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- EMDB-21611: Asymmetric vertex of the clathrin minicoat cage, subparticle refi... -

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Basic information

Entry
Database: EMDB / ID: EMD-21611
TitleAsymmetric vertex of the clathrin minicoat cage, subparticle refinement of the clathrin layer
Map dataSharpened asymmetric vertex
Sample
  • Complex: Natively assembled clathrin coated vesicles
    • Protein or peptide: Clathrin heavy chain 1
    • Protein or peptide: Clathrin light chain B
KeywordsClathrin coated vesicle / clathrin heavy chain / clathrin light chain / clathrin cage / ENDOCYTOSIS
Function / homology
Function and homology information


postsynaptic endocytic zone cytoplasmic component / Retrograde neurotrophin signalling / Recycling pathway of L1 / WNT5A-dependent internalization of FZD4 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / Gap junction degradation / Formation of annular gap junctions / Golgi Associated Vesicle Biogenesis / clathrin vesicle coat ...postsynaptic endocytic zone cytoplasmic component / Retrograde neurotrophin signalling / Recycling pathway of L1 / WNT5A-dependent internalization of FZD4 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / Gap junction degradation / Formation of annular gap junctions / Golgi Associated Vesicle Biogenesis / clathrin vesicle coat / RHOU GTPase cycle / RHOV GTPase cycle / clathrin coat of trans-Golgi network vesicle / Lysosome Vesicle Biogenesis / clathrin light chain binding / negative regulation of hyaluronan biosynthetic process / clathrin complex / clathrin coat / MHC class II antigen presentation / VLDLR internalisation and degradation / clathrin heavy chain binding / clathrin coat of coated pit / Cargo recognition for clathrin-mediated endocytosis / clathrin coat disassembly / clathrin coat assembly / clathrin-coated endocytic vesicle / membrane coat / Clathrin-mediated endocytosis / clathrin-dependent endocytosis / arrestin family protein binding / receptor-mediated endocytosis / intracellular protein transport / synaptic vesicle membrane / autophagy / spindle / disordered domain specific binding / melanosome / mitotic cell cycle / protein domain specific binding / cell division / structural molecule activity / mitochondrion / extracellular exosome / identical protein binding / plasma membrane
Similarity search - Function
Clathrin light chain / Clathrin light chain signature 1. / Clathrin light chain signature 2. / Clathrin light chain / Clathrin, heavy chain, linker, core motif / Clathrin heavy chain, N-terminal / Clathrin, heavy chain / Clathrin, heavy chain, propeller repeat / Clathrin propeller repeat / Clathrin, heavy-chain linker ...Clathrin light chain / Clathrin light chain signature 1. / Clathrin light chain signature 2. / Clathrin light chain / Clathrin, heavy chain, linker, core motif / Clathrin heavy chain, N-terminal / Clathrin, heavy chain / Clathrin, heavy chain, propeller repeat / Clathrin propeller repeat / Clathrin, heavy-chain linker / Clathrin-H-link / Region in Clathrin and VPS / Clathrin heavy chain repeat homology / Clathrin, heavy chain/VPS, 7-fold repeat / Clathrin heavy-chain (CHCR) repeat profile. / Tetratricopeptide-like helical domain superfamily / Armadillo-type fold
Similarity search - Domain/homology
Clathrin light chain B / Clathrin heavy chain 1
Similarity search - Component
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.3 Å
AuthorsParaan M / Mendez J / Sharum S / Kurtin D / He H / Stagg S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM108753 United States
CitationJournal: Sci Adv / Year: 2020
Title: The structures of natively assembled clathrin-coated vesicles.
Authors: Mohammadreza Paraan / Joshua Mendez / Savanna Sharum / Danielle Kurtin / Huan He / Scott M Stagg /
Abstract: Clathrin-coated vesicles mediate trafficking of proteins and nutrients in the cell and between organelles. Proteins included in the clathrin-coated vesicles (CCVs) category include clathrin heavy ...Clathrin-coated vesicles mediate trafficking of proteins and nutrients in the cell and between organelles. Proteins included in the clathrin-coated vesicles (CCVs) category include clathrin heavy chain (CHC), clathrin light chain (CLC), and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here, we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2 appendages crosslink adjacent CHC β-propellers and that the appendage densities are enriched in CCV hexagonal faces. We resolve how adaptor protein 2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.
History
DepositionMar 30, 2020-
Header (metadata) releaseApr 8, 2020-
Map releaseAug 19, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wcj
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6wcj
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21611.png

Downloads & links

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Map

FileDownload / File: emd_21611.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened asymmetric vertex
Voxel sizeX=Y=Z: 1.323 Å
Density
Contour LevelBy AUTHOR: 4.0 / Movie #1: 5
Minimum - Maximum-9.133832999999999 - 19.386827
Average (Standard dev.)0.008423435 (±0.23832925)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-300-300-300
Dimensions600600600
Spacing600600600
CellA=B=C: 793.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3231.3231.323
M x/y/z600600600
origin x/y/z0.0000.0000.000
length x/y/z793.800793.800793.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-205-205-205
NX/NY/NZ411411411
MAP C/R/S123
start NC/NR/NS-300-300-300
NC/NR/NS600600600
D min/max/mean-9.13419.3870.008

