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- EMDB-3385: Cryo-EM structure of the human APC/C-Cdc20-Hsl1 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-3385
TitleCryo-EM structure of the human APC/C-Cdc20-Hsl1 complex
Map dataSingle particle reconstruction of the human APC/C-Cdc20-Hsl1 complex
Sample
  • Sample: Recombinant human APC/C-Cdc20-Hsl1 complex
  • Protein or peptide: x 16 types
KeywordsCell cycle / phosphorylation / mitosis / ubiquitination
Function / homology
Function and homology information


protein localization to septin ring / mitotic morphogenesis checkpoint signaling / metaphase/anaphase transition of cell cycle / metaphase/anaphase transition of meiosis I / cellular bud neck septin ring / Inhibition of the proteolytic activity of APC/C required for the onset of anaphase by mitotic spindle checkpoint components / mitotic checkpoint complex / positive regulation of anaphase-promoting complex-dependent catabolic process / regulation of meiotic nuclear division / positive regulation of synapse maturation ...protein localization to septin ring / mitotic morphogenesis checkpoint signaling / metaphase/anaphase transition of cell cycle / metaphase/anaphase transition of meiosis I / cellular bud neck septin ring / Inhibition of the proteolytic activity of APC/C required for the onset of anaphase by mitotic spindle checkpoint components / mitotic checkpoint complex / positive regulation of anaphase-promoting complex-dependent catabolic process / regulation of meiotic nuclear division / positive regulation of synapse maturation / Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase / regulation of mitotic cell cycle spindle assembly checkpoint / protein branched polyubiquitination / Inactivation of APC/C via direct inhibition of the APC/C complex / APC/C:Cdc20 mediated degradation of mitotic proteins / Phosphorylation of Emi1 / anaphase-promoting complex / Aberrant regulation of mitotic exit in cancer due to RB1 defects / regulation of meiotic cell cycle / anaphase-promoting complex-dependent catabolic process / metaphase/anaphase transition of mitotic cell cycle / positive regulation of synaptic plasticity / regulation of exit from mitosis / anaphase-promoting complex binding / Phosphorylation of the APC/C / cellular bud neck / positive regulation of mitotic metaphase/anaphase transition / positive regulation of ubiquitin protein ligase activity / ubiquitin ligase activator activity / protein K11-linked ubiquitination / enzyme-substrate adaptor activity / positive regulation of dendrite morphogenesis / regulation of mitotic metaphase/anaphase transition / ubiquitin-ubiquitin ligase activity / mitotic sister chromatid cohesion / mitotic metaphase chromosome alignment / mitotic spindle assembly checkpoint signaling / Regulation of APC/C activators between G1/S and early anaphase / cullin family protein binding / Transcriptional Regulation by VENTX / mitotic spindle assembly / ubiquitin-like ligase-substrate adaptor activity / positive regulation of axon extension / protein K48-linked ubiquitination / heterochromatin / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / regulation of mitotic cell cycle / Resolution of Sister Chromatid Cohesion / APC/C:Cdc20 mediated degradation of Cyclin B / APC-Cdc20 mediated degradation of Nek2A / nuclear periphery / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / SCF-beta-TrCP mediated degradation of Emi1 / Assembly of the pre-replicative complex / RHO GTPases Activate Formins / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / brain development / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / protein catabolic process / CDK-mediated phosphorylation and removal of Cdc6 / mitotic spindle / kinetochore / spindle pole / spindle / ubiquitin-protein transferase activity / Separation of Sister Chromatids / G2/M transition of mitotic cell cycle / microtubule cytoskeleton / ubiquitin protein ligase activity / Antigen processing: Ubiquitination & Proteasome degradation / mitotic cell cycle / nervous system development / Senescence-Associated Secretory Phenotype (SASP) / ubiquitin-dependent protein catabolic process / protein phosphatase binding / molecular adaptor activity / cell differentiation / non-specific serine/threonine protein kinase / regulation of cell cycle / protein kinase activity / Ub-specific processing proteases / protein ubiquitination / cell division / negative regulation of gene expression / protein serine kinase activity / protein serine/threonine kinase activity / centrosome / ubiquitin protein ligase binding / nucleolus / zinc ion binding / nucleoplasm / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Anaphase-promoting complex subunit 15 / The WD repeat Cdc20/Fizzy family / Anaphase-promoting complex subunit 15 / Anaphase-promoting complex subunit 4, metazoa / Anaphase-promoting complex subunit 1, C-terminal / Anaphase-promoting complex subunit 1, middle domain / : / : / : / Anaphase-promoting complex subunit 1 WD40 beta-propeller domain ...Anaphase-promoting complex subunit 15 / The WD repeat Cdc20/Fizzy family / Anaphase-promoting complex subunit 15 / Anaphase-promoting complex subunit 4, metazoa / Anaphase-promoting complex subunit 1, C-terminal / Anaphase-promoting complex subunit 1, middle domain / : / : / : / Anaphase-promoting complex subunit 1 WD40 beta-propeller domain / Anaphase-promoting complex sub unit 1 C-terminal domain / Anaphase-promoting complex subunit 1 middle domain / APC1 beta sandwich domain / Anaphase-promoting complex subunit 5, N-terminal domain / Anaphase-promoting complex subunit 16 / Anaphase-promoting complex, subunit 16 / Cdc23 / Apc13 / Anaphase promoting complex subunit 8 / Cdc23 / Apc13p protein / Anaphase-promoting complex subunit 4 / Anaphase-promoting complex subunit 4 long domain / Anaphase-promoting complex subunit 1 / Anaphase-promoting complex subunit 5 domain / Anaphase-promoting complex subunit 5 / Anaphase-promoting complex subunit 5 / Anaphase-promoting complex, cyclosome, subunit 4 / Anaphase-promoting complex subunit APC10/Doc1 / Anaphase-promoting complex, subunit CDC26 / Anaphase-promoting complex APC subunit CDC26 / Anaphase-promoting complex subunit 11, RING-H2 finger / Anaphase-promoting complex subunit 11 RING-H2 finger / Anaphase-promoting complex subunit 2, C-terminal / Anaphase-promoting complex subunit 2 / Anaphase promoting complex (APC) subunit 2 / Anaphase promoting complex (APC) subunit 2 / APC10/DOC domain / Anaphase-promoting complex, subunit 10 (APC10) / DOC domain profile. / Anaphase-promoting complex, subunit 10 (APC10) / Anaphase-promoting complex, cyclosome, subunit 3 / TPR repeat / Tetratricopeptide repeat / : / Anaphase-promoting complex subunit 4, WD40 domain / Anaphase-promoting complex subunit 4 WD40 domain / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / Cullin family profile. / Tetratricopeptide repeat / Tetratricopeptide repeat / Tetratricopeptide repeat / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / Galactose-binding-like domain superfamily / Zinc finger RING-type profile. / Zinc finger, RING-type / Armadillo-like helical / Tetratricopeptide-like helical domain superfamily / Zinc finger, RING/FYVE/PHD-type / Winged helix DNA-binding domain superfamily / Trp-Asp (WD) repeats signature. / 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 / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Winged helix-like DNA-binding domain superfamily / Protein kinase domain / WD40/YVTN repeat-like-containing domain superfamily / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Cell division cycle protein 27 homolog / Probable serine/threonine-protein kinase HSL1 / Anaphase-promoting complex subunit 15 / Cell division cycle protein 20 homolog / Cell division cycle protein 16 homolog / Anaphase-promoting complex subunit CDC26 / Anaphase-promoting complex subunit 16 / Anaphase-promoting complex subunit 13 / Anaphase-promoting complex subunit 1 / Anaphase-promoting complex subunit 11 ...Cell division cycle protein 27 homolog / Probable serine/threonine-protein kinase HSL1 / Anaphase-promoting complex subunit 15 / Cell division cycle protein 20 homolog / Cell division cycle protein 16 homolog / Anaphase-promoting complex subunit CDC26 / Anaphase-promoting complex subunit 16 / Anaphase-promoting complex subunit 13 / Anaphase-promoting complex subunit 1 / Anaphase-promoting complex subunit 11 / Cell division cycle protein 23 homolog / Anaphase-promoting complex subunit 7 / Anaphase-promoting complex subunit 5 / Anaphase-promoting complex subunit 4 / Anaphase-promoting complex subunit 2 / Anaphase-promoting complex subunit 10
Similarity search - Component
Biological speciesHomo sapiens (human) / Saccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 3.9 Å
AuthorsZhang S / Chang L / Alfieri C / Zhang Z / Yang J / Maslen S / Skehel M / Barford D
CitationJournal: Nature / Year: 2016
Title: Molecular mechanism of APC/C activation by mitotic phosphorylation.
Authors: Suyang Zhang / Leifu Chang / Claudio Alfieri / Ziguo Zhang / Jing Yang / Sarah Maslen / Mark Skehel / David Barford /
Abstract: In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation ...In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our results reveal the mechanism for the regulation of mitotic APC/C by phosphorylation and provide a rationale for the development of selective inhibitors of this state.
History
DepositionMar 15, 2016-
Header (metadata) releaseApr 27, 2016-
Map releaseApr 27, 2016-
UpdateJun 1, 2016-
Current statusJun 1, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5g04
  • Surface level: 0.08
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3385.jpg

