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Yorodumi- EMDB-2870: Cryo-EM structure of the multimeric complex between Dark and Dron... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2870 | |||||||||
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Title | Cryo-EM structure of the multimeric complex between Dark and Dronc-CARD. | |||||||||
Map data | Reconstruction of the multimeric complex between Dark and Dronc-CARD. | |||||||||
Sample |
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Keywords | Cryo-EM / apoptosome / single particle analysis | |||||||||
Function / homology | Function and homology information hemocyte development / nurse cell apoptotic process / negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / Formation of apoptosome / Regulation of the apoptosome activity / head involution / embryonic development via the syncytial blastoderm / salivary gland histolysis / positive regulation of compound eye retinal cell programmed cell death ...hemocyte development / nurse cell apoptotic process / negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / Formation of apoptosome / Regulation of the apoptosome activity / head involution / embryonic development via the syncytial blastoderm / salivary gland histolysis / positive regulation of compound eye retinal cell programmed cell death / melanization defense response / sarcosine catabolic process / central nervous system formation / metamorphosis / : / compound eye development / chaeta development / sperm individualization / apoptosome / programmed cell death involved in cell development / S-adenosylmethionine cycle / Neutrophil degranulation / programmed necrotic cell death / CARD domain binding / programmed cell death / cysteine-type endopeptidase activity involved in apoptotic signaling pathway / triglyceride homeostasis / zymogen activation / dendrite morphogenesis / neuron remodeling / response to starvation / cysteine-type endopeptidase activator activity involved in apoptotic process / protein autoprocessing / ectopic germ cell programmed cell death / central nervous system development / regulation of autophagy / determination of adult lifespan / ADP binding / response to gamma radiation / neuron cellular homeostasis / endopeptidase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / positive regulation of apoptotic process / negative regulation of cell population proliferation / cysteine-type endopeptidase activity / apoptotic process / protein homodimerization activity / ATP binding / identical protein binding / nucleus / plasma membrane / cytoplasm Similarity search - Function | |||||||||
Biological species | Drosophila melanogaster (fruit fly) | |||||||||
Method | single particle reconstruction / cryo EM / negative staining / Resolution: 4.1 Å | |||||||||
Authors | Pang YX / Bai XC / Hao Q / Yan CY / Chen ZQ / Wang JW / Scheres SHW / Shi YG | |||||||||
Citation | Journal: Genes Dev / Year: 2015 Title: Structure of the apoptosome: mechanistic insights into activation of an initiator caspase from Drosophila. Authors: Yuxuan Pang / Xiao-chen Bai / Chuangye Yan / Qi Hao / Zheqin Chen / Jia-Wei Wang / Sjors H W Scheres / Yigong Shi / Abstract: Apoptosis is executed by a cascade of caspase activation. The autocatalytic activation of an initiator caspase, exemplified by caspase-9 in mammals or its ortholog, Dronc, in fruit flies, is ...Apoptosis is executed by a cascade of caspase activation. The autocatalytic activation of an initiator caspase, exemplified by caspase-9 in mammals or its ortholog, Dronc, in fruit flies, is facilitated by a multimeric adaptor complex known as the apoptosome. The underlying mechanism by which caspase-9 or Dronc is activated by the apoptosome remains unknown. Here we report the electron cryomicroscopic (cryo-EM) structure of the intact apoptosome from Drosophila melanogaster at 4.0 Å resolution. Analysis of the Drosophila apoptosome, which comprises 16 molecules of the Dark protein (Apaf-1 ortholog), reveals molecular determinants that support the assembly of the 2.5-MDa complex. In the absence of dATP or ATP, Dronc zymogen potently induces formation of the Dark apoptosome, within which Dronc is efficiently activated. At 4.1 Å resolution, the cryo-EM structure of the Dark apoptosome bound to the caspase recruitment domain (CARD) of Dronc (Dronc-CARD) reveals two stacked rings of Dronc-CARD that are sandwiched between two octameric rings of the Dark protein. The specific interactions between Dronc-CARD and both the CARD and the WD40 repeats of a nearby Dark protomer are indispensable for Dronc activation. These findings reveal important mechanistic insights into the activation of initiator caspase by the apoptosome. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2870.map.gz | 115.3 MB | EMDB map data format | |
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Header (meta data) | emd-2870-v30.xml emd-2870.xml | 10.7 KB 10.7 KB | Display Display | EMDB header |
Images | EMD_2870.png | 239.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2870 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2870 | HTTPS FTP |
-Related structure data
Related structure data | 3j9kMC 2871C 3j9lC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_2870.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of the multimeric complex between Dark and Dronc-CARD. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.34 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Dark apoptosome in complex with Dronc-CARD.
Entire | Name: Dark apoptosome in complex with Dronc-CARD. |
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Components |
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-Supramolecule #1000: Dark apoptosome in complex with Dronc-CARD.
Supramolecule | Name: Dark apoptosome in complex with Dronc-CARD. / type: sample / ID: 1000 / Oligomeric state: homo 16 mers / Number unique components: 2 |
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Molecular weight | Experimental: 3 MDa / Theoretical: 3 MDa |
-Macromolecule #1: Dark apoptosome
Macromolecule | Name: Dark apoptosome / type: protein_or_peptide / ID: 1 / Oligomeric state: 16 mers / Recombinant expression: Yes |
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Source (natural) | Organism: Drosophila melanogaster (fruit fly) / synonym: fruit fly / Cell: Hi-5 insect cells |
Molecular weight | Experimental: 3 MDa / Theoretical: 3 MDa |
Recombinant expression | Organism: Drosophila melanogaster (fruit fly) / Recombinant cell: Hi-5 insect cells / Recombinant plasmid: pFastBac1 |
-Macromolecule #2: Dronc-CARD
Macromolecule | Name: Dronc-CARD / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: No |
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Source (natural) | Organism: Drosophila melanogaster (fruit fly) / synonym: fruit fly |
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.15 mg/mL |
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Buffer | pH: 8 / Details: 25mM Tris, 150mM NaCl, 5mM dithiothreitol |
Staining | Type: NEGATIVE Details: Grids were blotted for 2 seconds and flash frozen in liquid ethane using an FEI Vitrobot. |
Grid | Details: Samples were placed on glow-discharged holey carbon grids (Quantifoil CuR2/2), on which a home-made continuous carbon film had previously been deposited. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 85 K / Instrument: FEI VITROBOT MARK II / Method: Blot for 2 seconds before plunging |
-Electron microscopy
Microscope | FEI POLARA 300 |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 104748 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 6.4 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 78000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
Temperature | Min: 80 K / Max: 90 K / Average: 85 K |
Date | Jan 8, 2014 |
Image recording | Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 1234 / Average electron dose: 28 e/Å2 Details: 16 video frames were recorded in 1s by FalconII detector. |
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: Each particle |
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Final reconstruction | Applied symmetry - Point group: D8 (2x8 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION Details: To correct for beam-induced movements, the 16 video frames for each micrograph were first aligned using whole-image motion correction. Second, particle based beam-induced movement correction ...Details: To correct for beam-induced movements, the 16 video frames for each micrograph were first aligned using whole-image motion correction. Second, particle based beam-induced movement correction was performed using statistical movie processing in RELION. Number images used: 11359 |