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- EMDB-74150: CryoEM structure of respiratory complex III intermediate from Aca... -

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Basic information

Entry
Database: EMDB / ID: EMD-74150
TitleCryoEM structure of respiratory complex III intermediate from Acanthamoeba castellanii
Map data
Sample
  • Complex: Acanthamoeba castellanii OXPHOS complex III intermediate assembly state missing the Rieske subunit
    • Protein or peptide: Peptidase M16 family protein
    • Protein or peptide: Cytochrome b-c1 complex subunit 8
    • Protein or peptide: mitochondrial processing peptidase,Mitochondrial-processing peptidase subunit beta
    • Protein or peptide: Ubiquinolcytochrome c reductase complex 14 kDa protein
    • Protein or peptide: Cytochrome b-c1 complex subunit 9
    • Protein or peptide: Cytochrome b-c1 complex subunit 6
    • Protein or peptide: Cytochrome c1, heme protein
    • Protein or peptide: Cytochrome b
    • Protein or peptide: Cytochrome b-c1 complex subunit 10
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
Keywordscomplex III / OXPHOS / cytochrome bc1 complex / OXIDOREDUCTASE
Function / homology
Function and homology information


pitrilysin / signal peptidase activity / respiratory chain complex III / quinol-cytochrome-c reductase activity / mitochondrial electron transport, ubiquinol to cytochrome c / metalloendopeptidase activity / mitochondrial inner membrane / mitochondrial matrix / metal ion binding / mitochondrion ...pitrilysin / signal peptidase activity / respiratory chain complex III / quinol-cytochrome-c reductase activity / mitochondrial electron transport, ubiquinol to cytochrome c / metalloendopeptidase activity / mitochondrial inner membrane / mitochondrial matrix / metal ion binding / mitochondrion / proteolysis / nucleus
Similarity search - Function
SAC3/GANP/THP3, conserved domain / SAC3/GANP/THP3 / SAC3/GANP family / Cytochrome b-c1 complex, subunit 6 / Cytochrome b-c1 complex subunit 8 / UcrQ family / Cytochrome b-c1 complex subunit 7 / Ubiquinol-cytochrome C reductase complex 14kD subunit / Cytochrome b-c1 complex subunit 7 superfamily / Cytochrome b-c1 complex subunit 8 superfamily ...SAC3/GANP/THP3, conserved domain / SAC3/GANP/THP3 / SAC3/GANP family / Cytochrome b-c1 complex, subunit 6 / Cytochrome b-c1 complex subunit 8 / UcrQ family / Cytochrome b-c1 complex subunit 7 / Ubiquinol-cytochrome C reductase complex 14kD subunit / Cytochrome b-c1 complex subunit 7 superfamily / Cytochrome b-c1 complex subunit 8 superfamily / Cytochrome b-c1 complex subunit 9 superfamily / Ubiquinol-cytochrome C reductase hinge domain / Ubiquinol-cytochrome C reductase hinge domain superfamily / Ubiquinol-cytochrome C reductase hinge protein / : / Cytochrome b / Peptidase M16, zinc-binding site / Insulinase family, zinc-binding region signature. / : / : / Cytochrome b/b6, C-terminal / Cytochrome b(C-terminal)/b6/petD / Cytochrome b/b6 C-terminal region profile. / Cytochrome b/b6, C-terminal domain superfamily / Cytochrome b/b6/petB / Cytochrome b/b6, N-terminal / Cytochrome b/b6-like domain superfamily / Cytochrome b/b6 N-terminal region profile. / Di-haem cytochrome, transmembrane / Peptidase M16, C-terminal / Peptidase M16 inactive domain / Peptidase M16, N-terminal / Insulinase (Peptidase family M16) / Metalloenzyme, LuxS/M16 peptidase-like
