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- EMDB-8177: Near atomic structure of the Dark apoptosome -

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Basic information

Entry
Database: EMDB / ID: EMD-8177
TitleNear atomic structure of the Dark apoptosome
Map dataNear atomic structure of the Dark apoptosome
Sample
  • Complex: Dark apoptosome
    • Protein or peptide: Apaf-1 related killer DARK
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
Function / homology
Function and homology information


negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / Formation of apoptosome / salivary gland histolysis / positive regulation of compound eye retinal cell programmed cell death / melanization defense response / sarcosine catabolic process / Activation of caspases through apoptosome-mediated cleavage / Regulation of the apoptosome activity / central nervous system formation ...negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / Formation of apoptosome / salivary gland histolysis / positive regulation of compound eye retinal cell programmed cell death / melanization defense response / sarcosine catabolic process / Activation of caspases through apoptosome-mediated cleavage / Regulation of the apoptosome activity / central nervous system formation / chaeta development / sperm individualization / apoptosome / autophagic cell death / Neutrophil degranulation / CARD domain binding / S-adenosylmethionine cycle / programmed cell death / triglyceride homeostasis / dendrite morphogenesis / response to starvation / cysteine-type endopeptidase activator activity involved in apoptotic process / response to gamma radiation / ADP binding / neuron cellular homeostasis / positive regulation of apoptotic process / ATP binding / identical protein binding
Similarity search - Function
: / : / Dark, CARD domain / Dark, winged-helix domain / APAF-1 helical domain / APAF-1 helical domain / NB-ARC / NB-ARC domain / WD40 repeats / WD40 repeat ...: / : / Dark, CARD domain / Dark, winged-helix domain / APAF-1 helical domain / APAF-1 helical domain / NB-ARC / NB-ARC domain / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Apaf-1 related killer DARK
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsCheng TC / Akey IV / Yuan S / Yu Z / Ludtke SJ / Akey CW
Funding support United States, 1 items
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
DepositionJun 17, 2016-
Header (metadata) releaseFeb 22, 2017-
Map releaseFeb 22, 2017-
UpdateDec 25, 2019-
Current statusDec 25, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5jul
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8177.png

Downloads & links

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Map

FileDownload / File: emd_8177.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNear atomic structure of the Dark apoptosome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 320 pix.
= 432. Å		1.35 Å/pix.
x 320 pix.
= 432. Å		1.35 Å/pix.
x 320 pix.
= 432. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.15139636 - 0.2221533
Average (Standard dev.)0.0009608244 (±0.0073119937)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 432.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z432.000432.000432.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1510.2220.001

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Supplemental data

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Additional map: Near atomic structure of the Dark apoptosome

Fileemd_8177_additional.map
AnnotationNear atomic structure of the Dark apoptosome
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dark apoptosome

EntireName: Dark apoptosome
Components
  • Complex: Dark apoptosome
    • Protein or peptide: Apaf-1 related killer DARK
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

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Supramolecule #1: Dark apoptosome

SupramoleculeName: Dark apoptosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBac
Molecular weightTheoretical: 2.3 MDa

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Macromolecule #1: Apaf-1 related killer DARK

