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- EMDB-11869: Structure of the actin filament Arp2/3 complex branch junction in... -

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Basic information

Entry
Database: EMDB / ID: EMD-11869
TitleStructure of the actin filament Arp2/3 complex branch junction in cells
Map dataStructure of the actin filament Arp2/3 complex branch junction
Sample
  • Cell: Actin Filament Arp2/3 Complex Branch Junction
    • Protein or peptide: Actin-related protein 2, Arp2
    • Protein or peptide: Actin-related protein 3, Arp3
    • Protein or peptide: Actin-related protein 2/3 complex subunit 1b, ArpC1b
    • Protein or peptide: Actin-related protein 2/3 complex subunit 2, ArpC2
    • Protein or peptide: Actin-related protein 2/3 complex subunit 3, ArpC3
    • Protein or peptide: Actin-related protein 2/3 complex subunit 4, ArpC4
    • Protein or peptide: Actin-related protein 2/3 complex subunit 5, ArpC5
    • Protein or peptide: Actin, alpha skeletal muscle, ACTA1
Function / homology
Function and homology information


podosome core / concave side of sperm head / actin filament branch point / ventral surface of cell / microtubule organizing center localization / negative regulation of bleb assembly / positive regulation of barbed-end actin filament capping / apical tubulobulbar complex / tubulobulbar complex / regulation of myosin II filament organization ...podosome core / concave side of sperm head / actin filament branch point / ventral surface of cell / microtubule organizing center localization / negative regulation of bleb assembly / positive regulation of barbed-end actin filament capping / apical tubulobulbar complex / tubulobulbar complex / regulation of myosin II filament organization / meiotic chromosome movement towards spindle pole / cytosolic transport / growth cone leading edge / peripheral region of growth cone / : / meiotic cytokinesis / muscle cell projection membrane / apical ectoplasmic specialization / basal ectoplasmic specialization / cellular response to rapamycin / lamellipodium organization / spindle localization / skeletal muscle fiber adaptation / cellular response to trichostatin A / leading edge of lamellipodium / Striated Muscle Contraction / EPHB-mediated forward signaling / hemidesmosome / RHO GTPases Activate WASPs and WAVEs / Arp2/3 protein complex / asymmetric cell division / protein kinase C signaling / Arp2/3 complex-mediated actin nucleation / actin filament network formation / podosome ring / orbitofrontal cortex development / actin cap / postsynaptic actin cytoskeleton organization / negative regulation of axon extension / Regulation of actin dynamics for phagocytic cup formation / positive regulation of dendritic spine morphogenesis / maintenance of cell polarity / positive regulation of astrocyte differentiation / Clathrin-mediated endocytosis / cellular response to organonitrogen compound / apical dendrite / response to extracellular stimulus / astrocyte differentiation / podosome / positive regulation of fibroblast migration / positive regulation of synapse assembly / positive regulation of podosome assembly / response to steroid hormone / positive regulation of dendrite morphogenesis / positive regulation of filopodium assembly / smooth muscle cell migration / establishment or maintenance of cell polarity / positive regulation of double-strand break repair via homologous recombination / positive regulation of actin filament polymerization / mesenchyme migration / cellular response to platelet-derived growth factor stimulus / positive regulation of smooth muscle cell migration / cell leading edge / filamentous actin / skeletal muscle thin filament assembly / brush border / associative learning / striated muscle thin filament / excitatory synapse / cilium assembly / positive regulation of protein targeting to membrane / response to immobilization stress / glutamate receptor binding / skeletal muscle fiber development / positive regulation of lamellipodium assembly / axon terminus / response to mechanical stimulus / positive regulation of substrate adhesion-dependent cell spreading / stress fiber / cellular response to epidermal growth factor stimulus / ruffle / cellular response to transforming growth factor beta stimulus / positive regulation of neuron differentiation / cytoskeletal protein binding / actin filament polymerization / Neutrophil degranulation / sarcomere / cellular response to nerve growth factor stimulus / dendritic shaft / filopodium / meiotic cell cycle / secretory granule / cell projection / actin filament / positive regulation of protein localization to plasma membrane / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / ruffle membrane / cellular response to type II interferon / actin filament binding
Similarity search - Function
Actin-related protein 3 / Actin-related protein 2/3 complex subunit 1B / Actin-related protein 2/3 complex subunit 5 / Actin-related protein 2/3 complex subunit 2 / Actin-related protein 2/3 complex subunit 3 / Actin-related protein 2/3 complex subunit 4 / Actin-related protein 2/3 complex subunit 1 / Arp2/3 complex subunit 2/4 / Actin-related protein 2/3 complex subunit 5 superfamily / Actin-related protein 2/3 complex subunit 3 superfamily ...Actin-related protein 3 / Actin-related protein 2/3 complex subunit 1B / Actin-related protein 2/3 complex subunit 5 / Actin-related protein 2/3 complex subunit 2 / Actin-related protein 2/3 complex subunit 3 / Actin-related protein 2/3 complex subunit 4 / Actin-related protein 2/3 complex subunit 1 / Arp2/3 complex subunit 2/4 / Actin-related protein 2/3 complex subunit 5 superfamily / Actin-related protein 2/3 complex subunit 3 superfamily / Arp2/3 complex, 34 kD subunit p34-Arc / ARP2/3 complex ARPC3 (21 kDa) subunit / ARP2/3 complex 16 kDa subunit (p16-Arc) / ARP2/3 complex 20 kDa subunit (ARPC4) / : / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Actin-related protein 2/3 complex subunit 4 / Actin-related protein 2 / Actin, alpha skeletal muscle / Actin-related protein 3 / Actin-related protein 2/3 complex subunit 5 / Actin-related protein 2/3 complex subunit 2 / Actin-related protein 2/3 complex subunit 3 / Actin-related protein 2/3 complex subunit 1B
Similarity search - Component
Biological speciesMus musculus (house mouse) / House mouse (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 9.0 Å
AuthorsFaessler F / Dimchev G / Hodirnau VV / Wan W / Schur FKM
Funding support Austria, 1 items
OrganizationGrant numberCountry
Austrian Science FundP33367 Austria
CitationJournal: Nat Commun / Year: 2020
Title: Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction.
Authors: Florian Fäßler / Georgi Dimchev / Victor-Valentin Hodirnau / William Wan / Florian K M Schur /
Abstract: The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves ...The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0 Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Notably, our model reveals a previously undescribed set of interactions of the Arp2/3 complex with the mother filament, significantly different to the previous branch junction model. Our structure also indicates a central role for the ArpC3 subunit in stabilizing the active conformation.
History
DepositionOct 22, 2020-
Header (metadata) releaseDec 2, 2020-
Map releaseDec 2, 2020-
UpdateJan 6, 2021-
Current statusJan 6, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00404
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.00404
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7aqk
  • Surface level: 0.00404
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7aqk
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11869.png

