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- EMDB-20770: CryoEM structure of human Arp2/3 complex with bound NPFs -

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Basic information

Entry
Database: EMDB / ID: EMD-20770
TitleCryoEM structure of human Arp2/3 complex with bound NPFs
Map dataCryo-EM structure of human Arp2/3 complex bound to two nucleation promoting factors (B-factor sharpened).
Sample
  • Complex: Cryo-EM structure of human Arp2/3 complex with bound NPFs
    • Protein or peptide: Actin-related protein 3
    • Protein or peptide: Actin-related protein 2
    • Protein or peptide: Actin-related protein 2/3 complex subunit 1B
    • Protein or peptide: Actin-related protein 2/3 complex subunit 2
    • Protein or peptide: Actin-related protein 2/3 complex subunit 3
    • Protein or peptide: Actin-related protein 2/3 complex subunit 4
    • Protein or peptide: Actin-related protein 2/3 complex subunit 5
    • Protein or peptide: Neural Wiskott-Aldrich syndrome protein
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
Keywordsactin / ATPase / actin related protein / arp / cytoskeleton / Arp2-3 complex / actin nucleation / actin branching / CONTRACTILE PROTEIN
Function / homology
Function and homology information


tubulobulbar complex / meiotic chromosome movement towards spindle pole / cytosolic transport / growth cone leading edge / negative regulation of membrane tubulation / meiotic cytokinesis / muscle cell projection membrane / spindle localization / membrane invagination / positive regulation of clathrin-dependent endocytosis ...tubulobulbar complex / meiotic chromosome movement towards spindle pole / cytosolic transport / growth cone leading edge / negative regulation of membrane tubulation / meiotic cytokinesis / muscle cell projection membrane / spindle localization / membrane invagination / positive regulation of clathrin-dependent endocytosis / actin polymerization-dependent cell motility / plasma membrane tubulation / Arp2/3 protein complex / asymmetric cell division / negative regulation of lymphocyte migration / Arp2/3 complex-mediated actin nucleation / actin nucleation / postsynapse organization / regulation of cell projection assembly / actin cap / vesicle organization / postsynaptic actin cytoskeleton organization / positive regulation of chemotaxis / regulation of actin filament polymerization / vesicle budding from membrane / vesicle transport along actin filament / dendritic spine morphogenesis / protein-containing complex localization / regulation of postsynapse organization / positive regulation of filopodium assembly / establishment or maintenance of cell polarity / positive regulation of double-strand break repair via homologous recombination / positive regulation of actin filament polymerization / cell leading edge / filamentous actin / brush border / cilium assembly / RHO GTPases Activate WASPs and WAVEs / positive regulation of lamellipodium assembly / positive regulation of substrate adhesion-dependent cell spreading / EPHB-mediated forward signaling / actin filament polymerization / cytoskeletal protein binding / cellular response to nerve growth factor stimulus / cell projection / response to bacterium / FCGR3A-mediated phagocytosis / structural constituent of cytoskeleton / cellular response to type II interferon / Regulation of actin dynamics for phagocytic cup formation / response to estrogen / regulation of protein localization / azurophil granule lumen / cell-cell junction / cell migration / actin cytoskeleton / response to estradiol / lamellipodium / site of double-strand break / Clathrin-mediated endocytosis / actin binding / cytoplasmic vesicle / actin cytoskeleton organization / postsynapse / secretory granule lumen / ficolin-1-rich granule lumen / endosome / neuron projection / cell division / Golgi membrane / focal adhesion / glutamatergic synapse / Neutrophil degranulation / protein-containing complex binding / enzyme binding / positive regulation of transcription by RNA polymerase II / extracellular exosome / extracellular region / nucleoplasm / ATP binding / membrane / nucleus / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Actin nucleation-promoting factor WAS, C-terminal / WASP family, EVH1 domain / WH2 motif / 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 nucleation-promoting factor WAS, C-terminal / WASP family, EVH1 domain / WH2 motif / 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) / Wiskott Aldrich syndrome homology region 2 / WH2 domain / WH2 domain profile. / WH1/EVH1 domain / WH1 domain / WH1 domain profile. / WASP homology region 1 / CRIB domain superfamily / P21-Rho-binding domain / CRIB domain profile. / P21-Rho-binding domain / CRIB domain / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain / PH-like domain superfamily / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Actin-related protein 2/3 complex subunit 1B / Actin-related protein 2/3 complex subunit 2 / Actin-related protein 2/3 complex subunit 3 / Actin-related protein 2/3 complex subunit 5 / Actin-related protein 2/3 complex subunit 4 / Actin-related protein 3 / Actin-related protein 2 / Actin nucleation-promoting factor WASL
Similarity search - Component
Biological speciesSpodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths) / Homo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsZimmet A / van Eeuwen T
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM07391 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32-GM008275 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)F31-HL146077 United States
CitationJournal: Sci Adv / Year: 2020
Title: Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism.
Authors: Austin Zimmet / Trevor Van Eeuwen / Malgorzata Boczkowska / Grzegorz Rebowski / Kenji Murakami / Roberto Dominguez /
Abstract: Actin-related protein (Arp) 2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including two actin-related subunits (Arp2 and Arp3). Two nucleation- ...Actin-related protein (Arp) 2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including two actin-related subunits (Arp2 and Arp3). Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions, and contribution of each NPF to activation are unresolved. Here, we report the cryo-electron microscopy structure of recombinantly expressed human Arp2/3 complex with two WASP family NPFs bound and address the mechanism of activation. A cross-linking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex.
History
DepositionSep 27, 2019-
Header (metadata) releaseNov 20, 2019-
Map releaseJul 1, 2020-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.475
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.475
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6uhc
  • Surface level: 0.475
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20770.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of human Arp2/3 complex bound to two nucleation promoting factors (B-factor sharpened).
Voxel sizeX=Y=Z: 0.836 Å
Density
Contour LevelBy AUTHOR: 0.475 / Movie #1: 0.475
Minimum - Maximum-1.97515 - 3.0070715
Average (Standard dev.)0.00638433 (±0.08849225)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 267.52002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8360.8360.836
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z267.520267.520267.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-1.9753.0070.006