Similarity search - Domain/homology
Peptidase M16 family protein / Cytochrome b-c1 complex subunit 8 / Ubiquinolcytochrome c reductase complex 14 kDa protein / Cytochrome b-c1 complex subunit 6 / Uncharacterized protein / Pitrilysin / Uncharacterized protein / Cytochrome b
Similarity search - Component
Biological speciesAcanthamoeba castellanii (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsFry MY / Luce BE / Stefely JA / Samuelson J / Mootha VK / Chao LH
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Cell / Year: 2026
Title: Acanthamoeba ATP synthase structure reveals the TCA cycle is tethered to OXPHOS.
Authors: Michelle Y Fry / Bridget E Luce / Jonathan A Stefely / Michael Z Chen / Felicia G Deng / Tiana M Issa / / Sarah E Calvo / John Samuelson / Vamsi K Mootha / Luke H Chao /
Abstract: Oxidative phosphorylation (OXPHOS) is a key metabolic process that couples redox energy to ATP production. While some core OXPHOS complex subunits are found across all domains of life, many have ...Oxidative phosphorylation (OXPHOS) is a key metabolic process that couples redox energy to ATP production. While some core OXPHOS complex subunits are found across all domains of life, many have diverged or expanded across evolution-as seen in the protozoan pathogen Acanthamoeba castellanii. By integrating cryo-electron microscopy of unenriched mitochondrial lysate with mass spectrometry proteomics, we resolved the structures of endogenous mitochondrial ATP synthase (complex V), Hsp60, and respiratory complex III from Acanthamoeba. We capture Acanthamoeba ATP synthase in an IF1-inhibited state and reveal how Acanthamoeba-specific subunits and extensions stabilize the molecular machine, which includes a β subunit extension that interfaces with the peripheral stalk. Additionally, we characterize an active malate dehydrogenase (MDH) dimer structurally integrated within the ATP synthase peripheral stalk, thus revealing a direct protein tether between OXPHOS and the tricarboxylic acid cycle. Together, these findings provide structural insight into lineage-specific adaptations in Acanthamoeba that may tune protozoan metabolism.
History
DepositionDec 2, 2025-
Header (metadata) releaseOct 14, 2026-
Map releaseOct 14, 2026-
UpdateOct 14, 2026-
Current statusOct 14, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_74150.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 400 pix.
= 328.8 Å
0.82 Å/pix.
x 400 pix.
= 328.8 Å
0.82 Å/pix.
x 400 pix.
= 328.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.822 Å
Density
Contour LevelBy AUTHOR: 0.111
Minimum - Maximum-0.2007559 - 0.37556854
Average (Standard dev.)0.0012860376 (±0.017442534)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 328.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_74150_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #2