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Supplemental data

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Mask #1

Fileemd_21611_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Nonsharpened asymmetric vertex

Fileemd_21611_additional.map
AnnotationNonsharpened asymmetric vertex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unmasked half map

Fileemd_21611_half_map_1.map
AnnotationUnmasked half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unmasked half map

Fileemd_21611_half_map_2.map
AnnotationUnmasked half 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 : Natively assembled clathrin coated vesicles

EntireName: Natively assembled clathrin coated vesicles
Components
  • Complex: Natively assembled clathrin coated vesicles
    • Protein or peptide: Clathrin heavy chain 1
    • Protein or peptide: Clathrin light chain B

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Supramolecule #1: Natively assembled clathrin coated vesicles

SupramoleculeName: Natively assembled clathrin coated vesicles / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bos taurus (cattle) / Organ: brain

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Macromolecule #1: Clathrin heavy chain 1

MacromoleculeName: Clathrin heavy chain 1 / type: protein_or_peptide / ID: 1 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 191.800312 KDa
SequenceString: MAQILPIRFQ EHLQLQNLGI NPANIGFSTL TMESDKFICI REKVGEQAQV VIIDMNDPSN PIRRPISADS AIMNPASKVI ALKAGKTLQ IFNIEMKSKM KAHTMTDDVT FWKWISLNTV ALVTDNAVYH WSMEGESQPV KMFDRHSSLA GCQIINYRTD A KQKWLLLT ...String:
MAQILPIRFQ EHLQLQNLGI NPANIGFSTL TMESDKFICI REKVGEQAQV VIIDMNDPSN PIRRPISADS AIMNPASKVI ALKAGKTLQ IFNIEMKSKM KAHTMTDDVT FWKWISLNTV ALVTDNAVYH WSMEGESQPV KMFDRHSSLA GCQIINYRTD A KQKWLLLT GISAQQNRVV GAMQLYSVDR KVSQPIEGHA ASFAQFKMEG NAEESTLFCF AVRGQAGGKL HIIEVGTPPT GN QPFPKKA VDVFFPPEAQ NDFPVAMQIS EKHDVVFLIT KYGYIHLYDL ETGTCIYMNR ISGETIFVTA PHEATAGIIG VNR KGQVLS VCVEEENIIP YITNVLQNPD LALRMAVRNN LAGAEELFAR KFNALFAQGN YSEAAKVAAN APKGILRTPD TIRR FQSVP AQPGQTSPLL QYFGILLDQG QLNKYESLEL CRPVLQQGRK QLLEKWLKED KLECSEELGD LVKSVDPTLA LSVYL RANV PNKVIQCFAE TGQVQKIVLY AKKVGYTPDW IFLLRNVMRI SPDQGQQFAQ MLVQDEEPLA DITQIVDVFM EYNLIQ QCT AFLLDALKNN RPSEGPLQTR LLEMNLMHAP QVADAILGNQ MFTHYDRAHI AQLCEKAGLL QRALEHFTDL YDIKRAV VH THLLNPEWLV NYFGSLSVED SLECLRAMLS ANIRQNLQIC VQVASKYHEQ LSTQSLIELF ESFKSFEGLF YFLGSIVN F SQDPDVHFKY IQAACKTGQI KEVERICRES NCYDPERVKN FLKEAKLTDQ LPLIIVCDRF DFVHDLVLYL YRNNLQKYI EIYVQKVNPS RLPVVIGGLL DVDCSEDVIK NLILVVRGQF STDELVAEVE KRNRLKLLLP WLEARIHEGC EEPATHNALA KIYIDSNNN PERFLRENPY YDSRVVGKYC EKRDPHLACV AYERGQCDLE LINVCNENSL FKSLSRYLVR RKDPELWGSV L LESNPYRR PLIDQVVQTA LSETQDPEEV SVTVKAFMTA DLPNELIELL EKIVLDNSVF SEHRNLQNLL ILTAIKADRT RV MEYINRL DNYDAPDIAN IAISNELFEE AFAIFRKFDV NTSAVQVLIE HIGNLDRAYE FAERCNEPAV WSQLAKAQLQ KGM VKEAID SYIKADDPSS YMEVVQAANT SGNWEELVKY LQMARKKARE SYVETELIFA LAKTNRLAEL EEFINGPNNA HIQQ VGDRC YDEKMYDAAK LLYNNVSNFG RLASTLVHLG EYQAAVDGAR KANSTRTWKE VCFACVDGKE FRLAQMCGLH IVVHA DELE ELINYYQDRG YFEELITMLE AALGLERAHM GMFTELAILY SKFKPQKMRE HLELFWSRVN IPKVLRAAEQ AHLWAE LVF LYDKYEEYDN AIITMMNHPT DAWKEGQFKD IITKVANVEL YYRAIQFYLE FKPLLLNDLL MVLSPRLDHT RAVNYFS KV KQLPLVKPYL RSVQNHNNKS VNESLNNLFI TEEDYQALRT SIDAYDNFDN ISLAQRLEKH ELIEFRRIAA YLFKGNNR W KQSVELCKKD SLYKDAMQYA SESKDTELAE ELLQWFLQEE KRECFGACLF TCYDLLRPDV VLETAWRHNI MDFAMPYFI QVMKEYLTKV DKLDASESLR KEEEQATETQ PIVYGQPQLM LTAGPSVAVP PQAPFGYGYT APAYGQPQPG FGYSM

