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- EMDB-21927: Structure of human Frizzled5 by fiducial-assisted cryo-EM -

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Basic information

Entry
Database: EMDB / ID: EMD-21927
TitleStructure of human Frizzled5 by fiducial-assisted cryo-EM
Map dataSharpen map of the Fzd5-BRIL/Fab/Nb complex.
Sample
  • Complex: Fzd5-BRIL/Fab/Nb complex
    • Complex: anti-BRIL Fab
      • Protein or peptide: anti-BRIL Fab Heavy chain
      • Protein or peptide: anti-BRIL Fab Light chain
    • Complex: anti-Fab Nanobody
      • Protein or peptide: anti-Fab Nanobody
    • Complex: Frizzled-5
      • Protein or peptide: Frizzled-5,Soluble cytochrome b562
Function / homology
Function and homology information


regulation of chorionic trophoblast cell proliferation / Spemann organizer formation / cellular response to molecule of bacterial origin / syncytiotrophoblast cell differentiation involved in labyrinthine layer development / chorionic trophoblast cell differentiation / embryonic camera-type eye morphogenesis / glandular epithelial cell maturation / Signaling by RNF43 mutants / post-embryonic camera-type eye development / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 ...regulation of chorionic trophoblast cell proliferation / Spemann organizer formation / cellular response to molecule of bacterial origin / syncytiotrophoblast cell differentiation involved in labyrinthine layer development / chorionic trophoblast cell differentiation / embryonic camera-type eye morphogenesis / glandular epithelial cell maturation / Signaling by RNF43 mutants / post-embryonic camera-type eye development / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / apoptotic process involved in morphogenesis / anterior/posterior axis specification, embryo / embryonic axis specification / Wnt receptor activity / regulation of mitophagy / non-canonical Wnt signaling pathway / intestinal epithelial cell maturation / Wnt-protein binding / branching involved in labyrinthine layer morphogenesis / Class B/2 (Secretin family receptors) / regulation of bicellular tight junction assembly / Disassembly of the destruction complex and recruitment of AXIN to the membrane / labyrinthine layer blood vessel development / positive regulation of protein targeting to mitochondrion / canonical Wnt signaling pathway / bicellular tight junction / synapse assembly / Regulation of FZD by ubiquitination / positive regulation of interleukin-1 beta production / Asymmetric localization of PCP proteins / G protein-coupled receptor activity / clathrin-coated endocytic vesicle membrane / neuron differentiation / positive regulation of T cell cytokine production / positive regulation of type II interferon production / positive regulation of tumor necrosis factor production / Ca2+ pathway / T cell differentiation in thymus / amyloid-beta binding / perikaryon / early endosome membrane / angiogenesis / electron transfer activity / periplasmic space / iron ion binding / axon / negative regulation of cell population proliferation / Golgi membrane / dendrite / synapse / lipid binding / ubiquitin protein ligase binding / heme binding / protein-containing complex binding / protein kinase binding / perinuclear region of cytoplasm / cell surface / positive regulation of transcription by RNA polymerase II / plasma membrane
Similarity search - Function
Frizzled-5, CRD domain / Frizzled/Smoothened, transmembrane domain / Frizzled/Smoothened family membrane region / Frizzled/Smoothened family membrane region / Frizzled/secreted frizzled-related protein / Frizzled / Frizzled domain / Frizzled cysteine-rich domain superfamily / Fz domain / Frizzled (fz) domain profile. ...Frizzled-5, CRD domain / Frizzled/Smoothened, transmembrane domain / Frizzled/Smoothened family membrane region / Frizzled/Smoothened family membrane region / Frizzled/secreted frizzled-related protein / Frizzled / Frizzled domain / Frizzled cysteine-rich domain superfamily / Fz domain / Frizzled (fz) domain profile. / GPCR, family 2-like / G-protein coupled receptors family 2 profile 2. / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562
Similarity search - Domain/homology
Soluble cytochrome b562 / Frizzled-5
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsTsutsumi N / Jude KM / Gati C / Garcia KC
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)1R01DK115728 United States
Howard Hughes Medical Institute (HHMI) United States
Department of Energy (DOE, United States)DE-AC02-76SF00515 United States
CitationJournal: Elife / Year: 2020
Title: Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling.
Authors: Naotaka Tsutsumi / Somnath Mukherjee / Deepa Waghray / Claudia Y Janda / Kevin M Jude / Yi Miao / John S Burg / Nanda Gowtham Aduri / Anthony A Kossiakoff / Cornelius Gati / K Christopher Garcia /
Abstract: Frizzleds (Fzd) are the primary receptors for Wnt morphogens, which are essential regulators of stem cell biology, yet the structural basis of Wnt signaling through Fzd remains poorly understood. ...Frizzleds (Fzd) are the primary receptors for Wnt morphogens, which are essential regulators of stem cell biology, yet the structural basis of Wnt signaling through Fzd remains poorly understood. Here we report the structure of an unliganded human Fzd5 determined by single-particle cryo-EM at 3.7 Å resolution, with the aid of an antibody chaperone acting as a fiducial marker. We also analyzed the topology of low-resolution XWnt8/Fzd5 complex particles, which revealed extreme flexibility between the Wnt/Fzd-CRD and the Fzd-TM regions. Analysis of Wnt/β-catenin signaling in response to Wnt3a versus a 'surrogate agonist' that cross-links Fzd to LRP6, revealed identical structure-activity relationships. Thus, canonical Wnt/β-catenin signaling appears to be principally reliant on ligand-induced Fzd/LRP6 heterodimerization, versus the allosteric mechanisms seen in structurally analogous class A G protein-coupled receptors, and Smoothened. These findings deepen our mechanistic understanding of Wnt signal transduction, and have implications for harnessing Wnt agonism in regenerative medicine.
History
DepositionMay 7, 2020-
Header (metadata) releaseAug 19, 2020-
Map releaseAug 19, 2020-
UpdateAug 19, 2020-
Current statusAug 19, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ww2
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6ww2
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21927.png

