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Structure of the Drosophila apoptosome

by single particle reconstruction, at 6.9 A resolution

Movie

Orientation:

#1: Depositted structure unit, Made by Jmol

#2: Superimposing with simplified surface model of EM map, EMDB-5235, Made by Jmol

#3: With PDB-3iz8, Made by Jmol

#4: Superimposing with EM 3D map: EMDB-5235 (with PDB-3iz8), Made by UCSF CHIMERA

Entry
Summary
Database / IDPORTEIN DATA BANK (PDB) / 1vt4
TitleStructure of the Drosophila apoptosome
DescriptorApaf-1 related killer DARK
KeywordsAPOPTOSIS, Drosophila apoptosome, apoptosis, programmed cell death
AuthorsYuan, S., Topf, M., Akey, C.W., Ludtke, S.J.
DateDeposition: 2010-11-09, Release: 2011-01-19
PDBj Mine pagesSummary, Structural Details, Experimental Details, Functional Details
Other databasesRCSB-PDB, PDBe, CATH, CE, FSSP, SCOP, VAST
Sequence detailsAUTHORS STATE THAT THE ACTUAL SEQUENCE FOR THE PROTEIN IS:
Sequence detailsMDFETGEHQYQYKDILSVFEDAFVDNFDCKDVQDMPKSILSKEEIDHIIM SKDAVSGTLR LFWTLLSKQEEMVQKFVEEVLRINYKFLMSPIKTEQRQPSMMTRMYIEQR DRLYNDNQVF AKYNVSRLQPYLKLRQALLELRPAKNVLIDGVLGSGKTWVALDVCLSYKV QCKMDFKIFW LNLKNCNSPETVLEMLQKLLYQIDPNWTSRSDHSSNIKLRIHSIQAELRR LLKSKPYENC LLVLLNVQNAKAWNAFNLSCKILLTTRFKQVTDFLSAATTTHISLDHHSM TLTPDEVKSL LLKYLDCRPQDLPREVLTTNPRRLSIIAESIRDGLATWDNWKHVNCDKLT TIIESSLNVL EPAEYRKMFDRLSVFPPSAHIPTILLSLIWFDVIKSDVMVVVNKLHKYSL VEKQPKESTI SIPSIYLELKVKLENEYALHRSIVDHYNIPKTFDSDDLIPPYLDQYFYSH IGHHLKNIEH PERMTLFRMVFLDFRFLEQKIRHDSTAWNASGSILNTLQQLKFYKPYICD NDPKYERLVN AILDFLPKIEENLICSKYTDLLRIALMAEDEAIFEEAHKQVQRFDDRVWF TNHGRFHQHR QIINLGDNEGRHAVYLHNDFCLIALASGQILLTDVSLEGEDTYLLRDESD SSDILRMAVF NQQKHLITLHCNGSVKLWSLWPDCPGRRHSGGSKQQLVNSVVKRFIGSYA NLKIVAFYLN EDAGLPEANIQLHVAFINGDVSILNWDEQDQEFKLSHVPVLKTMQSGIRC FVQVLKRYYV VCTSNCTLTVWDLTNGSSNTLELHVFNVENDTPLALDVFDERSKTATVLL IFKYSVWRLN FLPGLSVSLQSEAVQLPEGSFITCGKRSTDGRYLLLGTSEGLIVYDLKIS DPVLRSNVSE HIECVDIYELFDPVYKYIVLCGAKGKQVVHVHTLRSVSGSNSHQNREIAW VHSADEISVM TKACLEPNVYLRSLMDMTRERTQLLAVDSKERIHLIKPAISRISEWSTIT PTHAASNCKI NAISAFNDEQIFVGYVDGVIIDVIHDTALPQQFIEEPIDYLKQVSPNILV ASAHSAQKTV IFQLEKIDPLQPNDQWPLMMDVSTKYASLQEGQYIILFSDHGVCHLDIAN PSAFVKPKDS EEYIVGFDLKNSLLFLAYENNIIDVFRLIFSCNQLRYEQICEEEIAQKAK ISYLVATDDG TMLAMGFENGTLELFAVENRKVQLIYSIEEVHEHCIRQLLFSPCKLLLIS CAEQLCFWNV THMRNNQLEREQKRRRSRRHKQHSVTQEDAVDAAPIAADIDVDVTFVADE FHPVNRGTAE LWRNKRGNAIRPELLACVKFVGNEARQFFTDAHFSHFYAIDDEGVYYHLQ LLELSRLQPP PDPVTLDIANQYEDLKNLRILDSPLMQDSDSEGADVVGNLVLEKNGGVAR ATPILEEASS
Structure Visualization
MoviesMovie Page

