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- PDB-12pb: Single particle cryo-EM structure of human MTCH2 (hyperactive mut... -

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Basic information

Entry
Database: PDB / ID: 12pb
TitleSingle particle cryo-EM structure of human MTCH2 (hyperactive mutant K25E Y235A V238D)
Components
  • Mitochondrial carrier homolog 2,Mitochondrial brown fat uncoupling protein 1
  • UCP1 Nanobody
KeywordsMEMBRANE PROTEIN / mitochondrial outer membrane insertase / SLC25 carrier fold / hydrophilic groove / membrane protein biogenesis
Function / homology
Function and homology information


regulation of mitochondrial fusion / purine ribonucleotide binding / cellular response to dehydroepiandrosterone / Mitochondrial Uncoupling / The fatty acid cycling model / cellular response to radiation / oxidative phosphorylation uncoupler activity / : / mitochondrial transmembrane transport / hematopoietic stem cell migration ...regulation of mitochondrial fusion / purine ribonucleotide binding / cellular response to dehydroepiandrosterone / Mitochondrial Uncoupling / The fatty acid cycling model / cellular response to radiation / oxidative phosphorylation uncoupler activity / : / mitochondrial transmembrane transport / hematopoietic stem cell migration / membrane insertase activity / adaptive thermogenesis / protein insertion into mitochondrial outer membrane / hematopoietic stem cell homeostasis / positive regulation of stem cell differentiation / protein localization to mitochondrion / cardiolipin binding / lactate metabolic process / cellular response to fatty acid / regulation of mitochondrial membrane permeability involved in apoptotic process / positive regulation of intrinsic apoptotic signaling pathway in response to DNA damage / negative regulation of glycolytic process / regulation of reactive oxygen species biosynthetic process / long-chain fatty acid binding / response to temperature stimulus / diet induced thermogenesis / cellular response to cold / hepatocyte apoptotic process / proton transmembrane transporter activity / lipid homeostasis / negative regulation of mitochondrial membrane potential / mitochondrial ATP synthesis coupled electron transport / transmembrane transporter activity / Transcriptional regulation of brown and beige adipocyte differentiation by EBF2 / cellular response to hormone stimulus / proton transmembrane transport / brown fat cell differentiation / response to cold / cellular response to reactive oxygen species / response to nutrient levels / GDP binding / positive regulation of cold-induced thermogenesis / mitochondrial outer membrane / mitochondrial inner membrane / positive regulation of apoptotic process / regulation of transcription by RNA polymerase II / GTP binding / mitochondrion / membrane / nucleus
Similarity search - Function
: / Mitochondrial carrier protein / Mitochondrial substrate/solute carrier / Mitochondrial carrier domain superfamily / Mitochondrial carrier protein / Solute carrier (Solcar) repeat profile.
Similarity search - Domain/homology
Mitochondrial brown fat uncoupling protein 1 / Mitochondrial carrier homolog 2
Similarity search - Component
Biological speciessynthetic construct (others)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLuo, Z. / Stevens, T.A. / Voorhees, R.M.
Funding support United States, 8items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1K99GM151478 United States
Nagendranath Reddy Graduate Fellowship United States
Hevolution/AFAR new investigator award United States
Caltech Center for Evolutionary Science Grant United States
Caltech OTCCP Rothenberg Innovation Initiative Grant United States
Merkin Institute Translational Research Grant United States
Sontag Foundation United States
Howard Hughes Medical Institute (HHMI)Freeman Hrabowski Scholar United States
CitationJournal: To Be Published
Title: Structural evolution of the MTCH family of mitochondrial insertases
Authors: Luo, Z. / Stevens, T.A. / Lee, C.A. / Hazu, M. / Galatis, E.G. / Inglis, A.J. / Guna, A. / Voorhees, R.M.
History
DepositionApr 14, 2026Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 13, 2026Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: UCP1 Nanobody
A: Mitochondrial carrier homolog 2,Mitochondrial brown fat uncoupling protein 1


Theoretical massNumber of molelcules
Total (without water)47,3952
Polymers47,3952
Non-polymers00
Water00
1


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

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Components

#1: Antibody UCP1 Nanobody


Mass: 13913.538 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pETDuet / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Protein Mitochondrial carrier homolog 2,Mitochondrial brown fat uncoupling protein 1 / Met-induced mitochondrial protein / UCP 1 / Solute carrier family 25 member 7 / Thermogenin


Mass: 33480.988 Da / Num. of mol.: 1 / Mutation: K25E,Y235A,V238D,L199V,A200Y,T226A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MTCH2, MIMP, HSPC032, UCP1, SLC25A7, UCP / Plasmid: pFastBac1 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9Y6C9, UniProt: P25874
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: MTCH2 hyperactive mutant K25E Y235A V238D complexed with UCP1 nanobody
Type: COMPLEX
Details: Human MTCH2 hyperactive mutant K25E V238D Y235A with the cytosolic loop between TM4 and TM5 replaced by the anti-UCP1 nanobody recognition epitope from UCP1 in complex with the anti-UCP1 ...Details: Human MTCH2 hyperactive mutant K25E V238D Y235A with the cytosolic loop between TM4 and TM5 replaced by the anti-UCP1 nanobody recognition epitope from UCP1 in complex with the anti-UCP1 nanobody pMb65 (modified to permit NabFab binding).
Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human) / Cellular location: Mitochondrial outer membrane / Organelle: Mitochondrion
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Cell: Sf9 / Plasmid: pFastBac1
Buffer solutionpH: 7.5
Details: 50 mM HEPES/KOH pH 7.5, 200 mM NaCl, 2 mM Mg Acetate, and 0.07% UDM
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid/potassium hydroxideHEPES/KOH1
22 mMmagnesium acetateMgOAc1
30.07 %n-Undecyl-beta-D-MaltopyranosideUDM1
4200 mMsodium chlorideNaCl1
SpecimenConc.: 4.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: The grid was glow-discharged with a PELCO easiGlowTM (Ted Pella, Inc.) at 20 mA for 60 s.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 279.15 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.6 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10816
Image scansWidth: 11520 / Height: 8184

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.7.0particle selection
2SerialEMimage acquisition
4cryoSPARC4.7.0CTF correction
7Coot0.9.8.95model fitting
9PHENIX1.21.2_5419model refinement
10cryoSPARC4.7.0initial Euler assignment
11cryoSPARC4.7.0final Euler assignment
12cryoSPARC4.7.0classification
13cryoSPARC4.7.03D reconstruction
CTF correctionDetails: CTF parameters estimated using Patch CTF estimation in cryoSPARC. Global CTF correction for beam tilt was additionally performed.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3547162
Details: 9,633 out of 10,816 micrographs were selected for downstream processing. A total of 3,547,162 particles from blob picker and template picker were obtained. After running 7 rounds of ...Details: 9,633 out of 10,816 micrographs were selected for downstream processing. A total of 3,547,162 particles from blob picker and template picker were obtained. After running 7 rounds of heterogenous refinement, 612,877 particles were selected for further processing.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 612877 / Algorithm: FOURIER SPACE
Details: Final map generated by local refinement in cryoSPARC using a soft mask encompassing MTCH2 only.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 194 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Real-space correlation coefficient
Details: Initial fitting was done using Coot and refined by Phenix cryo-EM real space refinement.
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDChain-IDSource nameTypeAccession codeInitial refinement model-ID
1AAlphaFoldin silico model
28HBV

8hbv

BBPDBexperimental model8HBV2
RefinementHighest resolution: 3.1 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)

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