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- EMDB-7055: Cryo-EM structure of the NAIP5-NLRC4-flagellin inflammasome -

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Basic information

Entry
Database: EMDB / ID: EMD-7055
TitleCryo-EM structure of the NAIP5-NLRC4-flagellin inflammasome
Map dataPostprocessed (sharpened, filtered) final map of the NAIP5-NLRC4-flagellin inflammasome
Sample
  • Complex: NAIP5-NLRC4-flagellin inflammasome
    • Complex: NAIP5
      • Protein or peptide: Baculoviral IAP repeat-containing protein 1e
    • Complex: NLRC4
      • Protein or peptide: NLR family CARD domain-containing protein 4
    • Complex: flagellin
      • Protein or peptide: Flagellin
KeywordsInnate immunity / molecular complex / IMMUNE SYSTEM
Function / homology
Function and homology information


IPAF inflammasome complex / bacterial-type flagellum / pyroptotic inflammatory response / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / detection of bacterium / activation of innate immune response / positive regulation of interleukin-1 beta production / protein homooligomerization / perikaryon / regulation of apoptotic process ...IPAF inflammasome complex / bacterial-type flagellum / pyroptotic inflammatory response / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / detection of bacterium / activation of innate immune response / positive regulation of interleukin-1 beta production / protein homooligomerization / perikaryon / regulation of apoptotic process / defense response to Gram-negative bacterium / defense response to bacterium / inflammatory response / symbiont entry into host cell / innate immune response / neuronal cell body / negative regulation of apoptotic process / apoptotic process / structural molecule activity / protein homodimerization activity / extracellular region / ATP binding / identical protein binding / metal ion binding / cytosol
Similarity search - Function
Baculoviral IAP repeat-containing protein 1 / NLR family CARD domain-containing protein 4 / Flagellin hook, IN motif / NLRC4, helical domain / : / Flagellin hook IN motif / NLRC4 helical domain / Nlrc4-like, winged helix domain / Flagellin, C-terminal domain, subdomain 2 / Flagellin, C-terminal domain ...Baculoviral IAP repeat-containing protein 1 / NLR family CARD domain-containing protein 4 / Flagellin hook, IN motif / NLRC4, helical domain / : / Flagellin hook IN motif / NLRC4 helical domain / Nlrc4-like, winged helix domain / Flagellin, C-terminal domain, subdomain 2 / Flagellin, C-terminal domain / Bacterial flagellin C-terminal helical region / Flagellin / Flagellin, N-terminal domain / Bacterial flagellin N-terminal helical region / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / BIR repeat. / BIR repeat / Inhibitor of Apoptosis domain / BIR repeat profile. / Baculoviral inhibition of apoptosis protein repeat / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Death-like domain superfamily / Leucine-rich repeat domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
: / NLR family CARD domain-containing protein 4 / Flagellin / Baculoviral IAP repeat-containing protein 1e
Similarity search - Component
Biological speciesMus musculus (house mouse) / Legionella pneumophila (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.2 Å
AuthorsTenthorey JL / Haloupek N
Funding support Spain, United States, 3 items
OrganizationGrant numberCountry
MinecoBFU2016-76220-P Spain
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI075039 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI063302 United States
CitationJournal: Science / Year: 2017
Title: The structural basis of flagellin detection by NAIP5: A strategy to limit pathogen immune evasion.
Authors: Jeannette L Tenthorey / Nicole Haloupek / José Ramón López-Blanco / Patricia Grob / Elise Adamson / Ella Hartenian / Nicholas A Lind / Natasha M Bourgeois / Pablo Chacón / Eva Nogales / Russell E Vance /
Abstract: Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in ...Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in plants and animals. However, the structural basis for ligand-induced NLR activation has so far remained unknown. NAIP5 (NLR family, apoptosis inhibitory protein 5) binds the bacterial protein flagellin and assembles with NLRC4 to form a multiprotein complex called an inflammasome. Here we report the cryo-electron microscopy structure of the assembled ~1.4-megadalton flagellin-NAIP5-NLRC4 inflammasome, revealing how a ligand activates an NLR. Six distinct NAIP5 domains contact multiple conserved regions of flagellin, prying NAIP5 into an open and active conformation. We show that innate immune recognition of multiple ligand surfaces is a generalizable strategy that limits pathogen evolution and immune escape.
History
DepositionSep 29, 2017-
Header (metadata) releaseOct 25, 2017-
Map releaseNov 15, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6b5b
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7055.png