Downloads & links

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Map

FileDownload / File: emd_11869.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the actin filament Arp2/3 complex branch junction
Voxel sizeX=Y=Z: 2.137 Å
Density
Contour LevelBy AUTHOR: 0.00404 / Movie #1: 0.00404
Minimum - Maximum-0.0034114548 - 0.014956028
Average (Standard dev.)3.46011e-05 (±0.00058454473)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 512.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.1372.1372.137
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z512.880512.880512.880
α/β/γ90.00090.00090.000
start NX/NY/NZ79740
NX/NY/NZ93103213
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0030.0150.000

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Supplemental data

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Mask #1

Fileemd_11869_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_11869_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_11869_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Actin Filament Arp2/3 Complex Branch Junction

EntireName: Actin Filament Arp2/3 Complex Branch Junction
Components
  • Cell: Actin Filament Arp2/3 Complex Branch Junction
    • Protein or peptide: Actin-related protein 2, Arp2
    • Protein or peptide: Actin-related protein 3, Arp3
    • Protein or peptide: Actin-related protein 2/3 complex subunit 1b, ArpC1b
    • Protein or peptide: Actin-related protein 2/3 complex subunit 2, ArpC2
    • Protein or peptide: Actin-related protein 2/3 complex subunit 3, ArpC3
    • Protein or peptide: Actin-related protein 2/3 complex subunit 4, ArpC4
    • Protein or peptide: Actin-related protein 2/3 complex subunit 5, ArpC5
    • Protein or peptide: Actin, alpha skeletal muscle, ACTA1

