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- EMDB-15654: Structure of the giant inhibitor of apoptosis, BIRC6 bound to the... -

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Basic information

Entry
Database: EMDB / ID: EMD-15654
TitleStructure of the giant inhibitor of apoptosis, BIRC6 bound to the regulator SMAC
Map dataSharpened map from non-uniform refinement
Sample
  • Complex: Anti-parallel homodimer
    • Complex: Baculoviral IAP repeat-containing protein 6
      • Protein or peptide: Baculoviral IAP repeat-containing protein 6
    • Complex: Diablo IAP-binding mitochondrial protein
      • Protein or peptide: Diablo IAP-binding mitochondrial protein
Function / homology
Function and homology information


spongiotrophoblast layer development / activation of cysteine-type endopeptidase activity involved in apoptotic process by cytochrome c / Release of apoptotic factors from the mitochondria / CD40 receptor complex / labyrinthine layer development / ALK mutants bind TKIs / SMAC, XIAP-regulated apoptotic response / Flemming body / Regulation of the apoptosome activity / SMAC (DIABLO) binds to IAPs ...spongiotrophoblast layer development / activation of cysteine-type endopeptidase activity involved in apoptotic process by cytochrome c / Release of apoptotic factors from the mitochondria / CD40 receptor complex / labyrinthine layer development / ALK mutants bind TKIs / SMAC, XIAP-regulated apoptotic response / Flemming body / Regulation of the apoptosome activity / SMAC (DIABLO) binds to IAPs / SMAC(DIABLO)-mediated dissociation of IAP:caspase complexes / microtubule organizing center / intrinsic apoptotic signaling pathway in response to oxidative stress / cysteine-type endopeptidase inhibitor activity / ubiquitin conjugating enzyme activity / extrinsic apoptotic signaling pathway via death domain receptors / intrinsic apoptotic signaling pathway / regulation of cytokinesis / negative regulation of extrinsic apoptotic signaling pathway / RING-type E3 ubiquitin transferase / trans-Golgi network / mitochondrial intermembrane space / cytoplasmic side of plasma membrane / spindle pole / ubiquitin-protein transferase activity / activation of cysteine-type endopeptidase activity involved in apoptotic process / Signaling by ALK fusions and activated point mutants / regulation of cell population proliferation / midbody / neuron apoptotic process / cell population proliferation / protein ubiquitination / endosome / positive regulation of apoptotic process / cell cycle / cell division / protein phosphorylation / centrosome / apoptotic process / positive regulation of cell population proliferation / negative regulation of apoptotic process / mitochondrion / membrane / nucleus / cytosol
Similarity search - Function
Smac/DIABLO-like superfamily / Smac/DIABLO protein / Second Mitochondria-derived Activator of Caspases / Baculoviral IAP repeat-containing protein 6 / Baculoviral IAP repeat-containing protein 6 / BIR repeat / Inhibitor of Apoptosis domain / BIR repeat profile. / Baculoviral inhibition of apoptosis protein repeat / Ubiquitin-conjugating enzyme E2, catalytic domain homologues ...Smac/DIABLO-like superfamily / Smac/DIABLO protein / Second Mitochondria-derived Activator of Caspases / Baculoviral IAP repeat-containing protein 6 / Baculoviral IAP repeat-containing protein 6 / BIR repeat / Inhibitor of Apoptosis domain / BIR repeat profile. / Baculoviral inhibition of apoptosis protein repeat / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme/RWD-like
Similarity search - Domain/homology
Baculoviral IAP repeat-containing protein 6 / Diablo IAP-binding mitochondrial protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsDietz L / Elliott PR
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/R008582/1 United Kingdom
CitationJournal: Science / Year: 2023
Title: Structural basis for SMAC-mediated antagonism of caspase inhibition by the giant ubiquitin ligase BIRC6.
Authors: Larissa Dietz / Cara J Ellison / Carlos Riechmann / C Keith Cassidy / F Daniel Felfoldi / Adán Pinto-Fernández / Benedikt M Kessler / Paul R Elliott /
Abstract: Certain inhibitor of apoptosis (IAP) family members are sentinel proteins that prevent untimely cell death by inhibiting caspases. Antagonists, including second mitochondria-derived activator of ...Certain inhibitor of apoptosis (IAP) family members are sentinel proteins that prevent untimely cell death by inhibiting caspases. Antagonists, including second mitochondria-derived activator of caspases (SMAC), regulate IAPs and drive cell death. Baculoviral IAP repeat-containing protein 6 (BIRC6), a giant IAP with dual E2 and E3 ubiquitin ligase activity, regulates programmed cell death through unknown mechanisms. We show that BIRC6 directly restricts executioner caspase-3 and -7 and ubiquitinates caspase-3, -7, and -9, working exclusively with noncanonical E1, UBA6. Notably, we show that SMAC suppresses both mechanisms. Cryo-electron microscopy structures of BIRC6 alone and in complex with SMAC reveal that BIRC6 is an antiparallel dimer juxtaposing the substrate-binding module against the catalytic domain. Furthermore, we discover that SMAC multisite binding to BIRC6 results in a subnanomolar affinity interaction, enabling SMAC to competitively displace caspases, thus antagonizing BIRC6 anticaspase function.
History
DepositionAug 23, 2022-
Header (metadata) releaseMar 8, 2023-
Map releaseMar 8, 2023-
UpdateMar 29, 2023-
Current statusMar 29, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_15654.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map from non-uniform refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 300 pix.
= 248.7 Å
0.83 Å/pix.
x 300 pix.
= 248.7 Å
0.83 Å/pix.
x 300 pix.
= 248.7 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.829 Å
Density
Contour LevelBy AUTHOR: 0.144
Minimum - Maximum-0.7501397 - 1.4294059
Average (Standard dev.)0.0055784024 (±0.06963273)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 248.7 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15654_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: First half map non-uniform refinement

