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- PDB-9zfx: Mitochondrial malate dehydrogenase from Acanthamoeba castellanii -

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Basic information

Entry
Database: PDB / ID: 9zfx
TitleMitochondrial malate dehydrogenase from Acanthamoeba castellanii
Componentsmalate dehydrogenase
KeywordsOXIDOREDUCTASE / malate dehydrogenase / MDH / mitochondrial
Function / homology
Function and homology information


(S)-malate dehydrogenase (NAD+, oxaloacetate-forming) / L-malate dehydrogenase (NAD+) activity / malate metabolic process
Similarity search - Function
Malate dehydrogenase, type 2 / Lactate/malate dehydrogenase, N-terminal / Lactate/malate dehydrogenase, C-terminal / lactate/malate dehydrogenase, NAD binding domain / lactate/malate dehydrogenase, alpha/beta C-terminal domain / Lactate dehydrogenase/glycoside hydrolase, family 4, C-terminal / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
(S)-malate dehydrogenase (NAD(+), oxaloacetate-forming)
Similarity search - Component
Biological speciesAcanthamoeba castellanii (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.33 Å
AuthorsFry, M.Y. / Luce, B.E. / 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
D: malate dehydrogenase
A: malate dehydrogenase
B: malate dehydrogenase
C: malate dehydrogenase


Theoretical massNumber of molelcules
Total (without water)196,4084
Polymers196,4084
Non-polymers00
Water5,405300
1
D: malate dehydrogenase
B: malate dehydrogenase


Theoretical massNumber of molelcules
Total (without water)98,2042
Polymers98,2042
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3210 Å2
ΔGint-18 kcal/mol
Surface area26430 Å2
MethodPISA
2
A: malate dehydrogenase

C: malate dehydrogenase


Theoretical massNumber of molelcules
Total (without water)98,2042
Polymers98,2042
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_545-x,y-1/2,-z1
Buried area3280 Å2
ΔGint-17 kcal/mol
Surface area26570 Å2
MethodPISA
Unit cell
Length a, b, c (Å)50.409, 136.498, 129.548
Angle α, β, γ (deg.)90.00, 100.85, 90.00
Int Tables number4
Space group name H-MP1211
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein
malate dehydrogenase


Mass: 49101.977 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: ACANB11954 / Source: (gene. exp.) Acanthamoeba castellanii (eukaryote) / Gene: ACA1_152960 / Production host: Escherichia coli (E. coli)
References: UniProt: L8HFT9, (S)-malate dehydrogenase (NAD+, oxaloacetate-forming)
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 300 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.23 Å3/Da / Density % sol: 44.81 %
Crystal growTemperature: 277.15 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 100 mM Tris, pH 8, 25 mM magnesium chloride, 23% PEG3500

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS-II / Beamline: 17-ID-2 / Wavelength: 0.97911 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Sep 19, 2025
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97911 Å / Relative weight: 1
ReflectionResolution: 2.33→34.54 Å / Num. obs: 95755 / % possible obs: 96.2 % / Redundancy: 6.5 % / Biso Wilson estimate: 26.86 Å2 / CC1/2: 0.984 / CC star: 0.996 / Net I/σ(I): 1.56
Reflection shellResolution: 2.33→2.36 Å / Mean I/σ(I) obs: 0.73 / Num. unique obs: 3113 / CC1/2: 0.331

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Processing

Software
NameVersionClassification
PHENIX(1.21.2_5419: ???)refinement
autoPROCdata reduction
autoPROCdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.33→34.54 Å / SU ML: 0.29 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 30.93 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2587 3514 4.98 %
Rwork0.2044 --
obs0.2071 70594 96.18 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.33→34.54 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10135 0 0 300 10435
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01210337
X-RAY DIFFRACTIONf_angle_d1.17714011
X-RAY DIFFRACTIONf_dihedral_angle_d6.5751420
X-RAY DIFFRACTIONf_chiral_restr0.0581576
X-RAY DIFFRACTIONf_plane_restr0.0111832
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.33-2.360.29971230.25082802X-RAY DIFFRACTION100
2.36-2.40.33631450.26312825X-RAY DIFFRACTION100
2.4-2.430.32321280.23892723X-RAY DIFFRACTION100
2.43-2.470.29091680.24072760X-RAY DIFFRACTION100
2.47-2.510.31781670.22832814X-RAY DIFFRACTION100
2.51-2.550.3041470.22952739X-RAY DIFFRACTION100
2.55-2.60.32231450.24372767X-RAY DIFFRACTION100
2.6-2.650.30381250.23652873X-RAY DIFFRACTION100
2.65-2.70.2665770.25211733X-RAY DIFFRACTION63
2.7-2.760.27671580.22582772X-RAY DIFFRACTION100
2.76-2.830.29141790.22972766X-RAY DIFFRACTION100
2.83-2.90.28941350.22742780X-RAY DIFFRACTION100
2.9-2.980.27711400.23162807X-RAY DIFFRACTION100
2.98-3.060.30131230.22742742X-RAY DIFFRACTION100
3.06-3.160.29751860.21942811X-RAY DIFFRACTION100
3.16-3.270.27341510.21642728X-RAY DIFFRACTION100
3.27-3.410.2691530.21952814X-RAY DIFFRACTION100
3.41-3.560.3162910.21142273X-RAY DIFFRACTION81
3.56-3.750.2868910.20862114X-RAY DIFFRACTION76
3.75-3.980.27011210.19492407X-RAY DIFFRACTION86
3.98-4.290.221480.16952809X-RAY DIFFRACTION100
4.29-4.720.18661420.15482805X-RAY DIFFRACTION100
4.72-5.40.20131610.16472781X-RAY DIFFRACTION100
5.4-6.80.20951530.17792811X-RAY DIFFRACTION100
6.8-34.540.1891570.16532824X-RAY DIFFRACTION99

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