UniProtKB: Clathrin heavy chain 1

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Macromolecule #2: Clathrin light chain B

MacromoleculeName: Clathrin light chain B / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 25.109396 KDa
SequenceString: MADDFGFFSS SESGAPEAAE EDPAAAFLAQ QESEIAGIEN DEGFGAPAGS QGGLAQPGPA SGASEDMGAT VNGDVFQEAN GPADGYAAI AQADRLTQEP ESIRKWREEQ RKRLQELDAA SKVMEQEWRE KAKKDLEEWN QRQSEQVEKN KINNRIADKA F YQQPDADI ...String:
MADDFGFFSS SESGAPEAAE EDPAAAFLAQ QESEIAGIEN DEGFGAPAGS QGGLAQPGPA SGASEDMGAT VNGDVFQEAN GPADGYAAI AQADRLTQEP ESIRKWREEQ RKRLQELDAA SKVMEQEWRE KAKKDLEEWN QRQSEQVEKN KINNRIADKA F YQQPDADI IGYVASEEAF VKESKEETPG TEWEKVAQLC DFNPKSSKQC KDVSRLRSVL MSLKQTPLSR

UniProtKB: Clathrin light chain B

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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

Concentration6 mg/mL
BufferpH: 6.7
Component:
ConcentrationNameFormula
100.0 mMMES
1.0 mMMagnesium ChlorideMgCl2
1.0 mMEGTA
GridModel: C-flat-2/2 4C
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 800 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 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: 5.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 35000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 360000
Details: These are subparticle images that were extracted from particle images. Each minicoat particle has 24 asymmetric vertices. From a total of 15000 minicoat particles, 360000 vertex subparticles ...Details: These are subparticle images that were extracted from particle images. Each minicoat particle has 24 asymmetric vertices. From a total of 15000 minicoat particles, 360000 vertex subparticles were extracted. The position of subparticles was calculated by the localized reconstruction script in scipion, and the subparticle images were extracted with RELION extraction tools.
Startup modelType of model: OTHER
Details: An asymmetric vertex was cut out of the minicoat cage map from the same dataset.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1)
Details: After the final refinement in cisTEM, half-maps were generated in cisTEM and the FSC was calculated with EMAN FSC tools.
Number images used: 305413
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 2)
Details: Using the Euler angles of the mini-coat cage particles and the position of the pentagonal faces in the map, initial hexagonal face Eulers were calculated. Using 2D classification and ...Details: Using the Euler angles of the mini-coat cage particles and the position of the pentagonal faces in the map, initial hexagonal face Eulers were calculated. Using 2D classification and homogenous refinement tools of cryoSPARC shifts, Euler angles were refined for binned-by-2 subparticle images.
Final angle assignmentType: PROJECTION MATCHING / Software: (Name: cisTEM (ver. 1), RELION (ver. 3))
Details: The output of cryoSPARC was imported into cisTEM for unbinned refinement. Then, the initial output of cisTEM was used for RELION's per particle defocus estimation and beam tilt estimation. ...Details: The output of cryoSPARC was imported into cisTEM for unbinned refinement. Then, the initial output of cisTEM was used for RELION's per particle defocus estimation and beam tilt estimation. This was done step-by-step and after each estimation the map was refined in RELION. After defocus and beam tilt estimation with RELION converged, the vertex map was refined in cisTEM one last time.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6sct

chain_id: A, residue_range: 1627-1641, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: C, residue_range: 1627-1641, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: D, residue_range: 1627-1641, source_name: PDB, initial_model_type: experimental model
RefinementProtocol: AB INITIO MODEL
Output model

PDB-6wcj:
Asymmetric vertex of the clathrin minicoat cage

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Atomic model buiding 2

Initial model
PDB IDChain

6sct

chain_id: B, residue_range: 188-228, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: E, residue_range: 188-228, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: F, residue_range: 188-228, source_name: PDB, initial_model_type: experimental model
RefinementProtocol: AB INITIO MODEL
Output model

PDB-6wcj:
Asymmetric vertex of the clathrin minicoat cage

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Atomic model buiding 3

Initial model
PDB IDChain

6sct

chain_id: A, residue_range: 1248-1626, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: C, residue_range: 1248-1626, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: D, residue_range: 1248-1626, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: B, residue_range: 99-166, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: E, residue_range: 99-166, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: F, residue_range: 99-166, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: O, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: L, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: M, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: J, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: G, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: H, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: N, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: I, source_name: PDB, initial_model_type: experimental model

6sct

chain_id: K, source_name: PDB, initial_model_type: experimental model
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-6wcj:
Asymmetric vertex of the clathrin minicoat cage

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