Fileemd_15654_half_map_1.map
AnnotationFirst half map non-uniform refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Second half map non-uniform refinement

Fileemd_15654_half_map_2.map
AnnotationSecond half map non-uniform refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Anti-parallel homodimer

EntireName: Anti-parallel homodimer
Components
  • Complex: Anti-parallel homodimer
    • Complex: Baculoviral IAP repeat-containing protein 6
      • Protein or peptide: Baculoviral IAP repeat-containing protein 6
    • Complex: Diablo IAP-binding mitochondrial protein
      • Protein or peptide: Diablo IAP-binding mitochondrial protein

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Supramolecule #1: Anti-parallel homodimer

SupramoleculeName: Anti-parallel homodimer / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 1 MDa

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Supramolecule #2: Baculoviral IAP repeat-containing protein 6

SupramoleculeName: Baculoviral IAP repeat-containing protein 6 / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Diablo IAP-binding mitochondrial protein

SupramoleculeName: Diablo IAP-binding mitochondrial protein / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Baculoviral IAP repeat-containing protein 6

MacromoleculeName: Baculoviral IAP repeat-containing protein 6 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: RING-type E3 ubiquitin transferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 530.977 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GPMVTGGGAA PPGTVTEPLP SVIVLSAGRK MAAAAAAASG PGCSSAAGAG AAGVSEWLVL RDGCMHCDAD GLHSLSYHPA LNAILAVTS RGTIKVIDGT SGATLQASAL SAKPGGQVKC QYISAVDKVI FVDDYAVGCR KDLNGILLLD TALQTPVSKQ D DVVQLELP ...String:
GPMVTGGGAA PPGTVTEPLP SVIVLSAGRK MAAAAAAASG PGCSSAAGAG AAGVSEWLVL RDGCMHCDAD GLHSLSYHPA LNAILAVTS RGTIKVIDGT SGATLQASAL SAKPGGQVKC QYISAVDKVI FVDDYAVGCR KDLNGILLLD TALQTPVSKQ D DVVQLELP VTEAQQLLSA CLEKVDISST EGYDLFITQL KDGLKNTSHE TAANHKVAKW ATVTFHLPHH VLKSIASAIV NE LKKINQN VAALPVASSV MDRLSYLLPS ARPELGVGPG RSVDRSLMYS EANRRETFTS WPHVGYRWAQ PDPMAQAGFY