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Supramolecule #1: Actin Filament Arp2/3 Complex Branch Junction

SupramoleculeName: Actin Filament Arp2/3 Complex Branch Junction / type: cell / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Structure obtained from the actin network of extracted and fixed mouse fibroblast lamellipodia
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Actin-related protein 2, Arp2

MacromoleculeName: Actin-related protein 2, Arp2 / type: protein_or_peptide / ID: 1
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: House mouse (house mouse)
Molecular weightTheoretical: 44.818711 KDa
SequenceString: MDSQGRKVVV CDNGTGFVKC GYAGSNFPEH IFPALVGRPI IRSTTKVGNI EIKDLMVGDE ASELRSMLEV NYPMENGIVR NWDDMKHLW DYTFGPEKLN IDTRNCKILL TEPPMNPTKN REKIVEVMFE TYQFSGVYVA IQAVLTLYAQ GLLTGVVVDS G DGVTHICP ...String:
MDSQGRKVVV CDNGTGFVKC GYAGSNFPEH IFPALVGRPI IRSTTKVGNI EIKDLMVGDE ASELRSMLEV NYPMENGIVR NWDDMKHLW DYTFGPEKLN IDTRNCKILL TEPPMNPTKN REKIVEVMFE TYQFSGVYVA IQAVLTLYAQ GLLTGVVVDS G DGVTHICP VYEGFSLPHL TRRLDIAGRD ITRYLIKLLL LRGYAFNHSA DFETVRMIKE KLCYVGYNIE QEQKLALETT VL VESYTLP DGRIIKVGGE RFEAPEALFQ PHLINVEGVG VAELLFNTIQ AADIDTRSEF YKHIVLSGGS TMYPGLPSRL ERE LKQLYL ERVLKGDVEK LSKFKIRIED PPRRKHMVFL GGAVLADIMK DKDNFWMTRQ EYQEKGVRVL EKLGVTVR

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Macromolecule #2: Actin-related protein 3, Arp3

MacromoleculeName: Actin-related protein 3, Arp3 / type: protein_or_peptide / ID: 2
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: House mouse (house mouse)
Molecular weightTheoretical: 47.428031 KDa
SequenceString: MAGRLPACVV DCGTGYTKLG YAGNTEPQFI IPSCIAIKES AKVGDQAQRR VMKGVDDLDF FIGDEAIEKP TYATKWPIRH GIVEDWDLM ERFMEQVIFK YLRAEPEDHY FLLTEPPLNT PENREYTAEI MFESFNVPGL YIAVQAVLAL AASWTSRQVG E RTLTGTVI ...String:
MAGRLPACVV DCGTGYTKLG YAGNTEPQFI IPSCIAIKES AKVGDQAQRR VMKGVDDLDF FIGDEAIEKP TYATKWPIRH GIVEDWDLM ERFMEQVIFK YLRAEPEDHY FLLTEPPLNT PENREYTAEI MFESFNVPGL YIAVQAVLAL AASWTSRQVG E RTLTGTVI DSGDGVTHVI PVAEGYVIGS CIKHIPIAGR DITYFIQQLL RDREVGIPPE QSLETAKAVK ERYSYVCPDL VK EFNKYDT DGSKWIKQYT GINAISKKEF SIDVGYERFL GPEIFFHPEF ANPDFTQPIS EVVDEVIQNC PIDVRRPLYK NIV LSGGST MFRDFGRRLQ RDLKRTVDAR LKLSEELSGG RLKPKPIDVQ VITHHMQRYA VWFGGSMLAS TPEFYQVCHT KKDY EEIGP SICRHNPVFG VMS

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Macromolecule #3: Actin-related protein 2/3 complex subunit 1b, ArpC1b