#1: Depositted structure unit, Made by Jmol

#2: Superimposing with simplified surface model of EM map, EMDB-5235, Made by Jmol

#3: With PDB-3iz8, Made by Jmol

#4: Superimposing with EM 3D map: EMDB-5235 (with PDB-3iz8), Made by UCSF CHIMERA

Structure viewersYorodumi, jV4, Jmol, Biological unit (Images, jV)
Related Structure Data
Related Entries

EMDB-5235
CiteFit

PDB-3iz8
CiteSplit

Cite: data citing same article

Fit: target map of fitting

Split: split data constructing the same model

Similar strucutres (beta)

Diagram

PDB-3iz8

EMDB-2089

PDB-2cse

EMDB-5186
List of similar structure data about Omokage system
Article
Citation - primary
ArticleStructure, Vol. 19, Issue 1, Page 128-40, Year 2011
TitleStructure of the Drosophila apoptosome at 6.9 å resolution.
AuthorsShujun Yuan, Xinchao Yu, Maya Topf, Loretta Dorstyn, Sharad Kumar, Steven J Ludtke, Christopher W Akey
Department of Physiology and Biophysics, Boston University School of Medicine, 700 Albany Street, Boston, MA 02118, USA.
KeywordsAnimals, Apoptosomes (chemistry), Ark protein, Drosophila, Caspases (chemistry, 3.4.22.-), Cryoelectron Microscopy, Drosophila Proteins (chemistry), Drosophila melanogaster (chemistry), Ice protein, Drosophila (3.4.22.-), Nc protein, Drosophila (3.4.22.-), Protein Binding, Protein Interaction Domains and Motifs, Protein Multimerization, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Structure, Tertiary, Structural Homology, Protein
LinksPII: S0969-2126(10)00399-0, DOI: 10.1016/j.str.2010.10.009, PubMed: 21220123, PMC: PMC3053581
Components
ID 1 : Apaf-1-related-killer, isoform B, Apaf-1/CED-4-related caspase activator Dapaf-1L, Cell death protein HAC-1
Image
DescriptionApaf-1 related killer DARK
Typepolymer
Formula weight123196.281 Da
Number of molecules8
SourceMethod: Isolated from a genetically manipulated source
Gene: Fruit fly, Ark, Hac1, dapaf-1L, CG6829, Dmel_CG6829, ID:7227, Drosophila melanogaster
Host: ID:7108, Spodoptera frugiperda, sf21, baculovirus
Plasmid name: pFastBac
LinksUniProt: Q7KLI1, Sequence view
ID 2 : MAGNESIUM ION
Image
DescriptionMAGNESIUM ION
Typenon-polymer
Formula weight24.305 Da
Number of molecules16
SourceMethod: Obtained synthetically
ID 3 : 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
Image
Description2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
Typenon-polymer
Formula weight491.184 Da
Number of molecules16
SourceMethod: Obtained synthetically
Sample
Assembly
Aggregation statePARTICLE
Compositionhexadecamer of Dark molecules
DetailsSample assembled in low salt buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT) at about 0.5 mg per ml with dATP and additional EDTA
Theoretical Mass2.5
NameDrosophila apoptosome (double ring)
Num components1
Entity assembly
DetailsDark is assembled with 10 mM EDTA and 10 mM dATP in low salt buffer
NameApaf-1 related killer DARK
TypePROTEIN
Buffer
NameHEPES buffer
Experiment
Reconstruction methodSINGLE PARTICLE
Specimen typeVITREOUS ICE (CRYO EM)
Sample preparation