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Supplemental data

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Additional map: Cryo-EM structure of human Arp2/3 complex bound to...

Fileemd_20770_additional.map
AnnotationCryo-EM structure of human Arp2/3 complex bound to two nucleation promoting factors
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Cryo-EM structure of human Arp2/3 complex with bound NPFs

EntireName: Cryo-EM structure of human Arp2/3 complex with bound NPFs
Components
  • Complex: Cryo-EM structure of human Arp2/3 complex with bound NPFs
    • Protein or peptide: Actin-related protein 3
    • Protein or peptide: Actin-related protein 2
    • Protein or peptide: Actin-related protein 2/3 complex subunit 1B
    • Protein or peptide: Actin-related protein 2/3 complex subunit 2
    • Protein or peptide: Actin-related protein 2/3 complex subunit 3
    • Protein or peptide: Actin-related protein 2/3 complex subunit 4
    • Protein or peptide: Actin-related protein 2/3 complex subunit 5
    • Protein or peptide: Neural Wiskott-Aldrich syndrome protein
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Cryo-EM structure of human Arp2/3 complex with bound NPFs

SupramoleculeName: Cryo-EM structure of human Arp2/3 complex with bound NPFs
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
Molecular weightTheoretical: 243 kDa/nm

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Macromolecule #1: Actin-related protein 3

MacromoleculeName: Actin-related protein 3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 47.428031 KDa
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
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

UniProtKB: Actin-related protein 3

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Macromolecule #2: Actin-related protein 2

MacromoleculeName: Actin-related protein 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.818711 KDa
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
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

UniProtKB: Actin-related protein 2

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Macromolecule #3: Actin-related protein 2/3 complex subunit 1B