MacromoleculeName: Actin-related protein 2/3 complex subunit 1b, ArpC1b / type: protein_or_peptide / ID: 3
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: House mouse (house mouse)
Molecular weightTheoretical: 41.016738 KDa
SequenceString: MAYHSFLVEP ISCHAWNKDR TQIAICPNNH EVHIYEKSGN KWVQVHELKE HNGQVTGVDW APDSNRIVTC GTDRNAYVWT LKGRTWKPT LVILRINRAA RCVRWAPNEK KFAVGSGSRV ISICYFEQEN DWWVCKHIKK PIRSTVLSLD WHPNSVLLAA G SCDFKCRI ...String:
MAYHSFLVEP ISCHAWNKDR TQIAICPNNH EVHIYEKSGN KWVQVHELKE HNGQVTGVDW APDSNRIVTC GTDRNAYVWT LKGRTWKPT LVILRINRAA RCVRWAPNEK KFAVGSGSRV ISICYFEQEN DWWVCKHIKK PIRSTVLSLD WHPNSVLLAA G SCDFKCRI FSAYIKEVEE RPAPTPWGSK MPFGELMFES SSSCGWVHGV CFSANGSRVA WVSHDSTVCL ADADKKMAVA TL ASETLPL LAVTFITESS LVAAGHDCFP VLFTYDSAAG KLSFGGRLDV PKQSSQRGLT ARERFQNLDK KASSEGSAAA GAG LDSLHK NSVSQISVLS GGKAKCSQFC TTGMDGGMSI WDVRSLESAL KDLKIV

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Macromolecule #4: Actin-related protein 2/3 complex subunit 2, ArpC2

MacromoleculeName: Actin-related protein 2/3 complex subunit 2, ArpC2 / type: protein_or_peptide / ID: 4
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: House mouse (house mouse)
Molecular weightTheoretical: 34.402043 KDa
SequenceString: MILLEVNNRI IEETLALKFE NAAAGNKPEA VEVTFADFDG VLYHISNPNG DKTKVMVSIS LKFYKELQAH GADELLKRVY GSYLVNPES GYNVSLLYDL ENLPASKDSI VHQAGMLKRN CFASVFEKYF QFQEEGKEGE NRAVIHYRDD ETMYVESKKD R VTVVFSTV ...String:
MILLEVNNRI IEETLALKFE NAAAGNKPEA VEVTFADFDG VLYHISNPNG DKTKVMVSIS LKFYKELQAH GADELLKRVY GSYLVNPES GYNVSLLYDL ENLPASKDSI VHQAGMLKRN CFASVFEKYF QFQEEGKEGE NRAVIHYRDD ETMYVESKKD R VTVVFSTV FKDDDDVVIG KVFMQEFKEG RRASHTAPQV LFSHREPPLE LKDTDAAVGD NIGYITFVLF PRHTNASARD NT INLIHTF RDYLHYHIKC SKAYIHTRMR AKTSDFLKVL NRARPDAEKK EMKTITGKTF SSR

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Macromolecule #5: Actin-related protein 2/3 complex subunit 3, ArpC3

MacromoleculeName: Actin-related protein 2/3 complex subunit 3, ArpC3 / type: protein_or_peptide / ID: 5
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: House mouse (house mouse)
Molecular weightTheoretical: 20.572666 KDa
SequenceString:
MPAYHSSLMD PDTKLIGNMA LLPIRSQFKG PAPRETKDTD IVDEAIYYFK ANVFFKNYEI KNEADRTLIY ITLYISECLK KLQKCNSKS QGEKEMYTLG ITNFPIPGEP GFPLNAIYAK PANKQEDEVM RAYLQQLRQE TGLRLCEKVF DPQNDKPSKW W TCFVKRQF MNKSLSGPGQ

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Macromolecule #6: Actin-related protein 2/3 complex subunit 4, ArpC4

MacromoleculeName: Actin-related protein 2/3 complex subunit 4, ArpC4 / type: protein_or_peptide / ID: 6
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: House mouse (house mouse)
Molecular weightTheoretical: 19.697047 KDa
SequenceString:
MTATLRPYLS AVRATLQAAL CLENFSSQVV ERHNKPEVEV RSSKELLLQP VTISRNEKEK VLIEGSINSV RVSIAVKQAD EIEKILCHK FMRFMMMRAE NFFILRRKPV EGYDISFLIT NFHTEQMYKH KLVDFVIHFM EEIDKEISEM KLSVNARARI V AEEFLKNF

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Macromolecule #7: Actin-related protein 2/3 complex subunit 5, ArpC5