HQP ASSGDD RAMCFTCSVC LVCWEPTDEP WSEHERHSPN CPFVKGEHTQ NVPLSVTLAT SPAQFPCTDG TDRISCFGSG SCPH FLAAA TKRGKICIWD VSKLMKVHLK FEINAYDPAI VQQLILSGDP SSGVDSRRPT LAWLEDSSSC SDIPKLEGDS DDLLE DSDS EEHSRSDSVT GHTSQKEAME VSLDITALSI LQQPEKLQWE IVANVLEDTV KDLEELGANP CLTNSKSEKT KEKHQE QHN IPFPCLLAGG LLTYKSPATS PISSNSHRSL DGLSRTQGES ISEQGSTDNE SCTNSELNSP LVRRTLPVLL LYSIKES DE KAGKIFSQMN NIMSKSLHDD GFTVPQIIEM ELDSQEQLLL QDPPVTYIQQ FADAAANLTS PDSEKWNSVF PKPGTLVQ C LRLPKFAEEE NLCIDSITPC ADGIHLLVGL RTCPVESLSA INQVEALNNL NKLNSALCNR RKGELESNLA VVNGANISV IQHESPADVQ TPLIIQPEQR NVSGGYLVLY KMNYATRIVT LEEEPIKIQH IKDPQDTITS LILLPPDILD NREDDCEEPI EDMQLTSKN GFEREKTSDI STLGHLVITT QGGYVKILDL SNFEILAKVE PPKKEGTEEQ DTFVSVIYCS GTDRLCACTK G GELHFLQI GGTCDDIDEA DILVDGSLSK GIEPSSEGSK PLSNPSSPGI SGVDLLVDQP FTLEILTSLV ELTRFETLTP RF SATVPPC WVEVQQEQQQ RRHPQHLHQQ HHGDAAQHTR TWKLQTDSNS WDEHVFELVL PKACMVGHVD FKFVLNSNIT NIP QIQVTL LKNKAPGLGK VNALNIEVEQ NGKPSLVDLN EEMQHMDVEE SQCLRLCPFL EDHKEDILCG PVWLASGLDL SGHA GMLTL TSPKLVKGMA GGKYRSFLIH VKAVNERGTE EICNGGMRPV VRLPSLKHQS NKGYSLASLL AKVAAGKEKS SNVKN ENTS GTRKSENLRG CDLLQEVSVT IRRFKKTSIS KERVQRCAML QFSEFHEKLL NTLCRKTDDG QITEHAQSLV LDTLCW LAG VHSNGPGSSK EGNENLLSKT RKFLSDIVRV CFFEAGRSIA HKCARFLALC ISNGKCDPCQ PAFGPVLLKA LLDNMSF LP AATTGGSVYW YFVLLNYVKD EDLAGCSTAC ASLLTAVSRQ LQDRLTPMEA LLQTRYGLYS SPFDPVLFDL EMSGSSCK N VYNSSIGVQS DEIDLSDVLS GNGKVSSCTA AEGSFTSLTG LLEVEPLHFT CVSTSDGTRI ERDDAMSSFG VTPAVGGLS SGTVGEASTA LSSAAQVALQ SLSHAMASAE QQLQVLQEKQ QQLLKLQQQK AKLEAKLHQT TAAAAAAASA VGPVHNSVPS NPVAAPGFF IHPSDVIPPT PKTTPLFMTP PLTPPNEAVS VVINAELAQL FPGSVIDPPA VNLAAHNKNS NKSRMNPLGS G LALAISHA SHFLQPPPHQ SIIIERMHSG ARRFVTLDFG RPILLTDVLI PTCGDLASLS IDIWTLGEEV DGRRLVVATD IS THSLILH DLIPPPVCRF MKITVIGRYG STNARAKIPL GFYYGHTYIL PWESELKLMH DPLKGEGESA NQPEIDQHLA MMV ALQEDI QCRYNLACHR LETLLQSIDL PPLNSANNAQ YFLRKPDKAV EEDSRVFSAY QDCIQLQLQL NLAHNAVQRL KVAL GASRK MLSETSNPED LIQTSSTEQL RTIIRYLLDT LLSLLHASNG HSVPAVLQST FHAQACEELF KHLCISGTPK IRLHT GLLL VQLCGGERWW GQFLSNVLQE LYNSEQLLIF PQDRVFMLLS CIGQRSLSNS GVLESLLNLL DNLLSPLQPQ LPMHRR TEG VLDIPMISWV VMLVSRLLDY VATVEDEAAA