MacromoleculeName: Actin-related protein 2/3 complex subunit 1B / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.004781 KDa
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
SequenceString: MAYHSFLVEP ISCHAWNKDR TQIAICPNNH EVHIYEKSGA KWTKVHELKE HNGQVTGIDW APESNRIVTC GTDRNAYVWT LKGRTWKPT LVILRINRAA RCVRWAPNEN KFAVGSGSRV ISICYFEQEN DWWVCKHIKK PIRSTVLSLD WHPNNVLLAA G SCDFKCRI ...String:
MAYHSFLVEP ISCHAWNKDR TQIAICPNNH EVHIYEKSGA KWTKVHELKE HNGQVTGIDW APESNRIVTC GTDRNAYVWT LKGRTWKPT LVILRINRAA RCVRWAPNEN KFAVGSGSRV ISICYFEQEN DWWVCKHIKK PIRSTVLSLD WHPNNVLLAA G SCDFKCRI FSAYIKEVEE RPAPTPWGSK MPFGELMFES SSSCGWVHGV CFSASGSRVA WVSHDSTVCL ADADKKMAVA TL ASETLPL LALTFITDNS LVAAGHDCFP VLFTYDAAAG MLSFGGRLDV PKQSSQRGLT ARERFQNLDK KASSEGGTAA GAG LDSLHK NSVSQISVLS GGKAKCSQFC TTGMDGGMSI WDVKSLESAL KDLKIK

UniProtKB: Actin-related protein 2/3 complex subunit 1B

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

MacromoleculeName: Actin-related protein 2/3 complex subunit 2 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 34.386043 KDa
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
SequenceString: MILLEVNNRI IEETLALKFE NAAAGNKPEA VEVTFADFDG VLYHISNPNG DKTKVMVSIS LKFYKELQAH GADELLKRVY GSFLVNPES GYNVSLLYDL ENLPASKDSI VHQAGMLKRN CFASVFEKYF QFQEEGKEGE NRAVIHYRDD ETMYVESKKD R VTVVFSTV ...String:
MILLEVNNRI IEETLALKFE NAAAGNKPEA VEVTFADFDG VLYHISNPNG DKTKVMVSIS LKFYKELQAH GADELLKRVY GSFLVNPES GYNVSLLYDL ENLPASKDSI VHQAGMLKRN CFASVFEKYF QFQEEGKEGE NRAVIHYRDD ETMYVESKKD R VTVVFSTV FKDDDDVVIG KVFMQEFKEG RRASHTAPQV LFSHREPPLE LKDTDAAVGD NIGYITFVLF PRHTNASARD NT INLIHTF RDYLHYHIKC SKAYIHTRMR AKTSDFLKVL NRARPDAEKK EMKTITGKTF SSR

UniProtKB: Actin-related protein 2/3 complex subunit 2

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

MacromoleculeName: Actin-related protein 2/3 complex subunit 3 / type: protein_or_peptide / ID: 5 / Details: C-terminal Flag tag / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.19633 KDa
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
SequenceString: MPAYHSSLMD PDTKLIGNMA LLPIRSQFKG PAPRETKDTD IVDEAIYYFK ANVFFKNYEI KNEADRTLIY ITLYISECLK KLQKCNSKS QGEKEMYTLG ITNFPIPGEP GFPLNAIYAK PANKQEDEVM RAYLQQLRQE TGLRLCEKVF DPQNDKPSKW W TCFVKRQF ...String:
MPAYHSSLMD PDTKLIGNMA LLPIRSQFKG PAPRETKDTD IVDEAIYYFK ANVFFKNYEI KNEADRTLIY ITLYISECLK KLQKCNSKS QGEKEMYTLG ITNFPIPGEP GFPLNAIYAK PANKQEDEVM RAYLQQLRQE TGLRLCEKVF DPQNDKPSKW W TCFVKRQF MNKSLSGPGQ GTSENLYQGG GDYKDDDDKC GRASS

UniProtKB: Actin-related protein 2/3 complex subunit 3

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

MacromoleculeName: Actin-related protein 2/3 complex subunit 4 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 19.697047 KDa
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
SequenceString:
MTATLRPYLS AVRATLQAAL CLENFSSQVV ERHNKPEVEV RSSKELLLQP VTISRNEKEK VLIEGSINSV RVSIAVKQAD EIEKILCHK FMRFMMMRAE NFFILRRKPV EGYDISFLIT NFHTEQMYKH KLVDFVIHFM EEIDKEISEM KLSVNARARI V AEEFLKNF