MacromoleculeName: Actin-related protein 2/3 complex subunit 5, ArpC5 / type: protein_or_peptide / ID: 7
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: House mouse (house mouse)
Molecular weightTheoretical: 16.295317 KDa
SequenceString:
MSKNTVSSAR FRKVDVDEYD ENKFVDEDDG GDGQAGPDEG EVDSCLRQGN MTAALQAALK NPPINTKSQA VKDRAGSIVL KVLISFKAN DIEKAVQSLD KNGVDLLMKY IYKGFESPSD NSSAVLLQWH EKALAAGGVG SIVRVLTARK TV

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Macromolecule #8: Actin, alpha skeletal muscle, ACTA1

MacromoleculeName: Actin, alpha skeletal muscle, ACTA1 / type: protein_or_peptide / ID: 8
Details: As models derived from Oryctolagus cuniculus actin have been used for fitting, also the Oryctolagus cuniculus sequence (as well as the corresponding UniProt identifier) is given here, even ...Details: As models derived from Oryctolagus cuniculus actin have been used for fitting, also the Oryctolagus cuniculus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of copies: 11 / Enantiomer: LEVO
Source (natural)Organism: House mouse (house mouse)
Molecular weightTheoretical: 42.096953 KDa
SequenceString: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY ...String:
MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSSSL EK SYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVMSGGTTM YPGIADRMQK EIT ALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWITKQEYDE AGPSIVHRKC F

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 6.1
Component:
ConcentrationName
10.0 mMMES
150.0 mMsodium chloride
5.0 mMEGTA
5.0 mMGlucose
5.0 mMMagnesium chloride

Details: Adjust to pH 6.1 using NaOH
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Details: After glow discharging of the grid and prior to the seeding of cells, the grid was coated using 25ug/ml Fibronectin
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277 K
Details: Leica GP2, 3,5sec back-blotting, sensor on, 0,1mm movement after contact, manually pre-blotted within the chamber prior to the application of fiducials.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -0.0055 µm / Nominal defocus min: -0.00175 µm / Nominal magnification: 42000
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 BIOQUANTUM (6k x 4k) / Average exposure time: 1.21 sec. / Average electron dose: 2.79 e/Å2
Details: Images were collected in movie-mode at 7 frames per tilt
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 131 / Number images used: 39300
Reference model: Reference generated from manually selected particles
Method: Template Matching / Software: (Name: Dynamo (ver. 1.133), Warp (ver. 1.07))
Details: After first classification in Dynamo and re-extraction in Warp 17,146 subvolumes remained.
CTF correctionSoftware - Name: Warp (ver. 1.07)
Final 3D classificationNumber classes: 1 / Avg.num./class: 14296 / Software - Name: RELION (ver. 3.0.9)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.9)
Details: Multiple particle refinement in M version 1.0.9 was performed after 3D Refinement in RELION.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.9)
Details: Final reconstruction in RELION was performed after Multiple particle refinement in M version 1.0.9.
Number subtomograms used: 14296
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

1tyq

chain_id: A, residue_range: 2-39

1tyq

chain_id: A, residue_range: 52-353

1tyq

chain_id: A, residue_range: 360-418

1tyq

chain_id: B, residue_range: 151-345

1tyq

chain_id: C, residue_range: 1-288

1tyq

chain_id: C, residue_range: 319-372

1tyq

chain_id: D, residue_range: 1-208

1tyq

chain_id: D, residue_range: 217-281

1tyq

chain_id: E, residue_range: 2-150

1tyq

chain_id: E, residue_range: 155-174

1tyq

chain_id: F, residue_range: 3-168

1tyq

chain_id: G, residue_range: 36-151

4jd2

chain_id: B, residue_range: 4-150

4jd2

chain_id: B, residue_range: 346-387

1k8k

chain_id: C, residue_range: 297-309

6t20

chain_id: A, residue_range: 5-375

6t20

chain_id: B, residue_range: 5-375

6t20

chain_id: C, residue_range: 5-375

6t20

chain_id: D, residue_range: 5-375

6t20

chain_id: E, residue_range: 5-375
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7aqk:
Model of the actin filament Arp2/3 complex branch junction in cells

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