[English] 日本語
Yorodumi
- EMDB-7062: Cryo-EM structure of human insulin degrading enzyme in complex wi... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-7062
TitleCryo-EM structure of human insulin degrading enzyme in complex with FAB H11-E heavy chain, FAB H11-E light chain and insulin
Map dataInsulin degrading enzyme in complex with insulinInsulin-degrading enzyme
Sample
  • Complex: Insulin degrading enzyme/Insulin
    • Protein or peptide: Insulin-degrading enzyme
    • Protein or peptide: FAB H11-E heavy chain
    • Protein or peptide: FAB H11-E light chain
    • Protein or peptide: Insulin
Function / homology
Function and homology information


insulysin / ubiquitin recycling / insulin catabolic process / insulin metabolic process / amyloid-beta clearance by cellular catabolic process / hormone catabolic process / bradykinin catabolic process / ubiquitin-modified protein reader activity / insulin binding / negative regulation of NAD(P)H oxidase activity ...insulysin / ubiquitin recycling / insulin catabolic process / insulin metabolic process / amyloid-beta clearance by cellular catabolic process / hormone catabolic process / bradykinin catabolic process / ubiquitin-modified protein reader activity / insulin binding / negative regulation of NAD(P)H oxidase activity / regulation of aerobic respiration / immunoglobulin complex / negative regulation of glycogen catabolic process / regulation of cellular amino acid metabolic process / peptide catabolic process / Signaling by Insulin receptor / IRS activation / nitric oxide-cGMP-mediated signaling / negative regulation of fatty acid metabolic process / Insulin processing / negative regulation of feeding behavior / regulation of protein secretion / amyloid-beta clearance / positive regulation of peptide hormone secretion / Regulation of gene expression in beta cells / positive regulation of respiratory burst / peroxisomal matrix / positive regulation of dendritic spine maintenance / alpha-beta T cell activation / negative regulation of acute inflammatory response / negative regulation of respiratory burst involved in inflammatory response / negative regulation of protein secretion / fatty acid homeostasis / Synthesis, secretion, and deacylation of Ghrelin / positive regulation of glycogen biosynthetic process / positive regulation of lipid biosynthetic process / Signal attenuation / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / negative regulation of gluconeogenesis / positive regulation of nitric oxide mediated signal transduction / regulation of protein localization to plasma membrane / COPI-mediated anterograde transport / amyloid-beta metabolic process / negative regulation of lipid catabolic process / negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway / negative regulation of reactive oxygen species biosynthetic process / positive regulation of insulin receptor signaling pathway / transport vesicle / positive regulation of protein autophosphorylation / Insulin receptor recycling / insulin-like growth factor receptor binding / NPAS4 regulates expression of target genes / positive regulation of protein metabolic process / neuron projection maintenance / endoplasmic reticulum-Golgi intermediate compartment membrane / positive regulation of brown fat cell differentiation / activation of protein kinase B activity / positive regulation of glycolytic process / proteolysis involved in protein catabolic process / Insulin receptor signalling cascade / positive regulation of mitotic nuclear division / Regulation of insulin secretion / positive regulation of nitric-oxide synthase activity / positive regulation of long-term synaptic potentiation / endosome lumen / positive regulation of cytokine production / acute-phase response / positive regulation of protein secretion / regulation of transmembrane transporter activity / Peroxisomal protein import / positive regulation of cell differentiation / peptide binding / positive regulation of glucose import / negative regulation of proteolysis / regulation of synaptic plasticity / protein catabolic process / wound healing / insulin receptor binding / negative regulation of protein catabolic process / positive regulation of neuron projection development / hormone activity / metalloendopeptidase activity / antigen processing and presentation of endogenous peptide antigen via MHC class I / cognition / peroxisome / Golgi lumen / vasodilation / positive regulation of protein localization to nucleus / positive regulation of protein catabolic process / glucose metabolic process / regulation of protein localization / glucose homeostasis / virus receptor activity / cell-cell signaling / insulin receptor signaling pathway / positive regulation of NF-kappaB transcription factor activity / positive regulation of protein binding / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / positive regulation of cell growth / basolateral plasma membrane
Similarity search - Function
Peptidase M16, middle/third domain / Middle or third domain of peptidase_M16 / Peptidase M16, zinc-binding site / Insulinase family, zinc-binding region signature. / Peptidase M16, C-terminal / Peptidase M16 inactive domain / Peptidase M16, N-terminal / Insulinase (Peptidase family M16) / Metalloenzyme, LuxS/M16 peptidase-like / Insulin ...Peptidase M16, middle/third domain / Middle or third domain of peptidase_M16 / Peptidase M16, zinc-binding site / Insulinase family, zinc-binding region signature. / Peptidase M16, C-terminal / Peptidase M16 inactive domain / Peptidase M16, N-terminal / Insulinase (Peptidase family M16) / Metalloenzyme, LuxS/M16 peptidase-like / Insulin / Insulin family / Insulin/IGF/Relaxin family / Insulin, conserved site / Insulin family signature. / Insulin-like / Insulin / insulin-like growth factor / relaxin family. / Insulin-like superfamily / Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin V-set domain / Immunoglobulin subtype / Immunoglobulin / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin C-Type / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Insulin / Immunoglobulin gamma-1 heavy chain / Insulin-degrading enzyme / Ig-like domain-containing protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsZhang Z / Liang WG / Bailey LJ / Tan YZ / Wei H / Kossiakoff AA / Carragher B / Potter SC / Tang WJ
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM81539 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM121964 United States
Simons Foundation349247 United States
CitationJournal: Elife / Year: 2018
Title: Ensemble cryoEM elucidates the mechanism of insulin capture and degradation by human insulin degrading enzyme.
Authors: Zhening Zhang / Wenguang G Liang / Lucas J Bailey / Yong Zi Tan / Hui Wei / Andrew Wang / Mara Farcasanu / Virgil A Woods / Lauren A McCord / David Lee / Weifeng Shang / Rebecca Deprez- ...Authors: Zhening Zhang / Wenguang G Liang / Lucas J Bailey / Yong Zi Tan / Hui Wei / Andrew Wang / Mara Farcasanu / Virgil A Woods / Lauren A McCord / David Lee / Weifeng Shang / Rebecca Deprez-Poulain / Benoit Deprez / David R Liu / Akiko Koide / Shohei Koide / Anthony A Kossiakoff / Sheng Li / Bridget Carragher / Clinton S Potter / Wei-Jen Tang /
Abstract: Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes ...Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity. We also used cryoEM, X-ray crystallography, SAXS, and HDX-MS to elucidate the molecular basis of how amyloidogenic peptides stabilize the disordered IDE catalytic cleft, thereby inducing selective degradation by substrate-assisted catalysis. Furthermore, our insulin-bound IDE structures explain how IDE processively degrades insulin by stochastically cutting either chain without breaking disulfide bonds. Together, our studies provide a mechanism for how IDE selectively degrades amyloidogenic peptides and offers structural insights for developing IDE-based therapies.
History
DepositionOct 3, 2017-
Header (metadata) releaseDec 27, 2017-
Map releaseDec 27, 2017-
UpdateApr 28, 2021-
Current statusApr 28, 2021Processing site: RCSB / Status: Released

