[English] 日本語
Yorodumi
- EMDB-12437: 1918 H1N1 Viral influenza polymerase heterotrimer with Nb8207 -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-12437
Title1918 H1N1 Viral influenza polymerase heterotrimer with Nb8207
Map data
Sample
  • Complex: 1918 Influenza virus polymerase heterotirmer in complex with vRNA promoters and Nb8207
    • Complex: Nb8207
      • Protein or peptide: Nb8207
    • Complex: Polymerase heterotrimer
      • Protein or peptide: Polymerase acidic protein
      • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
      • Protein or peptide: Polymerase basic protein 2,Polymerase basic protein 2
    • Complex: RNA
      • RNA: RNA (5'-R(P*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*CP*C)-3')
      • RNA: RNA (5'-R(P*GP*GP*CP*CP*UP*GP*CP*U)-3')
KeywordsInfluenza / RNA polymerase / H1N1 / 1918 / VIRAL PROTEIN / nanobody
Function / homology
Function and homology information


cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / host cell mitochondrion / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / virion component / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds ...cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / host cell mitochondrion / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / virion component / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / viral translational frameshifting / RNA-directed RNA polymerase / viral RNA genome replication / RNA-directed RNA polymerase activity / nucleotide binding / DNA-templated transcription / : / host cell nucleus / RNA binding / metal ion binding
Similarity search - Function
Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / : / Influenza RNA polymerase PB2 N-terminal region / Influenza RNA polymerase PB2 second domain / : / Influenza RNA polymerase PB2 middle domain ...Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / : / Influenza RNA polymerase PB2 N-terminal region / Influenza RNA polymerase PB2 second domain / : / Influenza RNA polymerase PB2 middle domain / : / Influenza RNA polymerase PB2 C-terminal domain / : / Influenza RNA polymerase PB2 6th domain / : / Influenza RNA polymerase PB2 CAP binding domain / : / Influenza RNA polymerase PB2 helical domain / Polymerase acidic protein / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Influenza RNA-dependent RNA polymerase subunit PA / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile.
Similarity search - Domain/homology
Polymerase acidic protein / RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 2
Similarity search - Component
Biological speciesCamelidae mixed library (mammal) / Influenza A virus (A/Brevig Mission/1/1918(H1N1)) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.46 Å
AuthorsKeown JR / Carrique L
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Wellcome Trust200835/Z/16/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MR/R009945/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/K000241/1 United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: Mapping inhibitory sites on the RNA polymerase of the 1918 pandemic influenza virus using nanobodies.
Authors: Jeremy R Keown / Zihan Zhu / Loïc Carrique / Haitian Fan / Alexander P Walker / Itziar Serna Martin / Els Pardon / Jan Steyaert / Ervin Fodor / Jonathan M Grimes /
Abstract: Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral RNA- ...Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral RNA-dependent RNA polymerase which transcribes and replicates the viral RNA genome. The polymerase undergoes conformational rearrangements and interacts with viral and host proteins to perform these functions. Here we determine the structure of the 1918 influenza virus polymerase in transcriptase and replicase conformations using cryo-electron microscopy (cryo-EM). We then structurally and functionally characterise the binding of single-domain nanobodies to the polymerase of the 1918 pandemic influenza virus. Combining these functional and structural data we identify five sites on the polymerase which are sensitive to inhibition by nanobodies. We propose that the binding of nanobodies at these sites either prevents the polymerase from assuming particular functional conformations or interactions with viral or host factors. The polymerase is highly conserved across the influenza A subtypes, suggesting these sites as effective targets for potential influenza antiviral development.
History
DepositionFeb 17, 2021-
Header (metadata) releaseDec 1, 2021-
Map releaseDec 1, 2021-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBe / Status: Released

-
Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.651
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.651
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7nkc
  • Surface level: 0.651
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12437.png