Downloads & links

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Map

FileDownload / File: emd_7055.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed (sharpened, filtered) final map of the NAIP5-NLRC4-flagellin inflammasome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 448 pix.
= 586.88 Å		1.31 Å/pix.
x 448 pix.
= 586.88 Å		1.31 Å/pix.
x 448 pix.
= 586.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.10179346 - 0.18753581
Average (Standard dev.)-0.000043895485 (±0.004696659)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 586.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z448448448
origin x/y/z0.0000.0000.000
length x/y/z586.880586.880586.880
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS448448448
D min/max/mean-0.1020.188-0.000

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Supplemental data

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Mask #1

Fileemd_7055_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered, unsharpened half map #1 of the NAIP5-NLRC4-flagellin...

Fileemd_7055_half_map_1.map
AnnotationUnfiltered, unsharpened half map #1 of the NAIP5-NLRC4-flagellin inflammasome
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered, unsharpened half map #2 of the NAIP5-NLRC4-flagellin...

Fileemd_7055_half_map_2.map
AnnotationUnfiltered, unsharpened half map #2 of the NAIP5-NLRC4-flagellin inflammasome
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NAIP5-NLRC4-flagellin inflammasome

EntireName: NAIP5-NLRC4-flagellin inflammasome
Components
  • Complex: NAIP5-NLRC4-flagellin inflammasome
    • Complex: NAIP5
      • Protein or peptide: Baculoviral IAP repeat-containing protein 1e
    • Complex: NLRC4
      • Protein or peptide: NLR family CARD domain-containing protein 4
    • Complex: flagellin
      • Protein or peptide: Flagellin

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Supramolecule #1: NAIP5-NLRC4-flagellin inflammasome

SupramoleculeName: NAIP5-NLRC4-flagellin inflammasome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: NAIP5

SupramoleculeName: NAIP5 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: NLRC4

SupramoleculeName: NLRC4 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #4: flagellin

SupramoleculeName: flagellin / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Legionella pneumophila (bacteria)

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Macromolecule #1: Baculoviral IAP repeat-containing protein 1e

MacromoleculeName: Baculoviral IAP repeat-containing protein 1e / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 159.874828 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAEHGESSED RISEIDYEFL PELSALLGVD AFQVAKSQEE EEHKERMKMK KGFNSQMRSE AKRLKTFETY DTFRSWTPQE MAAAGFYHT GVKLGVQCFC CSLILFGNSL RKLPIERHKK LRPECEFLQG KDVGNIGKYD IRVKSPEKML RGGKARYHEE E ARLESFED ...String:
MAEHGESSED RISEIDYEFL PELSALLGVD AFQVAKSQEE EEHKERMKMK KGFNSQMRSE AKRLKTFETY DTFRSWTPQE MAAAGFYHT GVKLGVQCFC CSLILFGNSL RKLPIERHKK LRPECEFLQG KDVGNIGKYD IRVKSPEKML RGGKARYHEE E ARLESFED WPFYAHGTSP RVLSAAGFVF TGKRDTVQCF SCGGSLGNWE EGDDPWKEHA KWFPKCEFLQ SKKSSEEIAQ YI QSYEGFV HVTGEHFVKS WVRRELPMVS AYCNDSVFAN EELRMDMFKD WPQESPVGVE ALVRAGFFYT GKKDIVRCFS CGG CLEKWA EGDDPMEDHI KFFPECVFLQ TLKSSAEVIP TLQSQYALPE ATETTRESNH GDAAAVHSTV VDLGRSEAQW FQEA RSLSE QLRDNYTKAT FRHMNLPEVC SSLGTDHLLS CDVSIISKHI SQPVQEALTI PEVFSNLNSV MCVEGETGSG KTTFL KRIA FLWASGCCPL LYRFQLVFYL SLSSITPDQG LANIICAQLL GAGGCISEVC LSSSIQQLQH QVLFLLDDYS GLASLP QAL HTLITKNYLS RTCLLIAVHT NRVRDIRLYL GTSLEIQEFP FYNTVSVLRK FFSHDIICVE KLIIYFIDNK DLQGVYK TP LFVAAVCTDW IQNASAQDKF QDVTLFQSYM QYLSLKYKAT AEPLQATVSS CGQLALTGLF SSCFEFNSDD LAEAGVDE D EKLTTLLMSK FTAQRLRPVY RFLGPLFQEF LAAVRLTELL SSDRQEDQDL GLYYLRQIDS PLKAINSFNI FLYYVSSHS SSKAAPTVVS HLLQLVDEKE SLENMSENED YMKLHPQTFL WFQFVRGLWL VSPESSSSFV SEHLLRLALI FAYESNTVAE CSPFILQFL RGKTLALRVL NLQYFRDHPE SLLLLRSLKV SINGNKMSSY VDYSFKTYFE NLQPPAIDEE YTSAFEHISE W RRNFAQDE EIIKNYENIR PRALPDISEG YWKLSPKPCK IPKLEVQVNN TDAADQALLQ VLMEVFSASQ SIEFRLFNSS GF LESICPA LELSKASVTK CSMSRLELSR AEQELLLTLP ALQSLEVSET NQLPEQLFHN LHKFLGLKEL CVRLDGKPDV LSV LPGEFP NLHHMEKLSI RTSTESDLSK LVKFIQNFPN LHVFHLKCDF LSNCESLMAV LASCKKLREI EFSGRCFEAM TFVN ILPNF VSLKILNLKD QQFPDKETSE KFAQALGSLR NLEELLVPTG DGIHQVAKLI VRQCLQLPCL RVLTFHDILD DDSVI EIAR AATSGGFQKL ENLDISMNHK ITEEGYRNFF QALDNLPNLQ ELNICRNIPG RIQVQATTVK ALGQCVSRLP SLIRLH MLS WLLDEEDMKV INDVKERHPQ SKRLIIFWKL IVPFSPVILE