-
Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.024
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.024
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6b70
  • Surface level: 0.024
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7062.png

Downloads & links

-
Map

FileDownload / File: emd_7062.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationInsulin degrading enzyme in complex with insulin
Voxel sizeX=Y=Z: 1.073 Å
Density
Contour LevelBy AUTHOR: 0.024 / Movie #1: 0.024
Minimum - Maximum-0.057205748 - 0.14735058
Average (Standard dev.)0.00040003893 (±0.0041737817)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 343.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0731.0731.073
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z343.360343.360343.360
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0570.1470.000

-
Supplemental data

-
Mask #1

Fileemd_7062_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: Insulin degrading enzyme in complex with insulin

Fileemd_7062_half_map_1.map
AnnotationInsulin degrading enzyme in complex with insulin
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: Insulin degrading enzyme in complex with insulin

Fileemd_7062_half_map_2.map
AnnotationInsulin degrading enzyme in complex with insulin
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : Insulin degrading enzyme/Insulin

EntireName: Insulin degrading enzyme/Insulin
Components
  • Complex: Insulin degrading enzyme/Insulin
    • Protein or peptide: Insulin-degrading enzyme
    • Protein or peptide: FAB H11-E heavy chain
    • Protein or peptide: FAB H11-E light chain
    • Protein or peptide: Insulin

-
Supramolecule #1: Insulin degrading enzyme/Insulin

SupramoleculeName: Insulin degrading enzyme/Insulin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Cryo-EM structure of human insulin degrading enzyme in complex with insulin
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL
Molecular weightExperimental: 100 KDa

