SCSPLECRQF FLTQGALLND KHSNGTIKDR SPYRTLMSVP IGSVPSPYN ARFESIAWSA SACHDGINWL TIGITGPDSG AVAILKYNGI ITDTIKSWRN NILRTQESEC ACVNGSCFTI M TDGPSDGQ ...String:
VKLAGNSSLC PVSGWAPLSK DNSVRIGSKG DVFVIREPFI SCSPLECRQF FLTQGALLND KHSNGTIKDR SPYRTLMSVP IGSVPSPYN ARFESIAWSA SACHDGINWL TIGITGPDSG AVAILKYNGI ITDTIKSWRN NILRTQESEC ACVNGSCFTI M TDGPSDGQ ASYKIFRIEK GKIIKSVEMK APNYHYEECS CYPDSSEITC VCRDNWHGSN RPWVSFNQNL EYQMGYICSG VF GDNPRPN DKTGSCGPVS SNGANGVKGF SFKYGNGVWI GRTKSISSRK GFEMIWDPNG WTGTDNKFSI KQDIVGINEW SGY SGSFVM HPELTGLDCI VPCFWVELIR GRPEENTIWT SGSSISFCGV NSDTVGWSWP DGAELPFTID K

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Macromolecule #2: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 8 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER

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Image processing

Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 72106
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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