Downloads & links

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Map

FileDownload / File: emd_21927.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpen map of the Fzd5-BRIL/Fab/Nb complex.
Voxel sizeX=Y=Z: 1.078 Å
Density
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.4
Minimum - Maximum-0.8844181 - 2.6527205
Average (Standard dev.)0.0011502128 (±0.03884794)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 344.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0781.0781.078
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z344.960344.960344.960
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ401401401
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.8842.6530.001

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Supplemental data

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Additional map: Unsharpen map of the Fzd5-BRIL/Fab/Nb complex.

Fileemd_21927_additional.map
AnnotationUnsharpen map of the Fzd5-BRIL/Fab/Nb complex.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Fzd5-BRIL/Fab/Nb complex

EntireName: Fzd5-BRIL/Fab/Nb complex
Components
  • Complex: Fzd5-BRIL/Fab/Nb complex
    • Complex: anti-BRIL Fab
      • Protein or peptide: anti-BRIL Fab Heavy chain
      • Protein or peptide: anti-BRIL Fab Light chain
    • Complex: anti-Fab Nanobody
      • Protein or peptide: anti-Fab Nanobody
    • Complex: Frizzled-5
      • Protein or peptide: Frizzled-5,Soluble cytochrome b562

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Supramolecule #1: Fzd5-BRIL/Fab/Nb complex

SupramoleculeName: Fzd5-BRIL/Fab/Nb complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human) / Location in cell: Membrane
Molecular weightTheoretical: 100 KDa

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Supramolecule #2: anti-BRIL Fab

SupramoleculeName: anti-BRIL Fab / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #3
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293S GnTi-

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Supramolecule #3: anti-Fab Nanobody