UniProtKB: Baculoviral IAP repeat-containing protein 1e

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Macromolecule #2: NLR family CARD domain-containing protein 4

MacromoleculeName: NLR family CARD domain-containing protein 4 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 116.886 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MNFIRNNRRA LIQRMGLTVT KQICDDLFAL NVLNNQEANV IYCEPLEQEA ARKIIHMTMQ KGSAACNLFL KSLENWDYFV YQDLTGQNL SYQVTEEDLN VLAQNLKDLY NSPAFLNFYP LGEDIDIIFN LEKTFTEPIM WKKDHRHHRV EQLTLGSLLE A LKSPCLIE ...String:
MNFIRNNRRA LIQRMGLTVT KQICDDLFAL NVLNNQEANV IYCEPLEQEA ARKIIHMTMQ KGSAACNLFL KSLENWDYFV YQDLTGQNL SYQVTEEDLN VLAQNLKDLY NSPAFLNFYP LGEDIDIIFN LEKTFTEPIM WKKDHRHHRV EQLTLGSLLE A LKSPCLIE GESGKGKSTL LQRIAMLWAS GGCRALKGFR LVFFIHLRSA RGGLFETLYD QLLNIPDFIS KPTFKALLLK LH KEVLFLL DGYNEFHPQN CPEIEALIKE NHRFKNMVIV TTTTECLRHI RHVGALTAEV GDMTEDSAKD LIEAVLVPDQ VER LWAQIQ ESRCLRNLMK TPLFVVITCA IQMGRQEFQA HTQTMLFQTF YDLLIQKNSH RYRGGASGDF ARSLDYCGDL ALEG VFAHK FDFEPEHGSS MNEDVLVTIG LLCKYTAQRL KPTYKFFHKS FQEYTAGRRL SSLLTSKEPE EVSKGNSYLN KMVSI SDIT SLYGNLLLYT CGSSTEATRA VMRHLAMVYQ HGSLQGLSVT KRPLWRQESI QSLRNTTEQD VLKAINVNSF VECGIN LFS ESMSKSDLSQ EFEAFFQGKS LYINSENIPD YLFDFFEYLP NCASALDFVK LDFYERATES QDKAEENVPG VHTEGPS ET YIPPRAVSLF FNWKQEFKTL EVTLRDINKL NKQDIKYLGK IFSSATNLRL HIKRCAAMAG RLSSVLRTCK NMHTLMVE A SPLTTDDEQY ITSVTGLQNL SIHRLHTQQL PGGLIDSLGN LKNLERLILD DIRMNEEDAK NLAEGLRSLK KMRLLHLTH LSDIGEGMDY IVKSLSEESC DLQEMKLVAC CLTANSVKVL AQNLHNLIKL SILDISENYL EKDGNEALQE LIGRLGVLGE LTTLMLPWC WDVHTSLPKL LKQLEGTPGL AKLGLKNWRL RDEEIKSLGE FLEMNPLRDL QQLDLAGHCV SSDGWLYFMN V FENLKQLV FFDFSTEEFL PDAALVRKLS QVLSKLTLLQ EVKLTGWEFD DYDISAIKGT FKLVTA