MacromoleculeName: Apaf-1 related killer DARK / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 166.131 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDFETGEHQY QYKDILSVFE DAFVDNFDCK DVQDMPKSIL SKEEIDHIIM SKDAVSGTLR LFWTLLSKQE EMVQKFVEEV LRINYKFLM SPIKTEQRQP SMMTRMYIEQ RDRLYNDNQV FAKYNVSRLQ PYLKLRQALL ELRPAKNVLI DGVLGSGKTW V ALDVCLSY ...String:
MDFETGEHQY QYKDILSVFE DAFVDNFDCK DVQDMPKSIL SKEEIDHIIM SKDAVSGTLR LFWTLLSKQE EMVQKFVEEV LRINYKFLM SPIKTEQRQP SMMTRMYIEQ RDRLYNDNQV FAKYNVSRLQ PYLKLRQALL ELRPAKNVLI DGVLGSGKTW V ALDVCLSY KVQCKMDFKI FWLNLKNCNS PETVLEMLQK LLYQIDPNWT SRSDHSSNIK LRIHSIQAEL RRLLKSKPYE NC LLVLLNV QNA(APK)AWNAFN LSCKILLTTR FKQVTDFLSA ATTTHISLDH HSMTLTPDEV KSLLLKYLDC RPQDLPREV LTTNPRRLSI IAESIRDGLA TWDNWKHVNC DKLTTIIESS LNVLEPAEYR KMFDRLSVFP PSAHIPTILL SLIWFDVIKS DVMVVVNKL HKYSLVEKQP KESTISIPSI YLELKVKLEN EYALHRSIVD HYNIPKTFDS DDLIPPYLDQ YFYSHIGHHL K NIEHPERM TLFRMVFLDF RFLEQKIRHD STAWNASGSI LNTLQQLKFY KPYICDNDPK YERLVNAILD FLPKIEENLI CS KYTDLLR IALMAEDEAI FEEAHKQVQR FDDRVWFTNH GRFHQHRQII NLGDNEGRHA VYLHNDFCLI ALASGQILLT DVS LEGEDT YLLRDESDSS DILRMAVFNQ QKHLITLHCN GSVKLWSLWP DCPGRRHSGG SKQQLVNSVV KRFIGSYANL KIVA FYLNE DAGLPEANIQ LHVAFINGDV SILNWDEQDQ EFKLSHVPVL KTMQSGIRCF VQVLKRYYVV CTSNCTLTVW DLTNG SSNT LELHVFNVEN DTPLALDVFD ERSKTATVLL IFKYSVWRLN FLPGLSVSLQ SEAVQLPEGS FITCGKRSTD GRYLLL GTS EGLIVYDLKI SDPVLRSNVS EHIECVDIYE LFDPVYKYIV LCGAKGKQVV HVHTLRSVSG SNSHQNREIA WVHSADE IS VMTKACLEPN VYLRSLMDMT RERTQLLAVD SKERIHLIKP AISRISEWST ITPTHAASNC KINAISAFND EQIFVGYV D GVIIDVIHDT ALPQQFIEEP IDYLKQVSPN ILVASAHSAQ KTVIFQLEKI DPLQPNDQWP LMMDVSTKYA SLQEGQYII LFSDHGVCHL DIANPSAFVK PKDSEEYIVG FDLKNSLLFL AYENNIIDVF RLIFSCNQLR YEQICEEEIA QKAKISYLVA TDDGTMLAM GFENGTLELF AVENRKVQLI YSIEEVHEHC IRQLLFSPCK LLLISCAEQL CFWNVTHMRN NQLEREQKRR R SRRHKQHS VTQEDAVDAA PIAADIDVDV TFVADEFHPV NRGTAELWRN KRGNAIRPEL LACVKFVGNE ARQFFTDAHF SH FYAIDDE GVYYHLQLLE LSRLQPPPDP VTLDIANQYE DLKNLRILDS PLMQDSDSEG ADVVGNLVLE KNGGVARATP ILE EASS

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Macromolecule #2: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 16 / Formula: DTP
Molecular weightTheoretical: 491.182 Da
Chemical component information

ChemComp-DTP:
2'-DEOXYADENOSINE 5'-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

Concentration2.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
10.0 mMHepes
20.0 mMpotassium chlorideKCl
1.0 mMEDTA
1.0 mMEGTA
1.5 mMmagnesium chlorideMg2Cl2

Details: Buffer A
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK III
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 ...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

MicroscopeFEI TITAN KRIOS
Specialist opticsSpherical aberration corrector: FEI Cs corrector / Energy filter - Name: GIF
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 7476 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Frames/image: 2-23 / Number grids imaged: 1 / Number real images: 1991 / Average exposure time: 0.3 sec. / Average electron dose: 1.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 88485
CTF correctionSoftware - Name: CTFFIND3
Startup modelType of model: EMDB MAP
EMDB ID:

5235

Final reconstructionApplied symmetry - Point group: D8 (2x8 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 17769
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final 3D classificationSoftware - Name: RELION (ver. 1.3)

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Atomic model buiding 1

Detailslocal fitting with Chimera followed by flexible fitting with MDFF and refinement with Phenix.
RefinementProtocol: FLEXIBLE FIT
Output model

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

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