Fileemd_74150_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Acanthamoeba castellanii OXPHOS complex III intermediate assembly...

EntireName: Acanthamoeba castellanii OXPHOS complex III intermediate assembly state missing the Rieske subunit
Components
  • Complex: Acanthamoeba castellanii OXPHOS complex III intermediate assembly state missing the Rieske subunit
    • Protein or peptide: Peptidase M16 family protein
    • Protein or peptide: Cytochrome b-c1 complex subunit 8
    • Protein or peptide: mitochondrial processing peptidase,Mitochondrial-processing peptidase subunit beta
    • Protein or peptide: Ubiquinolcytochrome c reductase complex 14 kDa protein
    • Protein or peptide: Cytochrome b-c1 complex subunit 9
    • Protein or peptide: Cytochrome b-c1 complex subunit 6
    • Protein or peptide: Cytochrome c1, heme protein
    • Protein or peptide: Cytochrome b
    • Protein or peptide: Cytochrome b-c1 complex subunit 10
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE

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Supramolecule #1: Acanthamoeba castellanii OXPHOS complex III intermediate assembly...

SupramoleculeName: Acanthamoeba castellanii OXPHOS complex III intermediate assembly state missing the Rieske subunit
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9
Source (natural)Organism: Acanthamoeba castellanii (eukaryote) / Strain: Neff
Molecular weightTheoretical: 1.3 MDa

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Macromolecule #1: Peptidase M16 family protein

MacromoleculeName: Peptidase M16 family protein / type: protein_or_peptide / ID: 1 / Details: UQCRC2 | ACANB02868_t1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Acanthamoeba castellanii (eukaryote) / Strain: Neff
Molecular weightTheoretical: 53.933297 KDa
SequenceString: MKPQHAARLS LAKGVTRASS PSASVLGSKS YSSWTATSSL KQAHSSVPQT WEATPQTLDN VQTTTLDSGL KVTTVSSELP LAAVGLHVK AGARFETGES RGAAHFLRHL AFSRTSSRSP LTVTREMEVA TAAFDVSASR ENISYSGQLM PDYLEDYVWM L RDLMHPLA ...String:
MKPQHAARLS LAKGVTRASS PSASVLGSKS YSSWTATSSL KQAHSSVPQT WEATPQTLDN VQTTTLDSGL KVTTVSSELP LAAVGLHVK AGARFETGES RGAAHFLRHL AFSRTSSRSP LTVTREMEVA TAAFDVSASR ENISYSGQLM PDYLEDYVWM L RDLMHPLA WEYIVRDAAP QVAAEVHEAE TNPATALVEA IHREAYRDEG LGNSIYCPNY RVGAVTREAI IRYHHERYQA SN VALVGYG IKHEQLVAQA NKYFPADAFA EDKAPWTTLE AADRKPGAVY TAASSYTGGE LRLPGPGNSR VALAFEGASL ADP DVFAVR TLSSLLGGAA RFTRDGPGVG LRSRLARNVL AKGDYVLASS ALNASYSDSG LFGVFVEALP GHGADAARLL SAEL NSLAG SFSVDDAELT RAKNQAKASF FREVESRTGL VDYLARHTLA GTAPLAPAQY AARFDAVTRD DLARVARRVF SSPLT LVST GDIHGVPTKE ELRPKLKA

UniProtKB: Peptidase M16 family protein

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Macromolecule #2: Cytochrome b-c1 complex subunit 8

MacromoleculeName: Cytochrome b-c1 complex subunit 8 / type: protein_or_peptide / ID: 2 / Details: UQCRQ | ACANB04471 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Acanthamoeba castellanii (eukaryote) / Strain: Neff
Molecular weightTheoretical: 8.151524 KDa
SequenceString:
MGVRWTTYTL SPFRQKMFPG FWTGLIPRQI KRWKYNFPRV APPILAGIAI YTWGNWKHDQ IARSHWY

UniProtKB: Cytochrome b-c1 complex subunit 8

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Macromolecule #3: mitochondrial processing peptidase,Mitochondrial-processing pepti...

MacromoleculeName: mitochondrial processing peptidase,Mitochondrial-processing peptidase subunit beta
type: protein_or_peptide / ID: 3 / Details: PMPCB | ACANB00313_t1,PMPCB | ACANB00313_t1 / Number of copies: 2 / Enantiomer: LEVO / EC number: mitochondrial processing peptidase
Source (natural)Organism: Acanthamoeba castellanii (eukaryote) / Strain: Neff
Molecular weightTheoretical: 54.876777 KDa
SequenceString: MSSIRSRAPS MLLPTKLGTS GRLGAAARRL SSGQPQAARD VEKRLRLPEY LLNVPPTQVT ALPNKFRVAS EHKHGETATV GVWIDAGSV WETAENNGVA HFLEHLAFKG TKNRTKEQIE VEIENMGGQL NAYTSREQTV YHAHVFKKDV PKAVEIISDI I QNSNLKED ...String:
MSSIRSRAPS MLLPTKLGTS GRLGAAARRL SSGQPQAARD VEKRLRLPEY LLNVPPTQVT ALPNKFRVAS EHKHGETATV GVWIDAGSV WETAENNGVA HFLEHLAFKG TKNRTKEQIE VEIENMGGQL NAYTSREQTV YHAHVFKKDV PKAVEIISDI I QNSNLKED DVERERGVIL REMEEVESQT EEVIFDHLHS IAFQNTSLGY TILGPEKNIK KIKREDLVSY VGKHYTAPRM VL SAAGAVD HDELVKLAEK HFSGLSSETN VDYSNREKLF DFTGSMVQVR DTSIPLVHTT VAAKSVGWSD PDYFTFLVLQ QLV GSWDRS LGGAKNLSSN LAETFATEEL AHSLMSFNTC YHETGLFGAY FVGEMERTSD AIFEVLREWV RIGSGVSEVE VERA KNKLK STYLMQLDGT QAVAEDIGRQ LLTLGRRMPA AEAFMRIDAI DAKKVREVAY TYLNDVDVAV AAVGSVDSGL FPDYN VLRG WTYWNRL