Downloads & links

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Map

FileDownload / File: emd_3385.map.gz / Format: CCP4 / Size: 85.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle particle reconstruction of the human APC/C-Cdc20-Hsl1 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.36 Å/pix.
x 284 pix.
= 386.24 Å		1.36 Å/pix.
x 284 pix.
= 386.24 Å		1.36 Å/pix.
x 284 pix.
= 386.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.36 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.1960343 - 0.39152479
Average (Standard dev.)0.00180691 (±0.01470103)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions284284284
Spacing284284284
CellA=B=C: 386.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.361.361.36
M x/y/z284284284
origin x/y/z0.0000.0000.000
length x/y/z386.240386.240386.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS284284284
D min/max/mean-0.1960.3920.002

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Supplemental data

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Sample components

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Entire : Recombinant human APC/C-Cdc20-Hsl1 complex

EntireName: Recombinant human APC/C-Cdc20-Hsl1 complex
Components
  • Sample: Recombinant human APC/C-Cdc20-Hsl1 complex
  • Protein or peptide: Anaphase-promoting complex subunit 1
  • Protein or peptide: Anaphase-promoting complex subunit 2
  • Protein or peptide: Anaphase-promoting complex subunit 3
  • Protein or peptide: Anaphase-promoting complex subunit 4
  • Protein or peptide: Anaphase-promoting complex subunit 5
  • Protein or peptide: Anaphase-promoting complex subunit 6
  • Protein or peptide: Anaphase-promoting complex subunit 7
  • Protein or peptide: Anaphase-promoting complex subunit 8
  • Protein or peptide: Anaphase-promoting complex subunit 10
  • Protein or peptide: Anaphase-promoting complex subunit 11
  • Protein or peptide: Anaphase-promoting complex subunit 12
  • Protein or peptide: Anaphase-promoting complex subunit 13
  • Protein or peptide: Anaphase-promoting complex subunit 15
  • Protein or peptide: Anaphase-promoting complex subunit 16
  • Protein or peptide: Cell division cycle protein 20 homolog
  • Protein or peptide: Hsl1

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Supramolecule #1000: Recombinant human APC/C-Cdc20-Hsl1 complex

SupramoleculeName: Recombinant human APC/C-Cdc20-Hsl1 complex / type: sample / ID: 1000 / Number unique components: 16
Molecular weightExperimental: 1.2 MDa / Theoretical: 1.2 MDa

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Macromolecule #1: Anaphase-promoting complex subunit 1

MacromoleculeName: Anaphase-promoting complex subunit 1 / type: protein_or_peptide / ID: 1 / Name.synonym: Apc1 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 216.5 KDa / Theoretical: 216.5 KDa
Recombinant expressionOrganism: baculovirus-insect cells / Recombinant cell: High Five insect cells / Recombinant plasmid: pF1, pU1
SequenceUniProtKB: Anaphase-promoting complex subunit 1

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Macromolecule #2: Anaphase-promoting complex subunit 2