UniProtKB: Actin-related protein 2/3 complex subunit 4

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

MacromoleculeName: Actin-related protein 2/3 complex subunit 5 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 16.341407 KDa
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
SequenceString:
MSKNTVSSAR FRKVDVDEYD ENKFVDEEDG GDGQAGPDEG EVDSCLRQGN MTAALQAALK NPPINTKSQA VKDRAGSIVL KVLISFKAN DIEKAVQSLD KNGVDLLMKY IYKGFESPSD NSSAMLLQWH EKALAAGGVG SIVRVLTARK TV

UniProtKB: Actin-related protein 2/3 complex subunit 5

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Macromolecule #8: Neural Wiskott-Aldrich syndrome protein

MacromoleculeName: Neural Wiskott-Aldrich syndrome protein / type: protein_or_peptide / ID: 8 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 54.342023 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSSGQQPPRR VTNVGSLLLT PQENESLFSF LGKKCVTMSS AVVQLYAADR NCMWAKKCSG VACLVKDNPQ RSYFLRIFDI KDGKLLWEQ ELYNNFVYNS PRGYFHTFAG DTCQVALNFA NEEEAKKFRK AVTDLLGRRQ RKSEKRRDAP NGPNLPMATV D IKNPEITT ...String:
MSSGQQPPRR VTNVGSLLLT PQENESLFSF LGKKCVTMSS AVVQLYAADR NCMWAKKCSG VACLVKDNPQ RSYFLRIFDI KDGKLLWEQ ELYNNFVYNS PRGYFHTFAG DTCQVALNFA NEEEAKKFRK AVTDLLGRRQ RKSEKRRDAP NGPNLPMATV D IKNPEITT NRFYGSQVNN ISHTKEKKKG KAKKKRLTKA DIGTPSNFQH IGHVGWDPNT GFDLNNLDPE LKNLFDMCGI SE AQLKDRE TSKVIYDFIE KTGGVEAVKN ELRRQAPPPP PPSRGGPPPP PPPPHSSGPP PPPARGRGAP PPPPSRAPTA APP PPPPSR PGVVVPPPPP NRMYPPPPPA LPSSAPSGPP PPPPPSMAGS TAPPPPPPPP PPPGPPPPPG LPSDGDHQVP APSG NKAAL LDQIREGAQL KKVEQNSRPV SCSGRDALLD QIRQGIQLKS VSDGQESTPP TPAPTSGIVG ALMEVMQKRS KAIHS SDED EDDDDEEDFE DDDEWED

UniProtKB: Actin nucleation-promoting factor WASL

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #10: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 10 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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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

Concentration5 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
100.0 mMNaClSodium chloridesodium chloride
1.0 mMHSCH2CH(OH)CH(OH)CH2SHDithiothreitol
0.2 mMC10H14N5O13P3Na2Adenosine triphosphate disodium salt
0.2 mMMgCl2magnesium chloride
0.00025 V/VTERGITOL
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
Details: Grids glow discharged with easiGLOW glow discharger at 0.3 mBar, 25mA for 1 minute
VitrificationCryogen name: ETHANE / Instrument: LEICA EM CPC
Details: Grids manually blotted for 3 seconds with Whatman 41 filter paper and manually plunged on Leica EM CPC manual plunger..
DetailsThis sample was monodisperse

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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 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -3.5 µm / Nominal defocus min: -1.5 µm / Nominal magnification: 165000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsData collected in super resolution mode. Illuminated area of 1.01um. Nominal Dose of 40a/A^2 and a dose rate of 4.87 e-/s/pixel. 2 or 5 exposures per hole by image shift.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 2 / Number real images: 5004 / Average exposure time: 7.0 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 627163
Startup modelType of model: NONE / Details: ab initio model generated by cryoSPARC.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 4 / Avg.num./class: 100000 / Software - Name: cryoSPARC (ver. 2.9.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.9.0) / Number images used: 123198
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 132 / Target criteria: correlation
Output model

PDB-6uhc:
CryoEM structure of human Arp2/3 complex with bound NPFs

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  • 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

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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

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