-
Macromolecule #1: Insulin-degrading enzyme

MacromoleculeName: Insulin-degrading enzyme / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: insulysin
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 111.866484 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: AIKRIGNHIT KSPEDKREYR GLELANGIKV LLISDPTTDK SSAALDVHIG SLSDPPNIAG LSHFLEHMLF LGTKKYPKEN EYSQFLSEH AGSSNAFTSG EHTNYYFDVS HEHLEGALDR FAQFFLSPLF DESAKDREVN AVDSEHEKNV MNDAWRLFQL E KATGNPKH ...String:
AIKRIGNHIT KSPEDKREYR GLELANGIKV LLISDPTTDK SSAALDVHIG SLSDPPNIAG LSHFLEHMLF LGTKKYPKEN EYSQFLSEH AGSSNAFTSG EHTNYYFDVS HEHLEGALDR FAQFFLSPLF DESAKDREVN AVDSEHEKNV MNDAWRLFQL E KATGNPKH PFSKFGTGNK YTLETRPNQE GIDVRQELLK FHSAYYSSNL MAVVVLGRES LDDLTNLVVK LFSEVENKNV PL PEFPEHP FQEEHLKQLY KIVPIKDIRN LYVTFPIPDL QKYYKSNPGH YLGHLIGHEG PGSLLSELKS KGWVNTLVGG QKE GARGFM FFIINVDLTE EGLLHVEDII LHMFQYIQKL RAEGPQEWVF QELKDLNAVA FRFKDKERPR GYTSKIAGIL HYYP LEEVL TAEYLLEEFR PDLIEMVLDK LRPENVRVAI VSKSFEGKTD RTEEWYGTQY KQEAIPDEVI KKWQNADLNG KFKLP TKNE FIPTNFEILP LEKEATPYPA LIKDTAMSKL WFKQDDKFFL PKANLNFEFF SPFAYVDPLH SNMAYLYLEL LKDSLN EYA YAAELAGLSY DLQNTIYGMY LSVKGYNDKQ PILLKKIIEK MATFEIDEKR FEIIKEAYMR SLNNFRAEQP HQHAMYY LR LLMTEVAWTK DELKEALDDV TLPRLKAFIP QLLSRLHIEA LLHGNITKQA ALGIMQMVED TLIEHAHTKP LLPSQLVR Y REVQLPDRGW FVYQQRNEVH NNSGIEIYYQ TDMQSTSENM FLELFAQIIS EPAFNTLRTK EQLGYIVFSG PRRANGIQG LRFIIQSEKP PHYLESRVEA FLITMEKSIE DMTEEAFQKH IQALAIRRLD KPKKLSAESA KYWGEIISQQ YNFDRDNTEV AYLKTLTKE DIIKFYKEML AVDAPRRHKV SVHVLAREMD SCPVVGEFPC QNDINLSQAP ALPQPEVIQN MTEFKRGLPL F PLVKPH

-
Macromolecule #2: FAB H11-E heavy chain

MacromoleculeName: FAB H11-E heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.057748 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EVQLVESGGG LVQPGGSLRL SCAASGFNIS SSSIHWVRQA PGKGLEWVAS IYSYSGSTYY ADSVKGRFTI SADTSKNTAY LQMNSLRAE DTAVYYCARH YSAVAGLDYW GQGTLVTVFN QIKPPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW N SGALTSGV ...String:
EVQLVESGGG LVQPGGSLRL SCAASGFNIS SSSIHWVRQA PGKGLEWVAS IYSYSGSTYY ADSVKGRFTI SADTSKNTAY LQMNSLRAE DTAVYYCARH YSAVAGLDYW GQGTLVTVFN QIKPPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW N SGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP

-
Macromolecule #3: FAB H11-E light chain

MacromoleculeName: FAB H11-E light chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.087609 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD YTLTISSLQP EDFATYYCQ QSYFNPITFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS ...String:
DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD YTLTISSLQP EDFATYYCQ QSYFNPITFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNR

-
Macromolecule #4: Insulin

MacromoleculeName: Insulin / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.989862 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MALWMRLLPL LALLALWGPD PAAAFVNQHL CGSHLVEALY LVCGERGFFY TPKTRREAED LQVGQVELGG GPGAGSLQPL ALEGSLQKR GIVEQCCTSI CSLYQLENYC N

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.8
Component:
ConcentrationFormulaName
20.0 mmol/LC8H18N2O4SHEPES
300.0 mmol/LNaClSodium chlorideSodium chloride
20.0 mmol/LC10H16N2O8EDTAEthylenediaminetetraacetic acid
GridModel: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.001 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 298 K / Instrument: HOMEMADE PLUNGER
Details: The cryo grids were made using Spotiton and homemade plunger.
DetailsThe sample was monodisperse

-
Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 46598 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.9400000000000001 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
Alignment procedureComa free - Residual tilt: 10.0 mrad
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-50 / Number grids imaged: 3 / Number real images: 3085 / Average exposure time: 10.0 sec. / Average electron dose: 71.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

-
Image processing

Particle selectionNumber selected: 762283
CTF correctionSoftware - Name: Gctf (ver. Gctf-v0.50_sm_30_cu7.5_x86_64)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3qz2

Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 2
Software - Name: RELION (ver. 2.0)
Final 3D classificationNumber classes: 8 / Avg.num./class: 18549 / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 2
Software - Name: RELION (ver. 2.0)
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 116122
FSC plot (resolution estimation)

-
Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 92
Output model

PDB-6b70:
Cryo-EM structure of human insulin degrading enzyme in complex with FAB H11-E heavy chain, FAB H11-E light chain and insulin

+
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