Downloads & links

-
Map

FileDownload / File: emd_12437.map.gz / Format: CCP4 / Size: 18.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.4 Å/pix.
x 170 pix.
= 238. Å		1.4 Å/pix.
x 170 pix.
= 238. Å		1.4 Å/pix.
x 170 pix.
= 238. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.651 / Movie #1: 0.651
Minimum - Maximum-4.600016 - 7.427434
Average (Standard dev.)0.019610848 (±0.21148969)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions170170170
Spacing170170170
CellA=B=C: 238.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z170170170
origin x/y/z0.0000.0000.000
length x/y/z238.000238.000238.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ384384384
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS170170170
D min/max/mean-4.6007.4270.020

-
Supplemental data

-
Sample components

+
Entire : 1918 Influenza virus polymerase heterotirmer in complex with vRNA...

EntireName: 1918 Influenza virus polymerase heterotirmer in complex with vRNA promoters and Nb8207
Components
  • Complex: 1918 Influenza virus polymerase heterotirmer in complex with vRNA promoters and Nb8207
    • Complex: Nb8207
      • Protein or peptide: Nb8207
    • Complex: Polymerase heterotrimer
      • Protein or peptide: Polymerase acidic protein
      • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
      • Protein or peptide: Polymerase basic protein 2,Polymerase basic protein 2
    • Complex: RNA
      • RNA: RNA (5'-R(P*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*CP*C)-3')
      • RNA: RNA (5'-R(P*GP*GP*CP*CP*UP*GP*CP*U)-3')

+
Supramolecule #1: 1918 Influenza virus polymerase heterotirmer in complex with vRNA...

SupramoleculeName: 1918 Influenza virus polymerase heterotirmer in complex with vRNA promoters and Nb8207
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6

+
Supramolecule #2: Nb8207

SupramoleculeName: Nb8207 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #6
Source (natural)Organism: Camelidae mixed library (mammal)

+
Supramolecule #3: Polymerase heterotrimer

SupramoleculeName: Polymerase heterotrimer / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: Influenza A virus (A/Brevig Mission/1/1918(H1N1))

+
Supramolecule #4: RNA

SupramoleculeName: RNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4-#5
Source (natural)Organism: synthetic construct (others)

+
Macromolecule #1: Polymerase acidic protein

MacromoleculeName: Polymerase acidic protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Influenza A virus (A/Brevig Mission/1/1918(H1N1)) / Strain: A/Brevig Mission/1/1918(H1N1)
Molecular weightTheoretical: 82.707391 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEDFVRQCFN PMIVELAEKA MKEYGEDLKI ETNKFAAICT HLEVCFMYSD FHFINERGES IIVESGDPNA LLKHRFEIIE GRDRTMAWT VVNSICNTTG AEKPKFLPDL YDYKENRFIE IGVTRREVHI YYLEKANKIK SEKTHIHIFS FTGEEMATKA D YTLDEESR ...String:
MEDFVRQCFN PMIVELAEKA MKEYGEDLKI ETNKFAAICT HLEVCFMYSD FHFINERGES IIVESGDPNA LLKHRFEIIE GRDRTMAWT VVNSICNTTG AEKPKFLPDL YDYKENRFIE IGVTRREVHI YYLEKANKIK SEKTHIHIFS FTGEEMATKA D YTLDEESR ARIKTRLFTI RQEMASRGLW DSFRQSERGE ETIEERFEIT GTMRRLADQS LPPNFSSLEN FRAYVDGFEP NG YIEGKLS QMSKEVNARI EPFLKTTPRP LRLPDGPPCS QRSKFLLMDA LKLSIEDPSH EGEGIPLYDA IKCMRTFFGW KEP NVVKPH EKGINPNYLL AWKQVLAELQ DIENEEKIPK TKNMKKTSQL KWALGENMAP EKVDFDDCKD VSDLKQYDSD EPEL RSLAS WIQSEFNKAC ELTDSSWIEL DEIGEDVAPI EHIASMRRNY FTAEVSHCRA TEYIMKGVYI NTALLNASCA AMDDF QLIP MISKCRTKEG RRKTNLYGFI IKGRSHLRND TDVVNFVSME FSLTDPRLEP HKWEKYCVLE IGDMLLRSAI GQVSRP MFL YVRTNGTSKI KMKWGMEMRR CLLQSLQQIE SMIEAESSVK EKDMTKEFFE NKSETWPIGE SPKGVEEGSI GKVCRTL LA KSVFNSLYAS PQLEGFSAES RKLLLIVQAL RDNLEPGTFD LGGLYEAIEE CLINDPWVLL NASWFNSFLT HALR

