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- EMDB-21502: Structure of Dip1-activated Arp2/3 complex with nucleated actin f... -

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Basic information

Entry
Database: EMDB / ID: EMD-21502
TitleStructure of Dip1-activated Arp2/3 complex with nucleated actin filament
Map dataDip1 activated Arp2/3 complex with nucleated actin filament composite focused map
Sample
  • Complex: Complex consisting of actin-filament nucleator, Arp2/3 complex associated with nucleation promoting factor Dip1 and first four actin subunits from the pointed end of the nucleated actin-filament
    • Protein or peptide: x 10 types
  • Ligand: x 3 types
Function / homology
Function and homology information


protein localization to actin cortical patch / Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / actin cortical patch organization / cell cortex of cell tip / medial cortex / Neutrophil degranulation / establishment or maintenance of cell polarity regulating cell shape / actin cortical patch localization / positive regulation of Arp2/3 complex-mediated actin nucleation ...protein localization to actin cortical patch / Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / actin cortical patch organization / cell cortex of cell tip / medial cortex / Neutrophil degranulation / establishment or maintenance of cell polarity regulating cell shape / actin cortical patch localization / positive regulation of Arp2/3 complex-mediated actin nucleation / actin cortical patch assembly / Arp2/3 protein complex / Arp2/3 complex-mediated actin nucleation / Arp2/3 complex binding / cell tip / actin cortical patch / regulation of actin filament polymerization / cell septum / mating projection tip / cytoskeletal motor activator activity / cortical actin cytoskeleton organization / cell division site / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament / mitotic cytokinesis / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / endocytosis / calcium-dependent protein binding / actin filament binding / lamellipodium / cell body / toxin activity / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
SPIN90/Ldb17, leucine-rich domain / SPIN90/Ldb17 / SPIN90/Ldb17, leucine-rich domain / Amanitin/phalloidin toxin / 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 ...SPIN90/Ldb17, leucine-rich domain / SPIN90/Ldb17 / SPIN90/Ldb17, leucine-rich domain / Amanitin/phalloidin toxin / 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 2 / Protein dip1 / Phalloidin proprotein / Actin-related protein 3 / Actin, alpha skeletal muscle / Actin-related protein 2/3 complex subunit 1 / Actin-related protein 2/3 complex subunit 5 / Actin-related protein 2/3 complex subunit 4 / Actin-related protein 2 / Actin-related protein 2/3 complex subunit 3
Similarity search - Component
Biological speciesSchizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast) / Fission yeast (fission yeast) / Rabbit (rabbit) / death cap (death cap)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsShaaban M / Nolen BJ / Chowdhury S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM127440, R01GM092917, S10OD012272 United States
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Cryo-EM reveals the transition of Arp2/3 complex from inactive to nucleation-competent state.
Authors: Mohammed Shaaban / Saikat Chowdhury / Brad J Nolen /
Abstract: Arp2/3 complex, a crucial actin filament nucleator, undergoes structural rearrangements during activation by nucleation-promoting factors (NPFs). However, the conformational pathway leading to the ...Arp2/3 complex, a crucial actin filament nucleator, undergoes structural rearrangements during activation by nucleation-promoting factors (NPFs). However, the conformational pathway leading to the nucleation-competent state is unclear due to lack of high-resolution structures of the activated state. Here we report a ~3.9 Å resolution cryo-EM structure of activated Schizosaccharomyces pombe Arp2/3 complex bound to the S. pombe NPF Dip1 and attached to the end of the nucleated actin filament. The structure reveals global and local conformational changes that allow the two actin-related proteins in Arp2/3 complex to mimic a filamentous actin dimer and template nucleation. Activation occurs through a clamp-twisting mechanism, in which Dip1 forces two core subunits in Arp2/3 complex to pivot around one another, shifting half of the complex into a new activated position. By showing how Dip1 stimulates activation, the structure reveals how NPFs can activate Arp2/3 complex in diverse cellular processes.
History
DepositionMar 3, 2020-
Header (metadata) releaseApr 1, 2020-
Map releaseAug 12, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0269
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0269
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6w17
  • Surface level: 0.0269
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21502.png

Downloads & links

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Map

FileDownload / File: emd_21502.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDip1 activated Arp2/3 complex with nucleated actin filament composite focused map
Voxel sizeX=Y=Z: 1.10972 Å
Density
Contour LevelBy AUTHOR: 0.0269 / Movie #1: 0.0269
Minimum - Maximum-0.06679351 - 0.19063786
Average (Standard dev.)0.0008505606 (±0.0063711237)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 277.43106 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1097241.1097241.109724
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z277.431277.431277.431
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ250250250
MAP C/R/S213
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.0670.1910.001

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Supplemental data

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Mask #1

Fileemd_21502_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Focused map of Arps and actin filament

Fileemd_21502_additional_1.map
AnnotationFocused map of Arps and actin filament
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Focused map of Dip1 Arp2/3 complex

Fileemd_21502_additional_2.map
AnnotationFocused map of Dip1 Arp2/3 complex
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: Dip1 activated Arp2/3 complex with nucleated actin filament...