Details20mM HEPES, 10mM KCl, 1.5mM MgCl2, 1mM EDTA, 1mM EGTA, 1mM DTT
pH7.5
Sample concentration2 mg/ml
Sample support
DetailsC-flat 2/1 holey grids (400 mesh) covered with a thin carbon film
Vitrification
Cryogen nameETHANE
Detailsblotting at room temperature with sample at room temperature
Humidity100
InstrumentFEI Vitrobot Mark 3
MethodBlot for 2-2.5s before plunging
Temp77 Kelvin
Electron Microscopy
Imaging
Microscopemodel: FEI TECNAI F20
Date2009-09-15
Detailsactual magnification at the ccd 87000, camera pixel size 15um, 1.72 angstrom per pixel, data collected semi-automatically with EMTools (TVIPS)
Electron gun
Electron sourceFIELD EMISSION GUN
Accelerating voltage160 kV
Electron dose20 e/A**2
Illumination modeFLOOD BEAM
Lens
ModeBRIGHT FIELD
Magnificationcalibrated: 50000 X, nominal: 50000 X
Csnominal: 2.0 mm
Astigmatismobjective lens astigmatism was corrected at 200,000 times magnification
Detector distance0.0
Nominal defocusmax: 3000 nm, min: 1500 nm
Specimen holder
Specimen holdermodel: GATAN LIQUID NITROGEN, type: Side entry liquid nitrogen-cooled cryo specimen holder
Tilt anglemin: 0.0 degrees, max: 0.0 degrees
Temperature93 Kelvin
Recording temperaturemaximum: 100, minimum: 93
Detector
TypeTVIPS 4kx4k CCD camera
Processing
2D projection selection
Software nameModeller, Flex-EM, Chimera
Single particle entity
Symmetry typeDIHEDRAL
3D reconstruction
Actual pixel size1.72 A/pix
CTF correction methodeach CCD frame
DetailsProjection matching was done with Fourier ring correlation, model-based masking and SSNR weighting over an 80-6 angstrom resolution range. The final refinement steps used an angular step of 2.5 degrees and each of the 48,000 particles was matched to the best two projection classes (1353). In total, 45,000 particles were used in the final reconstruction and the 3D map was amplitude corrected then Gaussian low-pass filtered with a Fourier half-width of 0.12.
Methodprojection matching
Num class averages1353
Number of particles48271
Resolution6.9 A
Resolution methodFSC at 0.5 cut-off
SoftwareEMAN2
3D fitting
DetailsChimera was used to do initial domain fitting with Apaf-1 (1Z6T) and CED-4 (2A5Y)domains. Modeller and Flex-Em were then used to do local refinement of homology models of the various domains.
MethodFlexible fitting, local refinement
Refinement Protocolrigid body first, and then used individual helices
Refinement SpaceREAL
Software nameModeller, Flex-EM, Chimera
Target criteriaCross-correlation coefficient
Refine hist
Total atoms64336
Ligand atoms496
Protein atoms63840
Download
PDB format
Allpdb1vt4.ent.gz
pdb1vt4.ent (uncompressed file)
Header onlypdb1vt4.ent.gz
mmCIF format
mmCIF1vt4.cif.gz
XML format
All1vt4.xml.gz
No-atom1vt4-noatom.xml.gz
Ext-atom1vt4-extatom.xml.gz
Movie files
movie #1
.mp4 (H.264/MPEG-4 AVC format), 3 MB
.webm (WebM/VP8 format), 4 MB
movie #2
.mp4 (H.264/MPEG-4 AVC format), 3.6 MB
.webm (WebM/VP8 format), 5 MB
movie #3
.mp4 (H.264/MPEG-4 AVC format), 3.2 MB
.webm (WebM/VP8 format), 4.2 MB
movie #4
.mp4 (H.264/MPEG-4 AVC format), 3.7 MB
.webm (WebM/VP8 format), 5.7 MB