UniProtKB: Polymerase acidic protein

+
Macromolecule #2: RNA-directed RNA polymerase catalytic subunit

MacromoleculeName: RNA-directed RNA polymerase catalytic subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Influenza A virus (A/Brevig Mission/1/1918(H1N1)) / Strain: A/Brevig Mission/1/1918(H1N1)
Molecular weightTheoretical: 86.625211 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDVNPTLLFL KVPAQNAIST TFPYTGDPPY SHGTGTGYTM DTVNRTHQYS EKGRWTTNTE TGAPQLNPID GPLPEDNEPS GYAQTDCVL EAMAFLEESH PGIFENSCLE TMEVVQQTRV DKLTQGRQTY DWTLNRNQPA ATALANTIEV FRSNGLTANE S GRLIDFLK ...String:
MDVNPTLLFL KVPAQNAIST TFPYTGDPPY SHGTGTGYTM DTVNRTHQYS EKGRWTTNTE TGAPQLNPID GPLPEDNEPS GYAQTDCVL EAMAFLEESH PGIFENSCLE TMEVVQQTRV DKLTQGRQTY DWTLNRNQPA ATALANTIEV FRSNGLTANE S GRLIDFLK DVMESMDKEE MEITTHFQRK RRVRDNMTKK MVTQRTIGKK KQRLNKRSYL IRALTLNTMT KDAERGKLKR RA IATPGMQ IRGFVYFVET LARSICEKLE QSGLPVGGNE KKAKLANVVR KMMTNSQDTE LSFTITGDNT KWNENQNPRM FLA MITYIT RNQPEWFRNV LSIAPIMFSN KMARLGKGYM FESKSMKLRT QIPAEMLASI DLKYFNDSTR KKIEKIRPLL IDGT ASLSP GMMMGMFNML STVLGVSILN LGQKRYTKTT YWWDGLQSSD DFALIVNAPN HEGIQAGVDR FYRTCKLLGI NMSKK KSYI NRTGTFEFTS FFYRYGFVAN FSMELPSFGV SGINESADMS IGVTVIKNNM INNDLGPATA QMALQLFIKD YRYTYR CHR GDTQIQTRRS FEIKKLWEQT RSKAGLLVSD GGPNLYNIRN LHIPEVCLKW ELMDEDYQGR LCNPLNPFVS HKEIESV NN AVMMPAHGPA KNMEYDAVAT THSWIPKRNR SILNTSQRGI LEDEQMYQKC CNLFEKFFPS SSYRRPVGIS SMVEAMVS R ARIDARIDFE SGRIKKEEFA EIMKICSTIE ELRRQK