UniProtKB: NLR family CARD domain-containing protein 4

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Macromolecule #3: Flagellin

MacromoleculeName: Flagellin / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila (bacteria)
Molecular weightTheoretical: 58.49407 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MEQKLISEED LNEMEQKLIS EEDLNEMEQK LISEEDLNEM EQKLISEEDL NEMEQKLISE EDLNEMESLG DLTMEQKLIS EEDLNSGRP AAMAQVINTN VASLTAQRNL GVSGNMMQTS IQRLSSGLRI NSAKDDAAGL AISQRMTAQI RGMNQAVRNA N DGISLAQV ...String:
MEQKLISEED LNEMEQKLIS EEDLNEMEQK LISEEDLNEM EQKLISEEDL NEMEQKLISE EDLNEMESLG DLTMEQKLIS EEDLNSGRP AAMAQVINTN VASLTAQRNL GVSGNMMQTS IQRLSSGLRI NSAKDDAAGL AISQRMTAQI RGMNQAVRNA N DGISLAQV AEGAMQETTN ILQRMRELSV QAANSTNNSS DRSSIQSEIS QLKSELERIA QNTEFNGQRI LDGSFSGASF QV GANSNQT INFSIGSTKA SSLGGIATAT GTEVAGAAAA DITIAIGGGA ATSINSSANF TGALNGQDAT SAYAKAAAIN DAG IGGLSV TASTSGTQAV GAIGGTAGDT YNLTINGVAI YTNLNVATAL TNSDLRDAIN GVSNQTGVVA SLNGGNMTLT AADG RNITV TESGTGFTAG TDGLTVTGGA FDGALRGTLS ISAVDTIAIG GTVANIGLSA NISKDTVGID SLDVSTASGA QTAIK RIDA ALNSVNSNRA NMGALQNRFE STIANLQNVS DNLSAARSRI QDADYAAEMA SLTKNQILQQ AGTAMLAQAN SLPQSV LSL LGR

UniProtKB: UNIPROTKB: G8UUW9

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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: 7.6
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodium chloride
10.0 mMKClpotassium chloride
5.0 mMMgCl2magnesium chloride
5.0 %C3H8O3glycerol
0.02 %(C2H4O)multC1H24ONP-40
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Details: Carbon-coated
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Frames/image: 1-20 / Number grids imaged: 3 / Average exposure time: 6.0 sec. / Average electron dose: 45.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 22500
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 856358
Startup modelType of model: OTHER / Details: EMAN2 e2initialmodel
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 252214
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN2 (ver. 2.05) / Details: e2refine
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 1.4) / Details: maximum likelihood
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 1.4)

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Atomic model buiding 1

DetailsHomology models predicted by I-TASSER server were used as initial model sources. The main structural template identified by I-TASSER for NAIP5 was the crystal structure of NLRC4 in the inactive conformation (PDB ID: 4KXF) that covered all the domains except the N-terminal BIR region, where homology models from several BIR domains were recognized (PDB IDs: 1SE0, 2VM5, and 1OXQ for BIR1, BIR2, and BIR3, respectively). Predictions were first rigid body docked into the density map using Chimera and/or ADP_EM. Then, the docked models were flexibly fitted with iMODFIT, if necessary. Finally, all models were refined in Phenix.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 167 / Target criteria: Correlation coefficient
Output model

PDB-6b5b:
Cryo-EM structure of the NAIP5-NLRC4-flagellin inflammasome

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