UniProtKB: Pitrilysin

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Macromolecule #4: Ubiquinolcytochrome c reductase complex 14 kDa protein

MacromoleculeName: Ubiquinolcytochrome c reductase complex 14 kDa protein
type: protein_or_peptide / ID: 4 / Details: UQCRB | ACANB09944 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Acanthamoeba castellanii (eukaryote) / Strain: Neff
Molecular weightTheoretical: 14.371188 KDa
SequenceString:
MAARKGSSGF LSRISSSFKP VSGYQAYGLR LEDFYNAENP EIQEVLRRLP NKTKEERDLR IRRGHELHLK GTTLPESSWT TPEEDGQTY MEPYMSEVVQ EIEERKDFRA DFLLPVEKRH KRK

UniProtKB: Ubiquinolcytochrome c reductase complex 14 kDa protein

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Macromolecule #5: Cytochrome b-c1 complex subunit 9

MacromoleculeName: Cytochrome b-c1 complex subunit 9 / type: protein_or_peptide / ID: 5 / Details: UQCR10 | ACANB07539 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Acanthamoeba castellanii (eukaryote) / Strain: Neff
Molecular weightTheoretical: 9.28236 KDa
SequenceString:
MNALKSSGAS NWVYQNLFKR NSSYFALILV GAVVVESGVS RTVDFWWNSV NKGRTWNDFK RTQLPKIQAK KAAAAEEEES DE

UniProtKB: Uncharacterized protein

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Macromolecule #6: Cytochrome b-c1 complex subunit 6

MacromoleculeName: Cytochrome b-c1 complex subunit 6 / type: protein_or_peptide / ID: 6 / Details: UQCRH| ACANB09203_t1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Acanthamoeba castellanii (eukaryote) / Strain: Neff
Molecular weightTheoretical: 8.013151 KDa
SequenceString:
MSENWAGCAI KKSLEEKAHP KCLKKWAEYD ACGQRIKGDT TGEAQCSGQY FEYWHCVDAL VANDLFKRLK

UniProtKB: Cytochrome b-c1 complex subunit 6

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Macromolecule #7: Cytochrome c1, heme protein

MacromoleculeName: Cytochrome c1, heme protein / type: protein_or_peptide / ID: 7 / Details: ACANB07833_t1 | CYC1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Acanthamoeba castellanii (eukaryote) / Strain: Neff
Molecular weightTheoretical: 33.65225 KDa
SequenceString: MLRQRTLTSA TGMAFRSQTR SFATSAGASA KRLHPNVLGL VIGAGAVGAA AGTLISRARC SDDVLVAPAY PWSHKSEWKS YDVASLRRG HQVYKEVCAA CHSLNYIAWR NLIETLYTEE EVIAMAAETE VEDGPNDDGE MFTRPGKPTD YLPRPYPNEE A ARSANAGA ...String:
MLRQRTLTSA TGMAFRSQTR SFATSAGASA KRLHPNVLGL VIGAGAVGAA AGTLISRARC SDDVLVAPAY PWSHKSEWKS YDVASLRRG HQVYKEVCAA CHSLNYIAWR NLIETLYTEE EVIAMAAETE VEDGPNDDGE MFTRPGKPTD YLPRPYPNEE A ARSANAGA IPPDLTEMVK AREQGADYIF ALLTGYMPAP AGLNVREGLY YNPYFPGGAI GMPPPLNDGM LEYEDGTEAS VS QMAKDVS AFLCWTARPE QDERRVMGIK VLIYLGALFG CSWYIKRFKW SVLKNRKVQF PGKNWPTV