Fileemd_21502_additional_3.map
AnnotationDip1 activated Arp2/3 complex with nucleated actin filament unsharpened, non-filtered map
Projections & Slices
AxesZYX

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Half map: half map 1

Fileemd_21502_half_map_1.map
Annotationhalf map 1
Projections & Slices
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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_21502_half_map_2.map
Annotationhalf map 2
Projections & Slices
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Sample components

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Entire : Complex consisting of actin-filament nucleator, Arp2/3 complex as...

EntireName: Complex consisting of actin-filament nucleator, Arp2/3 complex associated with nucleation promoting factor Dip1 and first four actin subunits from the pointed end of the nucleated actin-filament
Components
  • Complex: Complex consisting of actin-filament nucleator, Arp2/3 complex associated with nucleation promoting factor Dip1 and first four actin subunits from the pointed end of the nucleated actin-filament
    • Protein or peptide: Actin-related protein 3
    • Protein or peptide: Actin-related protein 2
    • Protein or peptide: Actin-related protein 2/3 complex subunit 1
    • 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: Protein dip1
    • Protein or peptide: Actin, alpha skeletal muscle
    • Protein or peptide: Phalloidin
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Complex consisting of actin-filament nucleator, Arp2/3 complex as...

SupramoleculeName: Complex consisting of actin-filament nucleator, Arp2/3 complex associated with nucleation promoting factor Dip1 and first four actin subunits from the pointed end of the nucleated actin-filament
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#10
Source (natural)Organism: Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Strain: 972 / ATCC 24843
Molecular weightTheoretical: 446 KDa

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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: Fission yeast (fission yeast) / Strain: 972 / ATCC 24843
Molecular weightTheoretical: 47.427137 KDa
SequenceString: MASFNVPIIM DNGTGYSKLG YAGNDAPSYV FPTVIATRSA GASSGPAVSS KPSYMASKGS GHLSSKRATE DLDFFIGNDA LKKASAGYS LDYPIRHGQI ENWDHMERFW QQSLFKYLRC EPEDHYFLLT EPPLNPPENR ENTAEIMFES FNCAGLYIAV Q AVLALAAS ...String:
MASFNVPIIM DNGTGYSKLG YAGNDAPSYV FPTVIATRSA GASSGPAVSS KPSYMASKGS GHLSSKRATE DLDFFIGNDA LKKASAGYS LDYPIRHGQI ENWDHMERFW QQSLFKYLRC EPEDHYFLLT EPPLNPPENR ENTAEIMFES FNCAGLYIAV Q AVLALAAS WTSSKVTDRS LTGTVVDSGD GVTHIIPVAE GYVIGSSIKT MPLAGRDVTY FVQSLLRDRN EPDSSLKTAE RI KEECCYV CPDIVKEFSR FDREPDRYLK YASESITGHS TTIDVGFERF LAPEIFFNPE IASSDFLTPL PELVDNVVQS SPI DVRKGL YKNIVLSGGS TLFKNFGNRL QRDLKRIVDE RIHRSEMLSG AKSGGVDVNV ISHKRQRNAV WFGGSLLAQT PEFG SYCHT KADYEEYGAS IARRYQIFGN SL

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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: Fission yeast (fission yeast) / Strain: 972 / ATCC 24843
Molecular weightTheoretical: 44.286758 KDa
SequenceString: MESAPIVLDN GTGFVKVGYA KDNFPRFQFP SIVGRPILRA EEKTGNVQIK DVMVGDEAEA VRSLLQVKYP MENGIIRDFE EMNQLWDYT FFEKLKIDPR GRKILLTEPP MNPVANREKM CETMFERYGF GGVYVAIQAV LSLYAQGLSS GVVVDSGDGV T HIVPVYES ...String:
MESAPIVLDN GTGFVKVGYA KDNFPRFQFP SIVGRPILRA EEKTGNVQIK DVMVGDEAEA VRSLLQVKYP MENGIIRDFE EMNQLWDYT FFEKLKIDPR GRKILLTEPP MNPVANREKM CETMFERYGF GGVYVAIQAV LSLYAQGLSS GVVVDSGDGV T HIVPVYES VVLNHLVGRL DVAGRDATRY LISLLLRKGY AFNRTADFET VREMKEKLCY VSYDLELDHK LSEETTVLMR NY TLPDGRV IKVGSERYEC PECLFQPHLV GSEQPGLSEF IFDTIQAADV DIRKYLYRAI VLSGGSSMYA GLPSRLEKEI KQL WFERVL HGDPARLPNF KVKIEDAPRR RHAVFIGGAV LADIMAQNDH MWVSKAEWEE YGVRALDKLG PRTT