AKKPLNGNQW SFINNNLHTQ SLNRSSKGSS SLDRLYSRKI RKQLVHH KQ QLNLLKAKQK ALVEQMEKEK IQSNKGSSYK LLVEQAKLKQ ATSKHFKDLI RLRRTAEWSR SNLDTEVTTA KESPEIEP L PFTLAHERCI SVVQKLVLFL LSMDFTCHAD LLLFVCKVLA RIANATRPTI HLCEIVNEPQ LERLLLLLVG TDFNRGDIS WGGAWAQYSL TCMLQDILAG ELLAPVAAEA MEEGTVGDDV GATAGDSDDS LQQSSVQLLE TIDEPLTHDI TGAPPLSSLE KDKEIDLEL LQDLMEVDID PLDIDLEKDP LAAKVFKPIS STWYDYWGAD YGTYNYNPYI GGLGIPVAKP PANTEKNGSQ T VSVSVSQA LDARLEVGLE QQAELMLKMM STLEADSILQ ALTNTSPTLS QSPTGTDDSL LGGLQAANQT SQLIIQLSSV PM LNVCFNK LFSMLQVHHV QLESLLQLWL TLSLNSSSTG NKENGADIFL YNANRIPVIS LNQASITSFL TVLAWYPNTL LRT WCLVLH SLTLMTNMQL NSGSSSAIGT QESTAHLLVS DPNLIHVLVK FLSGTSPHGT NQHSPQVGPT ATQAMQEFLT RLQV HLSST CPQIFSEFLL KLIHILSTER GAFQTGQGPL DAQVKLLEFT LEQNFEVVSV STISAVIESV TFLVHHYITC SDKVM SRSG SDSSVGARAC FGGLFANLIR PGDAKAVCGE MTRDQLMFDL LKLVNILVQL PLSGNREYSA RVSVTTNTTD SVSDEE KVS GGKDGNGSST SVQGSPAYVA DLVLANQQIM SQILSALGLC NSSAMAMIIG ASGLHLTKHE NFHGGLDAIS VGDGLFT IL TTLSKKASTV HMMLQPILTY MACGYMGRQG SLATCQLSEP LLWFILRVLD TSDALKAFHD MGGVQLICNN MVTSTRAI V NTARSMVSTI MKFLDSGPNK AVDSTLKTRI LASEPDNAEG IHNFAPLGTI TSSSPTAQPA EVLLQATPPH RRARSAAWS YIFLPEEAWC DLTIHLPAAV LLKEIHIQPH LASLATCPSS VSVEVSADGV NMLPLSTPVV TSGLTYIKIQ LVKAEVASAV CLRLHRPRD ASTLGLSQIK LLGLTAFGTT SSATVNNPFL PSEDQVSKTS IGWLRLLHHC LTHISDLEGM MASAAAPTAN L LQTCAALL MSPYCGMHSP NIEVVLVKIG LQSTRIGLKL IDILLRNCAA SGSDPTDLNS PLLFGRLNGL SSDSTIDILY QL GTTQDPG TKDRIQALLK WVSDSARVAA MKRSGRMNYM CPNSSTVEYG LLMPSPSHLH CVAAILWHSY ELLVEYDLPA LLD QELFEL LFNWSMSLPC NMVLKKAVDS LLCSMCHVHP NYFSLLMGWM GITPPPVQCH HRLSMTDDSK KQDLSSSLTD DSKN AQAPL ALTESHLATL ASSSQSPEAI KQLLDSGLPS LLVRSLASFC FSHISSSESI AQSIDISQDK LRRHHVPQQC NKMPI TADL VAPILRFLTE VGNSHIMKDW LGGSEVNPLW TALLFLLCHS GSTSGSHNLG AQQTSARSAS LSSAATTGLT TQQRTA IEN ATVAFFLQCI SCHPNNQKLM AQVLCELFQT SPQRGNLPTS GNISGFIRRL FLQLMLEDEK VTMFLQSPCP LYKGRIN AT SHVIQHPMYG AGHKFRTLHL PVSTTLSDVL DRVSDTPSIT AKLISEQKDD KEKKNHEEKE KVKAENGFQD NYSVVVAS G LKSQSKRAVS ATPPRPPSRR GRTIPDKIGS TSGAEAANKI ITVPVFHLFH KLLAGQPLPA EMTLAQLLTL LYDRKLPQG YRSIDLTVKL GSRVITDPSL SKTDSYKRLH PEKDHGDLLA SCPEDEALTP GDECMDGILD ESLLETCPIQ SPLQVFAGMG