UniProtKB: RNA-directed RNA polymerase catalytic subunit

+
Macromolecule #3: Polymerase basic protein 2,Polymerase basic protein 2

MacromoleculeName: Polymerase basic protein 2,Polymerase basic protein 2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus (A/Brevig Mission/1/1918(H1N1))
Molecular weightTheoretical: 102.377219 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MERIKELRDL MSQSRTREIL TKTTVDHMAI IKKYTSGRQE KNPALRMKWM MAMKYPITAD KRIMEMIPER NEQGQTLWSK TNDAGSDRV MVSPLAVTWW NRNGPTTSAV HYPKIYKTYF EKVERLKHGT FGPVHFRNQV KIRRRVDINP GHADLSAKEA Q DVIMEVVF ...String:
MERIKELRDL MSQSRTREIL TKTTVDHMAI IKKYTSGRQE KNPALRMKWM MAMKYPITAD KRIMEMIPER NEQGQTLWSK TNDAGSDRV MVSPLAVTWW NRNGPTTSAV HYPKIYKTYF EKVERLKHGT FGPVHFRNQV KIRRRVDINP GHADLSAKEA Q DVIMEVVF PNEVGARILT SESQLTITKE KKEELQDCKI SPLMVAYMLE RELVRKTRFL PVAGGTSSVY IEVLHLTQGT CW EQMYTPG GEVRNDDVDQ SLIIAARNIV RRATVSADPL ASLLEMCHST QIGGIRMVDI LRQNPTEEQA VDICKAAMGL RIS SSFSFG GFTFKRTSGS SVKREEEVLT GNLQTLKIRV HEGYEEFTMV GRRATAILRK ATRRLIQLIV SGRDEQSIAE AIIV AMVFS QEDCMIKAVR GDLNFVNRAN QRLNPMHQLL RHFQKDAKVL FQNWGIEPID NVMGMIGILP DMTPSTEMSM RGVRV SKMG VDEYSSTERV VVSIDRFLRV RDQRGNVLLS PEEVSETQGT EKLTITYSSS MMWEVNGPES VLVNTYQWII RNWETV KIQ WSQNPTMLYN KMEFEPFQSL VPKAARGQYS GFVRTLFQQM RDVLGTFDTV QIIKLLPFAA APPKQSRMQF SSLTVNV RG SGMRILVRGN SPVFNYNKAT KRLTVLGKDA GALTEDPDEG TAGVESAVLR GFLILGKEDR RYGPALSINE LSNLAKGE K ANVLIGQGDV VLVMKRKRDS SILTDSQTAT KRIRMAINEN LYFQGELKTA ALAQHDEAVD NKFNKEQQNA FYEILHLPN LNEEQRNAFI QSLKDDPSQS ANLLAEAKKL NDAQAPKVDN KFNKEQQNAF YEILHLPNLN EEQRNAFIQS LKADPSQSAN LLAEAKKLN GAQAPKVDAN SAGKST

UniProtKB: Polymerase basic protein 2

+
Macromolecule #6: Nb8207

MacromoleculeName: Nb8207 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Camelidae mixed library (mammal)
Molecular weightTheoretical: 15.286715 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQAGASLRL SCTASGRTSS TYPMGWFRQG PGKEREFVAA ISWAGSSTYY GDFVKGRFTI SRDNAKNTVY LQMNSLKPE DTAVYYCARA ARPYAYGLDY STEASYDYWG QGTQVTVSSH HHHHHEPEA

+
Macromolecule #4: RNA (5'-R(P*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*CP*C)-3')

MacromoleculeName: RNA (5'-R(P*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*CP*C)-3')
type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.862017 KDa
SequenceString:
AGUAGAAACA AGGCC

+
Macromolecule #5: RNA (5'-R(P*GP*GP*CP*CP*UP*GP*CP*U)-3')

MacromoleculeName: RNA (5'-R(P*GP*GP*CP*CP*UP*GP*CP*U)-3') / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.335125 KDa
SequenceString:
GGCCUGCUUU UGCUAUU

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
166.0 mMNaClSodium chloride
25.0 mMNaSCNSodium thiocynate
20.0 mMHEPES-NaHepes
0.005 %Tween-20Tween-20
VitrificationCryogen name: ETHANE

-
Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Average electron dose: 72.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

-
Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.46 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Number images used: 90450
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2.15)
Final angle assignmentType: MAXIMUM LIKELIHOOD

-
Atomic model buiding 1

Initial modelPDB ID:

7hna


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7nkc:
1918 H1N1 Viral influenza polymerase heterotrimer with Nb8207

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more