MacromoleculeName: Anaphase-promoting complex subunit 2 / type: protein_or_peptide / ID: 2 / Name.synonym: Apc2 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 93.8 KDa / Theoretical: 93.8 KDa
Recombinant expressionOrganism: baculovirus-insect cells / Recombinant cell: High Five insect cells / Recombinant plasmid: pF1, pU1
SequenceUniProtKB: Anaphase-promoting complex subunit 2

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Macromolecule #3: Anaphase-promoting complex subunit 3

MacromoleculeName: Anaphase-promoting complex subunit 3 / type: protein_or_peptide / ID: 3 / Name.synonym: Apc3 / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 91.8 KDa / Theoretical: 91.8 KDa
Recombinant expressionOrganism: baculovirus-insect cells / Recombinant cell: High Five insect cells / Recombinant plasmid: pF1, pU1
SequenceUniProtKB: Cell division cycle protein 27 homolog

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Macromolecule #4: Anaphase-promoting complex subunit 4

MacromoleculeName: Anaphase-promoting complex subunit 4 / type: protein_or_peptide / ID: 4 / Name.synonym: Apc4 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 92.1 KDa / Theoretical: 92.1 KDa
Recombinant expressionOrganism: baculovirus-insect cells / Recombinant cell: High Five insect cells / Recombinant plasmid: pF1, pU1
SequenceUniProtKB: Anaphase-promoting complex subunit 4

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Macromolecule #5: Anaphase-promoting complex subunit 5

MacromoleculeName: Anaphase-promoting complex subunit 5 / type: protein_or_peptide / ID: 5 / Name.synonym: Apc5 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 85.1 KDa / Theoretical: 85.1 KDa
Recombinant expressionOrganism: baculovirus-insect cells / Recombinant cell: High Five insect cells / Recombinant plasmid: pF1, pU1
SequenceUniProtKB: Anaphase-promoting complex subunit 5

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Macromolecule #6: Anaphase-promoting complex subunit 6

MacromoleculeName: Anaphase-promoting complex subunit 6 / type: protein_or_peptide / ID: 6 / Name.synonym: Apc6 / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 71.7 KDa / Theoretical: 71.7 KDa
Recombinant expressionOrganism: baculovirus-insect cells / Recombinant cell: High Five insect cells / Recombinant plasmid: pF1, pU1
SequenceUniProtKB: Cell division cycle protein 16 homolog

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Macromolecule #7: Anaphase-promoting complex subunit 7

MacromoleculeName: Anaphase-promoting complex subunit 7 / type: protein_or_peptide / ID: 7 / Name.synonym: Apc7 / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 66.9 KDa / Theoretical: 66.9 KDa
Recombinant expressionOrganism: baculovirus-insect cells / Recombinant cell: High Five insect cells / Recombinant plasmid: pF1, pU1
SequenceUniProtKB: Anaphase-promoting complex subunit 7

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Macromolecule #8: Anaphase-promoting complex subunit 8

MacromoleculeName: Anaphase-promoting complex subunit 8 / type: protein_or_peptide / ID: 8 / Name.synonym: Apc8 / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 68.8 KDa / Theoretical: 68.8 KDa
Recombinant expressionOrganism: baculovirus-insect cells / Recombinant cell: High Five insect cells / Recombinant plasmid: pF1, pU1
SequenceUniProtKB: Cell division cycle protein 23 homolog

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Macromolecule #9: Anaphase-promoting complex subunit 10

MacromoleculeName: Anaphase-promoting complex subunit 10 / type: protein_or_peptide / ID: 9 / Name.synonym: Apc10 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 21.3 KDa / Theoretical: 21.3 KDa
Recombinant expressionOrganism: baculovirus-insect cells / Recombinant cell: High Five insect cells / Recombinant plasmid: pF1, pU1
SequenceUniProtKB: Anaphase-promoting complex subunit 10

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Macromolecule #10: Anaphase-promoting complex subunit 11

MacromoleculeName: Anaphase-promoting complex subunit 11 / type: protein_or_peptide / ID: 10 / Name.synonym: Apc11 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 9.8 KDa / Theoretical: 9.8 KDa
Recombinant expressionOrganism: baculovirus-insect cells / Recombinant cell: High Five insect cells / Recombinant plasmid: pF1, pU1
SequenceUniProtKB: Anaphase-promoting complex subunit 11

