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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-3385 | |||||||||
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Title | Cryo-EM structure of the human APC/C-Cdc20-Hsl1 complex | |||||||||
![]() | Single particle reconstruction of the human APC/C-Cdc20-Hsl1 complex | |||||||||
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![]() | Cell cycle / phosphorylation / mitosis / ubiquitination | |||||||||
Function / homology | ![]() 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 / Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase ...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 / Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase / regulation of mitotic cell cycle spindle assembly checkpoint / positive regulation of synapse maturation / 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 / metaphase/anaphase transition of mitotic cell cycle / anaphase-promoting complex-dependent catabolic process / 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 / Regulation of APC/C activators between G1/S and early anaphase / 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 mitotic cell cycle / cullin family protein binding / Transcriptional Regulation by VENTX / ubiquitin-like ligase-substrate adaptor activity / mitotic spindle assembly / 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 / 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 / CDK-mediated phosphorylation and removal of Cdc6 / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / protein catabolic process / brain development / mitotic spindle / kinetochore / G2/M transition of mitotic cell cycle / spindle pole / spindle / Separation of Sister Chromatids / ubiquitin-protein transferase activity / microtubule cytoskeleton / ubiquitin protein ligase activity / Antigen processing: Ubiquitination & Proteasome degradation / Senescence-Associated Secretory Phenotype (SASP) / mitotic cell cycle / nervous system development / protein phosphatase binding / ubiquitin-dependent protein catabolic process / cell differentiation / molecular adaptor activity / regulation of cell cycle / non-specific serine/threonine protein kinase / 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 / cytoplasm / cytosol Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / negative staining / Resolution: 3.9 Å | |||||||||
![]() | Zhang S / Chang L / Alfieri C / Zhang Z / Yang J / Maslen S / Skehel M / Barford D | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 10 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 26.1 KB 26.1 KB | Display Display | ![]() |
Images | ![]() | 76.9 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 5g04MC ![]() 3386C ![]() 3387C ![]() 3388C ![]() 3389C ![]() 3390C ![]() 5g05C M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Single particle reconstruction of the human APC/C-Cdc20-Hsl1 complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.36 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
+Entire : Recombinant human APC/C-Cdc20-Hsl1 complex
+Supramolecule #1000: Recombinant human APC/C-Cdc20-Hsl1 complex
+Macromolecule #1: Anaphase-promoting complex subunit 1
+Macromolecule #2: Anaphase-promoting complex subunit 2
+Macromolecule #3: Anaphase-promoting complex subunit 3
+Macromolecule #4: Anaphase-promoting complex subunit 4
+Macromolecule #5: Anaphase-promoting complex subunit 5
+Macromolecule #6: Anaphase-promoting complex subunit 6
+Macromolecule #7: Anaphase-promoting complex subunit 7
+Macromolecule #8: Anaphase-promoting complex subunit 8
+Macromolecule #9: Anaphase-promoting complex subunit 10
+Macromolecule #10: Anaphase-promoting complex subunit 11
+Macromolecule #11: Anaphase-promoting complex subunit 12
+Macromolecule #12: Anaphase-promoting complex subunit 13
+Macromolecule #13: Anaphase-promoting complex subunit 15
+Macromolecule #14: Anaphase-promoting complex subunit 16
+Macromolecule #15: Cell division cycle protein 20 homolog
+Macromolecule #16: Hsl1
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.15 mg/mL |
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Buffer | pH: 8 / Details: 20mM Hepes, 150mM NaCl, 0.005mM TCEP |
Staining | Type: NEGATIVE / Details: Vitrification in liquid ethane |
Grid | Details: 200 mesh Quantifoil R2/2 copper grid with thin carbon support, treated with a 9:1 argon:oxygen plasma cleaner for 20-40s |
Vitrification | Cryogen 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
Microscope | FEI POLARA 300 |
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Temperature | Min: 90 K / Max: 120 K / Average: 105 K |
Specialist optics | Energy filter - Name: FEI |
Date | Oct 30, 2015 |
Image recording | Category: 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 beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination 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 stage | Specimen holder: liquid nitrogen cooled / Specimen holder model: OTHER |
Experimental equipment | ![]() Model: Tecnai Polara / Image courtesy: FEI Company |
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Image processing
Details | Particles were selected by automatic particle picking in RELION |
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CTF correction | Details: Each micrograph |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 179660 |