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- EMDB-42787: Arp2/3 branch junction complex, ADP state -

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Basic information

Entry
Database: EMDB / ID: EMD-42787
TitleArp2/3 branch junction complex, ADP state
Map dataCryosparc final map, sharpened
Sample
  • Complex: Arp2/3 branch complex (ADP state)
    • Complex: Arp2/3 complex
      • Protein or peptide: x 7 types
    • Organelle or cellular component: Actin
      • Protein or peptide: x 1 types
  • Ligand: x 3 types
Keywordsactin / arp2/3 / cytoskeleton / branch / CYTOSOLIC PROTEIN
Function / homology
Function and homology information


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 assembly / Arp2/3 protein complex / Arp2/3 complex-mediated actin nucleation ...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 assembly / Arp2/3 protein complex / Arp2/3 complex-mediated actin nucleation / cell tip / actin cortical patch / regulation of actin filament polymerization / 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 / skeletal muscle thin filament assembly / actin filament bundle 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 / 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
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 ...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 / 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 (fission yeast) / Gallus gallus (chicken) / Oryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsChavali SS / Chou SZ / Sindelar CV
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM136656 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM026338 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM110530 United States
CitationJournal: Nat Commun / Year: 2024
Title: Cryo-EM structures reveal how phosphate release from Arp3 weakens actin filament branches formed by Arp2/3 complex.
Authors: Sai Shashank Chavali / Steven Z Chou / Wenxiang Cao / Thomas D Pollard / Enrique M De La Cruz / Charles V Sindelar /
Abstract: Arp2/3 complex nucleates branched actin filaments for cell and organelle movements. Here we report a 2.7 Å resolution cryo-EM structure of the mature branch junction formed by S. pombe Arp2/3 ...Arp2/3 complex nucleates branched actin filaments for cell and organelle movements. Here we report a 2.7 Å resolution cryo-EM structure of the mature branch junction formed by S. pombe Arp2/3 complex that provides details about interactions with both mother and daughter filaments. We determine a second structure at 3.2 Å resolution with the phosphate analog BeF bound with ADP to Arp3 and ATP bound to Arp2. In this ADP-BeF transition state the outer domain of Arp3 is rotated 2° toward the mother filament compared with the ADP state and makes slightly broader contacts with actin in both the mother and daughter filaments. Thus, dissociation of P from the ADP-P transition state reduces the interactions of Arp2/3 complex with the actin filaments and may contribute to the lower mechanical stability of mature branch junctions with ADP bound to the Arps. Our structures also reveal that the mother filament in contact with Arp2/3 complex is slightly bent and twisted, consistent with the preference of Arp2/3 complex binding curved actin filaments. The small degree of twisting constrains models of actin filament mechanics.
History
DepositionNov 11, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42787.png

Downloads & links

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Map

FileDownload / File: emd_42787.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryosparc final map, sharpened
Voxel sizeX=Y=Z: 0.673 Å
Density
Contour LevelBy EMDB: 0.32
Minimum - Maximum-1.9361758 - 3.6229525
Average (Standard dev.)0.0024116829 (±0.089961566)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 344.576 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Cryosparc final map, no sharpening

Fileemd_42787_additional_1.map
AnnotationCryosparc final map, no sharpening
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryosparc half map A

Fileemd_42787_half_map_1.map
AnnotationCryosparc half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryosparc half map B

Fileemd_42787_half_map_2.map
AnnotationCryosparc half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Arp2/3 branch complex (ADP state)

EntireName: Arp2/3 branch complex (ADP state)
Components
  • Complex: Arp2/3 branch complex (ADP state)
    • Complex: Arp2/3 complex
      • 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
    • Organelle or cellular component: Actin
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Arp2/3 branch complex (ADP state)

SupramoleculeName: Arp2/3 branch complex (ADP state) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Details: S. pombe arp2/3 complex (ADP state) in a branch junction with chicken skeletal actin mother and daughter filaments (ADP state)

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Supramolecule #2: Arp2/3 complex

SupramoleculeName: Arp2/3 complex / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#7 / Details: S. pombe arp2/3 complex (ADP state)
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast)

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Supramolecule #3: Actin

SupramoleculeName: Actin / type: organelle_or_cellular_component / ID: 3 / Parent: 1 / Macromolecule list: #8 / Details: Actin mother and daughter filaments
Source (natural)Organism: Gallus gallus (chicken)

