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- PDB-9zfs: CryoEM structure of respiratory complex III intermediate from Aca... -

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Basic information

Entry
Database: PDB / ID: 9zfs
TitleCryoEM structure of respiratory complex III intermediate from Acanthamoeba castellanii
Components
  • (Cytochrome b-c1 complex subunit ...) x 4
  • Cytochrome b
  • Cytochrome c1, heme protein
  • Peptidase M16 family protein
  • Ubiquinolcytochrome c reductase complex 14 kDa protein
  • mitochondrial processing peptidase,Mitochondrial-processing peptidase subunit beta
KeywordsOXIDOREDUCTASE / complex III / OXPHOS / cytochrome bc1 complex
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
PROTOPORPHYRIN IX CONTAINING FE / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsFry, M.Y. / Luce, B.E. / Stefely, J.A. / Samuelson, J. / Mootha, V.K. / Chao, L.H.
Funding support United States, 1items
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, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 14, 2026Provider: repository / Type: Initial release
Revision 1.0Oct 14, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Peptidase M16 family protein
E: Cytochrome b-c1 complex subunit 8
J: mitochondrial processing peptidase,Mitochondrial-processing peptidase subunit beta
M: Ubiquinolcytochrome c reductase complex 14 kDa protein
O: Cytochrome b-c1 complex subunit 9
A: Cytochrome b-c1 complex subunit 6
G: Cytochrome c1, heme protein
Q: Cytochrome b
L: Cytochrome b-c1 complex subunit 10
C: Peptidase M16 family protein
F: Cytochrome b-c1 complex subunit 8
H: mitochondrial processing peptidase,Mitochondrial-processing peptidase subunit beta
I: Ubiquinolcytochrome c reductase complex 14 kDa protein
K: Cytochrome b-c1 complex subunit 9
N: Cytochrome b-c1 complex subunit 6
R: Cytochrome c1, heme protein
T: Cytochrome b
W: Cytochrome b-c1 complex subunit 10
hetero molecules


Theoretical massNumber of molelcules
Total (without water)476,35622
Polymers473,89018
Non-polymers2,4664
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 10 molecules BCJHMIGRQT

#1: Protein Peptidase M16 family protein


Mass: 53933.297 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: UQCRC2 | ACANB02868_t1 / Source: (natural) Acanthamoeba castellanii (eukaryote) / Strain: Neff / References: UniProt: L8GTW2
#3: Protein mitochondrial processing peptidase,Mitochondrial-processing peptidase subunit beta / Beta-MPP


Mass: 54876.777 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: PMPCB | ACANB00313_t1,PMPCB | ACANB00313_t1 / Source: (natural) Acanthamoeba castellanii (eukaryote) / Strain: Neff
References: UniProt: L8HK40, mitochondrial processing peptidase
#4: Protein Ubiquinolcytochrome c reductase complex 14 kDa protein


Mass: 14371.188 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: UQCRB | ACANB09944 / Source: (natural) Acanthamoeba castellanii (eukaryote) / Strain: Neff / References: UniProt: L8H0Y7
#7: Protein Cytochrome c1, heme protein


Mass: 33652.250 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: ACANB07833_t1 | CYC1 / Source: (natural) Acanthamoeba castellanii (eukaryote) / Strain: Neff
#8: Protein Cytochrome b / Complex III subunit 3 / Complex III subunit III / Cytochrome b-c1 complex subunit 3 / Ubiquinol- ...Complex III subunit 3 / Complex III subunit III / Cytochrome b-c1 complex subunit 3 / Ubiquinol-cytochrome-c reductase complex cytochrome b subunit


Mass: 43958.941 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: MT CYB | ACANB15918 / Source: (natural) Acanthamoeba castellanii (eukaryote) / Strain: Neff / References: UniProt: Q37378

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Cytochrome b-c1 complex subunit ... , 4 types, 8 molecules EFOKANLW

#2: Protein Cytochrome b-c1 complex subunit 8 / Complex III subunit 8


Mass: 8151.524 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: UQCRQ | ACANB04471 / Source: (natural) Acanthamoeba castellanii (eukaryote) / Strain: Neff / References: UniProt: L8GXE4
#5: Protein Cytochrome b-c1 complex subunit 9


Mass: 9282.360 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: UQCR10 | ACANB07539 / Source: (natural) Acanthamoeba castellanii (eukaryote) / Strain: Neff / References: UniProt: L8HKA7
#6: Protein Cytochrome b-c1 complex subunit 6


Mass: 8013.151 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: UQCRH| ACANB09203_t1 / Source: (natural) Acanthamoeba castellanii (eukaryote) / Strain: Neff / References: UniProt: L8H1V3
#9: Protein Cytochrome b-c1 complex subunit 10


Mass: 10705.308 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: UQCR11 | ACANB09854_t1 / Source: (natural) Acanthamoeba castellanii (eukaryote) / Strain: Neff / References: UniProt: L8HFA4

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Non-polymers , 1 types, 4 molecules

#10: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationY

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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: Acanthamoeba castellanii OXPHOS complex III intermediate assembly state missing the Rieske subunit
Type: COMPLEX / Entity ID: #1-#9 / Source: NATURAL
Molecular weightValue: 1.3 MDa / Experimental value: NO
Source (natural)Organism: Acanthamoeba castellanii (eukaryote) / Strain: Neff
Buffer solutionpH: 7.9
Buffer component
IDConc.NameBuffer-ID
150 mMHEPES1
2300 mMsodium chloride1
34 mMmagnesium chloride1
420 uMCalcium Chloride1
51 mMDithiothreitol1
60.5 % (w/v)DDM1
SpecimenConc.: 2.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was not purified. Instead it was solved from imaging mitochondrial lysate.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277.14 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: DARK FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 0.99 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1cryoSPARCv.4.7.0particle selection
2PHENIX1.21.2_5419model refinement
3EPUimage acquisition
5cryoSPARCv.4.7.0CTF correctionPatch CTF estimation
10cryoSPARCinitial Euler assignmentab initio reconstruction
13cryoSPARC3D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 5208 / Symmetry type: POINT
RefinementHighest resolution: 3.65 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00327006
ELECTRON MICROSCOPYf_angle_d0.58336720
ELECTRON MICROSCOPYf_dihedral_angle_d7.7243628
ELECTRON MICROSCOPYf_chiral_restr0.0393998
ELECTRON MICROSCOPYf_plane_restr0.0054649

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