GLALIAERL PMLYPEVIQQ VSAPVVTSTT QEKPKDSDQF EWVTIEQSGE LVYEAPETVA AEPPPIKSAV QTMSPIPAHS L AAFGLFLR LPGYAEVLLK ERKHAQCLLR LVLGVTDDGE GSHILQSPSA NVLPTLPFHV LRSLFSTTPL TTDDGVLLRR MA LEIGALH LILVCLSALS HHSPRVPNSS VNQTEPQVSS SHNPTSTEEQ QLYWAKGTGF GTGSTASGWD VEQALTKQRL EEE HVTCLL QVLASYINPV SSAVNGEAQS SHETRGQNSN ALPSVLLELL SQSCLIPAMS SYLRNDSVLD MARHVPLYRA LLEL LRAIA SCAAMVPLLL PLSTENGEEE EEQSECQTSV GTLLAKMKTC VDTYTNRLRS KRENVKTGVK PDASDQEPEG LTLLV PDIQ KTAEIVYAAT TSLRQANQEK KLGEYSKKAA MKPKPLSVLK SLEEKYVAVM KKLQFDTFEM VSEDEDGKLG FKVNYH YMS QVKNANDANS AARARRLAQE AVTLSTSLPL SSSSSVFVRC DEERLDIMKV LITGPADTPY ANGCFEFDVY FPQDYPS SP PLVNLETTGG HSVRFNPNLY NDGKVCLSIL NTWHGRPEEK WNPQTSSFLQ VLVSVQSLIL VAEPYFNEPG YERSRGTP S GTQSSREYDG NIRQATVKWA MLEQIRNPSP CFKEVIHKHF YLKRVEIMAQ CEEWIADIQQ YSSDKRVGRT MSHHAAALK RHTAQLREEL LKLPCPEGLD PDTDDAPEVC RATTGAEETL MHDQVKPSSS KELPSDFQL

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Macromolecule #2: Diablo IAP-binding mitochondrial protein

MacromoleculeName: Diablo IAP-binding mitochondrial protein / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 20.787098 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
AVPIAQKSEP HSLSSEALMR RAVSLVTDST STFLSQTTYA LIEAITEYTK AVYTLTSLYR QYTSLLGKMN SEEEDEVWQV IIGARAEMT SKHQEYLKLE TTWMTAVGLS EMAAEAAYQT GADQASITAR NHIQLVKLQV EEVHQLSRKA ETKLAEAQIE E LRQKTQEE GEERAESEQE AYLRED

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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

Concentration4 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 2.3 sec. / Average electron dose: 47.27 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.5 µm / Calibrated defocus min: 0.75 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 105000
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: 1417739
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 36872
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER / Overall B value: 191.06
Output model

PDB-8ato:
Structure of the giant inhibitor of apoptosis, BIRC6 bound to the regulator SMAC

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