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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: Schizosaccharomyces pombe (fission yeast)
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

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: Schizosaccharomyces pombe (fission yeast)
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

UniProtKB: Actin-related protein 2

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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: Schizosaccharomyces pombe (fission yeast)
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

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

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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: Schizosaccharomyces pombe (fission yeast)
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

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 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast)
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

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: Schizosaccharomyces pombe (fission yeast)
Molecular weightTheoretical: 19.637695 KDa
SequenceString:
MSNTLRPYLN AVRSTLTASL ALEEFSSEIV ERQSQPEVEV GRSPEILLKP LVVSRNEQEQ CLIESSVNSV RFSIRIKQVD EIERILVRK FMQFLMGRAE SFFILRRKPV QGYDISFLIT NYHTEEMLKH KLVDFIIEFM EEVDAEISEM KLFLNGRARL V AETYLSCF

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: Schizosaccharomyces pombe (fission yeast)
Molecular weightTheoretical: 16.922059 KDa
SequenceString:
MTFRTLDVDS ITEPVLTEQD IFPIRNETAE QVQAAVSQLI PQARSAIQTG NALQGLKTLL SYVPYGNDVQ EVRTQYLNAF VDVLSNIRA ADIPAFVKEC STEEIDNIVN FIYRGLANPQ AYNSSVLLNW HEKVVEISGI GCIVRVLNSR PDL

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

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Macromolecule #8: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 8 / Number of copies: 8 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Oryctolagus cuniculus (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

UniProtKB: Actin, alpha skeletal muscle

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Macromolecule #9: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 9 / Number of copies: 9 / 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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Macromolecule #10: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 10 / Number of copies: 10 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #11: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 11 / Number of copies: 1 / 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

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Details: Grids were not glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: The samples were incubated on the grid for 50 s and the extra solution was blotted using two Vitrobot filter papers (0.55/20 mm, Grade 595, Ted Pella) for 4 s at 0 blot force. The grids were ...Details: The samples were incubated on the grid for 50 s and the extra solution was blotted using two Vitrobot filter papers (0.55/20 mm, Grade 595, Ted Pella) for 4 s at 0 blot force. The grids were plunged into liquid ethane at ~180 degrees C with a wait time of 0.5 s..
DetailsActin monomers with bound Ca2+ were converted to Mg2+-actin by equilibrating with 50 micromolar MgCl2 and 0.2 mM EGTA (pH 7.5) for 10 min on ice. Actin was polymerized in the presence of capping protein (CP) by sequentially mixing 8.75 micromolar Mg-ATP-actin monomers with 0.75 micromolar CP and equilibrated at room temperature for 1 h. In parallel, Arp2/3 complex (0.4 micromolar) in QB buffer was activated by mixing 0.85 micromolar GCN4-VCA and 50 micromolar ATP and incubated at 4 degrees for 1 h. The capped actin filaments sample was then gently mixed with an equal volume of activated Arp2/3 complex sample using cut pipette tips and equilibrated at 4 degrees for 5 min. Daughter filaments were formed and elongated in the presence of CP by adding 0.25 micromolar Mg-actin monomers, 50 micromolar ATP, and 40 nM CP and incubated for 5 min at room temperature. This step was subsequently repeated 4 more times, then aged for ~90 min before preparing grids for cryo-EM data collection.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 64000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Details: Electron micrographs for image reconstructions were collected using Titan Krios equipped with X-cold field emission gun at 300 kV, Gatan image filter with slit width of 20 eV and a nanoprobe.
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsThe vitrified grids were screened for sample homogeneity and ice thickness in a Glacios 200 kV transmission electron microscope equipped with Gatan K2 summit camera. Electron micrographs for image reconstructions were collected using Titan Krios equipped with X-cold field emission gun at 300 kV, Gatan image filter with slit width of 20 eV and a nanoprobe.
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 8519 / Average exposure time: 3.3 sec. / Average electron dose: 51.4 e/Å2
Details: Each movie contains 41 frames with a frame time of 0.08 s. A dose rate of 28.4 counts/pixel/s and a physical pixel size of 1.346 Angstroms was used.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3000000
Startup modelType of model: NONE
Details: These particles were subjected to 3-dimensional structure refinement and classification using 'Ab-initio 3D reconstruction' (10 classes)
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0) / Number images used: 400000
FSC plot (resolution estimation)

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