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

MacromoleculeName: Actin-related protein 2/3 complex subunit 1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Fission yeast (fission yeast) / Strain: 972 / ATCC 24843
Molecular weightTheoretical: 41.643465 KDa
SequenceString: MATSQVLHIL PKPSYEHAFN SQRTEFVTTT ATNQVELYEQ DGNGWKHART FSDHDKIVTC VDWAPKSNRI VTCSQDRNAY VYEKRPDGT WKQTLVLLRL NRAATFVRWS PNEDKFAVGS GARVISVCYF EQENDWWVSK HLKRPLRSTI LSLDWHPNNV L LAAGCADR ...String:
MATSQVLHIL PKPSYEHAFN SQRTEFVTTT ATNQVELYEQ DGNGWKHART FSDHDKIVTC VDWAPKSNRI VTCSQDRNAY VYEKRPDGT WKQTLVLLRL NRAATFVRWS PNEDKFAVGS GARVISVCYF EQENDWWVSK HLKRPLRSTI LSLDWHPNNV L LAAGCADR KAYVLSAYVR DVDAKPEASV WGSRLPFNTV CAEYPSGGWV HAVGFSPSGN ALAYAGHDSS VTIAYPSAPE QP PRALITV KLSQLPLRSL LWANESAIVA AGYNYSPILL QGNESGWAHT RDLDAGTSKT SFTHTGNTGE GREEEGPVSF TAL RSTFRN MDLKGSSQSI SSLPTVHQNM IATLRPYAGT PGNITAFTSS GTDGRVVLWT L

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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: Fission yeast (fission yeast) / Strain: 972 / ATCC 24843
Molecular weightTheoretical: 37.02523 KDa
SequenceString: MLSLDYNNIF IYELLTERFS SENPSSIDQV VTDFDGVTFH ISTPEEKTKI LISLSMKCYP ELVNYGTLDL LKQIYGAYVH EPEMGYNFS ILIDLQQLPA TDEEKEQLAM SISMLKRNVL AAPFHRAFTK QAELADLARK DPENAPMLDK QATSQELMAI H YRDEETIV ...String:
MLSLDYNNIF IYELLTERFS SENPSSIDQV VTDFDGVTFH ISTPEEKTKI LISLSMKCYP ELVNYGTLDL LKQIYGAYVH EPEMGYNFS ILIDLQQLPA TDEEKEQLAM SISMLKRNVL AAPFHRAFTK QAELADLARK DPENAPMLDK QATSQELMAI H YRDEETIV LWPEHDRVTV VFSTKFREET DRIFGKVFLQ EFVDARRRPA IQTAPQVLFS YRDPPLEIRD IQGIQKGDDF GF VTFVLFE RHFTPQNRED CISHIQVFRN TLHFHIKASK AYMHQRMRKR VADFQKVLNR AKPDVELERK TATGRSFVRA

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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 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Fission yeast (fission yeast) / Strain: 972 / ATCC 24843
Molecular weightTheoretical: 19.865746 KDa
SequenceString:
MPAYHSSFLS LTDVPTTGNI AMLPLKTKFR GPAYPADESQ MDIIDECIGL FRANCFFRNF EIKGPADRTL IYGTLFISEC LGRVNGLNY RDAERQLNSL ALENFSIPGS AGFPLNALYA PPLSPQDAEI MRTYLTQFRQ ELAYRLLSHV YATEKDHPSK W WTCFSKRR FMNKAL

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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: Fission yeast (fission yeast) / Strain: 972 / ATCC 24843
Molecular weightTheoretical: 19.637695 KDa
SequenceString:
MSNTLRPYLN AVRSTLTASL ALEEFSSEIV ERQSQPEVEV GRSPEILLKP LVVSRNEQEQ CLIESSVNSV RFSIRIKQVD EIERILVRK FMQFLMGRAE SFFILRRKPV QGYDISFLIT NYHTEEMLKH KLVDFIIEFM EEVDAEISEM KLFLNGRARL V AETYLSCF