SupramoleculeName: anti-Fab Nanobody / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Recombinant expressionOrganism: synthetic construct (others)

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Supramolecule #4: Frizzled-5

SupramoleculeName: Frizzled-5 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: anti-BRIL Fab Heavy chain

MacromoleculeName: anti-BRIL Fab Heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.321084 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK S

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Macromolecule #2: anti-Fab Nanobody

MacromoleculeName: anti-Fab Nanobody / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.390644 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSQVQLQESG GGLVQPGGSL RLSCAASGRT ISRYAMSWFR QAPGKEREFV AVARRSGDGA FYADSVQGRF TVSRDDAKNT VYLQMNSLK PEDTAVYYCA IDSDTFYSGS YDYWGQGTQV TVSS

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Macromolecule #3: anti-BRIL Fab Light chain

MacromoleculeName: anti-BRIL Fab Light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.353947 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYLYYSLVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYLYYSLVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRG

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Macromolecule #4: Frizzled-5,Soluble cytochrome b562

MacromoleculeName: Frizzled-5,Soluble cytochrome b562 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 74.75418 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DYKDDDDASK APVCQEITVP MCRGIGYNLT HMPNQFNHDT QDEAGLEVHQ FWPLVEIQCS PDLRFFLCSM YTPICLPDYH KPLPPCRSV CERAKAGCSP LMRQYGFAWP ERMSCDRLPV LGRDAEVLCM DYNRSEATTA PPRPFPAKPT LPGPPGAPAS G GECPAGGP ...String:
DYKDDDDASK APVCQEITVP MCRGIGYNLT HMPNQFNHDT QDEAGLEVHQ FWPLVEIQCS PDLRFFLCSM YTPICLPDYH KPLPPCRSV CERAKAGCSP LMRQYGFAWP ERMSCDRLPV LGRDAEVLCM DYNRSEATTA PPRPFPAKPT LPGPPGAPAS G GECPAGGP FVCKCREPFV PILKESHPLY NKVRTGQVPN CAVPCYQPSF SADERTFATF WIGLWSVLCF ISTSTTVATF LI DMERFRY PERPIIFLSA CYLCVSLGFL VRLVVGHASV ACSREHNHIH YETTGPALCT IVFLLVYFFG MASSIWWVIL SLT WFLAAG MKWGNEAIAG YAQYFHLAAW LIPSVKSITA LALSSVDGDP VAGICYVGNQ NLNSLRGFVL GPLVLYLLVG TLFL LAGFV SLFRARRQLA DLEDNWETLN DNLKVIEKAD NAAQVKDALT KMRAAALDAQ KATPPKLEDK SPDSPEMKDF RHGFD ILVG QIDDALKLAN EGKVKEAQAA AEQLKTTRNA YIQKYLERAR STLDKLEKLM IRIGIFTLLY TVPASIVVAC YLYEQH YRE SWEAALTCAC PGHDTGQPRA KPEYWVLMLK YFMCLVVGIT SGVWIWSGKT VESWRRFTSR CCCRPRRGHK AAALEVL FQ GPGAAEDQVD PRLIDGKHHH HHHHH

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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

Concentration8 mg/mL
BufferpH: 7.2
Component:
ConcentrationName
10.0 mMHEPES
150.0 mMSodium chloride
0.001 % (w/v)GDN
0.0001 % (w/v)CHS
0.05 % (w/v)Digitonin
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 281 K / Instrument: LEICA EM GP / Details: 5s blotting.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 81000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -2.0 µm / Nominal defocus min: -0.8 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 10898 / Average exposure time: 0.1225 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6483398
CTF correctionSoftware - Name: cryoSPARC (ver. 2.12.14)
Startup modelType of model: OTHER / Details: Initial model generation in cryoSPARC.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.12.14)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 2.12.14)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.12.14)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.12.14) / Number images used: 207021

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6ww2:
Structure of human Frizzled5 by fiducial-assisted cryo-EM

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