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Macromolecule #8: Cytochrome b

MacromoleculeName: Cytochrome b / type: protein_or_peptide / ID: 8 / Details: MT CYB | ACANB15918 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Acanthamoeba castellanii (eukaryote) / Strain: Neff
Molecular weightTheoretical: 43.958941 KDa
SequenceString: MRFIKKYPLL NIVNSFLIDY PAPPNISYLW NFGALAAFCL GIQIVTGIFL AMFYVPSIES AFSSVQYIMR DVNYGWLIRY IHANGASFF FICVYIHVFR GLYYNSYITP RELLWNLGVT ILIVMILTAF LGYVLPWGQM SFWAATVITS LFSAIPVVGE Y IVIWLWGG ...String:
MRFIKKYPLL NIVNSFLIDY PAPPNISYLW NFGALAAFCL GIQIVTGIFL AMFYVPSIES AFSSVQYIMR DVNYGWLIRY IHANGASFF FICVYIHVFR GLYYNSYITP RELLWNLGVT ILIVMILTAF LGYVLPWGQM SFWAATVITS LFSAIPVVGE Y IVIWLWGG FSVDNATLNR FFSLHYLLPF VIALLSLIHV AVLHESGSSN PLNIALSVAN KVPFHPYFIF KDLLGIIFFL IV FCYAVFF KPDSIIDPIN NVPANPLVTP THIVPEWYFL PFYAILRSIP NKLGGVITLG LALIVLFLLP FITNNVFKGS FFE ISKTIL FWSFFSVCVL LGWIGFKPIE DPYLMLGQML TVLYFFYFFS LAVIVPTLQA YTFKNVFAK

UniProtKB: Cytochrome b

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Macromolecule #9: Cytochrome b-c1 complex subunit 10

MacromoleculeName: Cytochrome b-c1 complex subunit 10 / type: protein_or_peptide / ID: 9 / Details: UQCR11 | ACANB09854_t1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Acanthamoeba castellanii (eukaryote) / Strain: Neff
Molecular weightTheoretical: 10.705308 KDa
SequenceString:
MSIHKLDKTH ATVNLEEAAT RTGTIRSVPY HETIAYQRFY ANVFTKATVF GKRLIPLTKN YAPTAFRWGA LAGLAAAYVI EPSFLTKYF TKSESK

UniProtKB: Uncharacterized protein

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Macromolecule #10: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 10 / Number of copies: 4 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

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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

Concentration2.25 mg/mL
BufferpH: 7.9
Component:
ConcentrationName
50.0 mMHEPES
300.0 mMsodium chloride
4.0 mMmagnesium chloride
20.0 uMCalcium Chloride
1.0 mMDithiothreitol
0.5 % (w/v)DDM
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277.14 K / Instrument: FEI VITROBOT MARK IV
DetailsSample was not purified. Instead it was solved from imaging mitochondrial lysate.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 0.99 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: DARK FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 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

CTF correctionSoftware - Name: cryoSPARC (ver. v.4.7.0) / Software - details: Patch CTF estimation / Type: NONE
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 5208
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC / Software - details: ab initio reconstruction
Final angle assignmentType: PROJECTION MATCHING

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