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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: Fission yeast (fission yeast) / Strain: 972 / ATCC 24843
Molecular weightTheoretical: 16.922059 KDa
SequenceString:
MTFRTLDVDS ITEPVLTEQD IFPIRNETAE QVQAAVSQLI PQARSAIQTG NALQGLKTLL SYVPYGNDVQ EVRTQYLNAF VDVLSNIRA ADIPAFVKEC STEEIDNIVN FIYRGLANPQ AYNSSVLLNW HEKVVEISGI GCIVRVLNSR PDL

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Macromolecule #8: Protein dip1

MacromoleculeName: Protein dip1 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Strain: 972 / ATCC 24843
Molecular weightTheoretical: 43.715137 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEFTDVFYNL ENEKQFLAEI DELLTIPYEK NELEIGVSKF LSFINKYGEP YLITEFQLQR CMEKFLNSPL YQLDENAVLS IFYDCFFFL KEQQTLKFII IVFQSEIQEN EYCLRLLAST GFIPVLIKAM KQFKDRSENV AFTSFRYALF LVYYICRSRR L SPTDLVAI ...String:
MEFTDVFYNL ENEKQFLAEI DELLTIPYEK NELEIGVSKF LSFINKYGEP YLITEFQLQR CMEKFLNSPL YQLDENAVLS IFYDCFFFL KEQQTLKFII IVFQSEIQEN EYCLRLLAST GFIPVLIKAM KQFKDRSENV AFTSFRYALF LVYYICRSRR L SPTDLVAI DEYFLVNLFK TSEEAWNDED MDSFGMCVLT LLSINEQFML VRLASKGSFE IANGIMDLLS SSKVDNGIYS EG LVFTLNR EKDPRSRMLI LKQLFLLFTT PATYEIFYTN DLNVLIDIFI REINNIPDEL SDLRYAYLQV LIPLLENTQV RHP PHYKTK CIVDAVNNVL ISHSKSSNME DARTTDVAIR VLEVPWLQQE MKSLGTAQ

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Macromolecule #9: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 9 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Rabbit (rabbit)
Molecular weightTheoretical: 42.109973 KDa
SequenceString: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIE(HIC)G IIT NWDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLD SG DGVTHNVPIY ...String:
MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIE(HIC)G IIT NWDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLD SG DGVTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSS S LEKSYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVMSGGTTM YPGIADRMQ KEITALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWITKQEYDE AGPSIVHRKC F

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Macromolecule #10: Phalloidin

MacromoleculeName: Phalloidin / type: protein_or_peptide / ID: 10 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: death cap (death cap)
Molecular weightTheoretical: 808.899 Da
SequenceString:
(HYP)AW(G5G)A(ALO)C

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Macromolecule #11: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 11 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #12: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 12 / 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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Macromolecule #13: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 13 / Number of copies: 4 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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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

Concentration3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMKClpotassium chloride
1.0 mMC14H24N2O10egtazic acid
1.0 mMMgCl2magnesium chloride
0.2 mMC10H16N5O13P3adenosine triphosphate
20.0 mMC8H18N2O4S4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
1.0 mMC4H10O2S2dithiothreitol
10.0 microMC35H48N8O11Sphalloidin
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.019 kPa / Details: 20mA current
VitrificationCryogen name: ETHANE / Chamber humidity: 98 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -1.75 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 92000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsData were collected by stage shifting to targeted exposure positions.
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4000 pixel / Digitization - Dimensions - Height: 4000 pixel / Digitization - Sampling interval: 14.0 µm / Number grids imaged: 2 / Number real images: 5109 / Average exposure time: 60.0 sec. / Average electron dose: 36.35 e/Å2
Details: Each micrograph was collected as dose-fractionated movies consisting of 45 fractions per movie.
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3500000
Details: Particles were picked using Laplacian Gaussian auto-picking.
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Details: Particles were CTF corrected during projection matching and back projection.
Startup model#0 - Type of model: INSILICO MODEL
#0 - In silico model: Ab-initio initial model was determined using CryoSPARCv2.
#1 - Type of model: INSILICO MODEL
#1 - In silico model: Ab-initio initial model was determined using CryoSPARCv2.
Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.0.6)
Details: Classification was performed without image alignments
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.6)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.6) / Number images used: 110433
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

3dwl


3j8i


6d8c

chain_id: N
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6w17:
Structure of Dip1-activated Arp2/3 complex with nucleated actin filament

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