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- PDB-5jul: Near atomic structure of the Dark apoptosome -

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Basic information

Entry
Database: PDB / ID: 5jul
TitleNear atomic structure of the Dark apoptosome
ComponentsApaf-1 related killer DARK
KeywordsAPOPTOSIS / Dark / apoptosome / apotosis / AAA+ ATPase
Function / homology
Function and homology information


negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / Formation of apoptosome / Regulation of the apoptosome activity / salivary gland histolysis / positive regulation of compound eye retinal cell programmed cell death / melanization defense response / sarcosine catabolic process / central nervous system formation / : ...negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / Formation of apoptosome / Regulation of the apoptosome activity / salivary gland histolysis / positive regulation of compound eye retinal cell programmed cell death / melanization defense response / sarcosine catabolic process / central nervous system formation / : / chaeta development / sperm individualization / apoptosome / S-adenosylmethionine cycle / Neutrophil degranulation / CARD domain binding / programmed cell death / triglyceride homeostasis / dendrite morphogenesis / response to starvation / cysteine-type endopeptidase activator activity involved in apoptotic process / ADP binding / response to gamma radiation / neuron cellular homeostasis / positive regulation of apoptotic process / ATP binding / identical protein binding
Similarity search - Function
APAF-1 helical domain / APAF-1 helical domain / NB-ARC / NB-ARC domain / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / Apaf-1 related killer DARK
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsCheng, T.C. / Akey, I.V. / Yuan, S. / Yu, Z. / Ludtke, S.J. / Akey, C.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RO1 GM63834 United States
CitationJournal: Structure / Year: 2017
Title: A Near-Atomic Structure of the Dark Apoptosome Provides Insight into Assembly and Activation.
Authors: Tat Cheung Cheng / Ildikó V Akey / Shujun Yuan / Zhiheng Yu / Steven J Ludtke / Christopher W Akey /
Abstract: In Drosophila, the Apaf-1-related killer (Dark) forms an apoptosome that activates procaspases. To investigate function, we have determined a near-atomic structure of Dark double rings using cryo- ...In Drosophila, the Apaf-1-related killer (Dark) forms an apoptosome that activates procaspases. To investigate function, we have determined a near-atomic structure of Dark double rings using cryo-electron microscopy. We then built a nearly complete model of the apoptosome that includes 7- and 8-blade β-propellers. We find that the preference for dATP during Dark assembly may be governed by Ser325, which is in close proximity to the 2' carbon of the deoxyribose ring. Interestingly, β-propellers in V-shaped domains of the Dark apoptosome are more widely separated, relative to these features in the Apaf-1 apoptosome. This wider spacing may be responsible for the lack of cytochrome c binding to β-propellers in the Dark apoptosome. Our structure also highlights the roles of two loss-of-function mutations that may block Dark assembly. Finally, the improved model provides a framework to understand apical procaspase activation in the intrinsic cell death pathway.
History
DepositionMay 10, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 22, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 27, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Jul 18, 2018Group: Data collection / Experimental preparation / Category: em_sample_support / Item: _em_sample_support.grid_type
Revision 1.3Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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  • Deposited structure unit
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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Apaf-1 related killer DARK
B: Apaf-1 related killer DARK
C: Apaf-1 related killer DARK
D: Apaf-1 related killer DARK
E: Apaf-1 related killer DARK
F: Apaf-1 related killer DARK
G: Apaf-1 related killer DARK
H: Apaf-1 related killer DARK
I: Apaf-1 related killer DARK
J: Apaf-1 related killer DARK
K: Apaf-1 related killer DARK
L: Apaf-1 related killer DARK
M: Apaf-1 related killer DARK
N: Apaf-1 related killer DARK
O: Apaf-1 related killer DARK
P: Apaf-1 related killer DARK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,665,95532
Polymers2,658,09616
Non-polymers7,85916
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Apaf-1 related killer DARK / Apaf-1/CED-4-related caspase activator Dapaf-1L / Cell death protein HAC-1 / Death-associated APAF1- ...Apaf-1/CED-4-related caspase activator Dapaf-1L / Cell death protein HAC-1 / Death-associated APAF1-related killer / isoform B / FI21208p1


Mass: 166131.000 Da / Num. of mol.: 16
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: Dark, Ark, Ark-RB, dapaf-1L, Hac1, CG6829, Dmel_CG6829
Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q7KLI1
#2: Chemical
ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / Deoxyadenosine triphosphate


Mass: 491.182 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C10H16N5O12P3

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Dark apoptosome / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 2.3 MDa / Experimental value: NO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Plasmid: pFastBac
Buffer solutionpH: 7.5 / Details: Buffer A
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHepes1
220 mMpotassium chlorideKCl1
31 mMEDTAEthylenediaminetetraacetic acid1
41 mMEGTA1
51.5 mMmagnesium chlorideMg2Cl21
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K
Details: 2.5ul sample blot additives present at the indicated final concentrations: NP-40 (0.025%), DHPG (diheptanoylphosphatidylglycerol; 0.05%), cytochrome c (0.5 mg/ml), DeoxybigChaps (0.01%), and lysine (0.03 mg/ml)

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1500 nm / Cs: 0.01 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.3 sec. / Electron dose: 1.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1991
EM imaging opticsEnergyfilter name: GIF / Spherical aberration corrector: FEI Cs corrector
Image scansWidth: 7476 / Height: 7420 / Movie frames/image: 23 / Used frames/image: 2-23

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Processing

EM software
IDNameVersionCategory
1e2boxerparticle selection
2SerialEMimage acquisition
4CTFFIND3CTF correction
7MDFFmodel fitting
9RELION1.3initial Euler assignment
10RELION1.3final Euler assignment
11RELION1.3classification
12RELION1.33D reconstruction
19PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 88485
SymmetryPoint symmetry: D8 (2x8 fold dihedral)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 17769 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Details: local fitting with Chimera followed by flexible fitting with MDFF and refinement with Phenix.

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