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Macromolecule #11: Anaphase-promoting complex subunit 12

MacromoleculeName: Anaphase-promoting complex subunit 12 / type: protein_or_peptide / ID: 11 / Name.synonym: Apc12 / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 9.8 KDa / Theoretical: 9.8 KDa
Recombinant expressionOrganism: baculovirus-insect cells / Recombinant cell: High Five insect cells / Recombinant plasmid: pF1, pU1
SequenceUniProtKB: Anaphase-promoting complex subunit CDC26

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Macromolecule #12: Anaphase-promoting complex subunit 13

MacromoleculeName: Anaphase-promoting complex subunit 13 / type: protein_or_peptide / ID: 12 / Name.synonym: Apc13 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 8.5 KDa / Theoretical: 8.5 KDa
Recombinant expressionOrganism: baculovirus-insect cells / Recombinant cell: High Five insect cells / Recombinant plasmid: pF1, pU1
SequenceUniProtKB: Anaphase-promoting complex subunit 13

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Macromolecule #13: Anaphase-promoting complex subunit 15

MacromoleculeName: Anaphase-promoting complex subunit 15 / type: protein_or_peptide / ID: 13 / Name.synonym: Apc15 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 14.3 KDa / Theoretical: 14.3 KDa
Recombinant expressionOrganism: baculovirus-insect cells / Recombinant cell: High Five insect cells / Recombinant plasmid: pF1, pU1
SequenceUniProtKB: Anaphase-promoting complex subunit 15

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Macromolecule #14: Anaphase-promoting complex subunit 16

MacromoleculeName: Anaphase-promoting complex subunit 16 / type: protein_or_peptide / ID: 14 / Name.synonym: Apc16 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 11.7 KDa / Theoretical: 11.7 KDa
Recombinant expressionOrganism: baculovirus-insect cells / Recombinant cell: High Five insect cells / Recombinant plasmid: pF1, pU1
SequenceUniProtKB: Anaphase-promoting complex subunit 16

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Macromolecule #15: Cell division cycle protein 20 homolog

MacromoleculeName: Cell division cycle protein 20 homolog / type: protein_or_peptide / ID: 15 / Name.synonym: Cdc20 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 54.7 KDa / Theoretical: 54.7 KDa
Recombinant expressionOrganism: baculovirus-insect cells / Recombinant cell: High Five insect cells / Recombinant plasmid: modified pFastBac HTa
SequenceUniProtKB: Cell division cycle protein 20 homolog

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Macromolecule #16: Hsl1

MacromoleculeName: Hsl1 / type: protein_or_peptide / ID: 16 / Name.synonym: Hsl1 / Details: truncated construct of Hsl1 (residues 667-872) / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 24.6 KDa / Theoretical: 24.6 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 Star / Recombinant plasmid: pET28a
SequenceUniProtKB: Probable serine/threonine-protein kinase HSL1

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.15 mg/mL
BufferpH: 8 / Details: 20mM Hepes, 150mM NaCl, 0.005mM TCEP
StainingType: NEGATIVE / Details: Vitrification in liquid ethane
GridDetails: 200 mesh Quantifoil R2/2 copper grid with thin carbon support, treated with a 9:1 argon:oxygen plasma cleaner for 20-40s
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 100 K / Instrument: FEI VITROBOT MARK III
Method: The grids were incubated for 30s at 4 degree and 100% humidity before blotting for 5s and plunging into liquid ethane

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 90 K / Max: 120 K / Average: 105 K
Specialist opticsEnergy filter - Name: FEI
DateOct 30, 2015
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 11000 / Average electron dose: 27 e/Å2
Details: The exposure time for each micrograph was 2s at a dose rate of 27 electrons/angstrom2/s. 34 movie frames were recorded for each micrograph.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 78000
Sample stageSpecimen holder: liquid nitrogen cooled / Specimen holder model: OTHER
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsParticles were selected by automatic particle picking in RELION
CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 179660

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  • 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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