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- EMDB-52632: Structure of the Chaetomium thermophilum Pmt4 homodimer (C1 symmetry) -

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Basic information

Entry
Database: EMDB / ID: EMD-52632
TitleStructure of the Chaetomium thermophilum Pmt4 homodimer (C1 symmetry)
Map datafinal map used for model building
Sample
  • Complex: Chaetomium thermophilum Pmt4 homodimer bound to dolichol-phosphate and dolichol-phosphate-mannose
    • Protein or peptide: Dolichyl-phosphate-mannose--protein mannosyltransferase
  • Ligand: dolichol-phosphate-mannose (n=2)
  • Ligand: dolichol-phosphate (n=4)
Keywordshomodimer / glycosylation / ER / biogenesis / MEMBRANE PROTEIN
Function / homology
Function and homology information


dolichyl-phosphate-mannose-protein mannosyltransferase / dolichyl-phosphate-mannose-protein mannosyltransferase activity / endoplasmic reticulum membrane
Similarity search - Function
Glycosyl transferase family 39/83 / Glycosyltransferase 39-like / Protein O-mannosyl-transferase, C-terminal four TM domain / Dolichyl-phosphate-mannose-protein mannosyltransferase / C-terminal four TMM region of protein-O-mannosyltransferase / MIR motif / MIR domain / MIR domain profile. / Domain in ryanodine and inositol trisphosphate receptors and protein O-mannosyltransferases / Mir domain superfamily
Similarity search - Domain/homology
Dolichyl-phosphate-mannose--protein mannosyltransferase
Similarity search - Component
Biological speciesThermochaetoides thermophila (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsMcDowell MA / Wild K / Sinning I
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)FOR2509 (P07) Germany
CitationJournal: Nat Commun / Year: 2025
Title: Structural characterisation of the fungal Pmt4 homodimer.
Authors: Melanie A McDowell / Klemens Wild / Francesco Fiorentino / Daniela Bausewein / Anke Metschies / Antonella Chiapparino / Yvonne Hackmann / Florestan L Bilsing / David Brenske / Sofia ...Authors: Melanie A McDowell / Klemens Wild / Francesco Fiorentino / Daniela Bausewein / Anke Metschies / Antonella Chiapparino / Yvonne Hackmann / Florestan L Bilsing / David Brenske / Sofia Mortensen / Di Wu / Carol V Robinson / Sabine Strahl / Irmgard Sinning /
Abstract: Protein O-mannosyltransferases (PMTs) are conserved endoplasmic reticulum membrane-embedded enzymes responsible for the transfer of mannose from dolichol phosphate-mannose (Dol-P-Man) to ...Protein O-mannosyltransferases (PMTs) are conserved endoplasmic reticulum membrane-embedded enzymes responsible for the transfer of mannose from dolichol phosphate-mannose (Dol-P-Man) to serine/threonine-rich protein substrates or unfolded proteins. PMTs from three subfamilies form obligate dimers with different substrate specificities and require the concerted action of their transmembrane domains (TMDs) and a luminal MIR domain for catalysis. Here, we present structures, native mass spectrometry, and structure-based mutagenesis of the fungal Pmt4 homodimer. The core fold of the TMDs and MIR domain is conserved with the Pmt1-Pmt2 heterodimer, indicating a shared catalytic mechanism. Distinct from Pmt4, the MIR domain interacts in cis with the TMDs of the same subunit and has a β-hairpin insertion required for O-mannosylation of substrates. We further identify a cytosolic binding site for substrate Dol-P-Man within the Pmt4 TMDs, which is conserved amongst PMTs and important for in vivo activity. Thus, we provide a framework to understand the substrate specificity and regulation of the Pmt4 homodimer.
History
DepositionJan 28, 2025-
Header (metadata) releaseDec 24, 2025-
Map releaseDec 24, 2025-
UpdateDec 24, 2025-
Current statusDec 24, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52632.png

Downloads & links

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Map

FileDownload / File: emd_52632.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfinal map used for model building
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 211.712 Å		0.83 Å/pix.
x 256 pix.
= 211.712 Å		0.83 Å/pix.
x 256 pix.
= 211.712 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.827 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.053151924 - 0.08785172
Average (Standard dev.)0.000022085069 (±0.002965799)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.712 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_52632_msk_1.map
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Additional map: map low-pass filtered to 6 A

Fileemd_52632_additional_1.map
Annotationmap low-pass filtered to 6 A
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Additional map: local density sharpening with LocScale to represent each...

Fileemd_52632_additional_2.map
Annotationlocal density sharpening with LocScale to represent each region at its local resolution
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Half map: #2

Fileemd_52632_half_map_1.map
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Half map: #1

Fileemd_52632_half_map_2.map
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Sample components

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Entire : Chaetomium thermophilum Pmt4 homodimer bound to dolichol-phosphat...

EntireName: Chaetomium thermophilum Pmt4 homodimer bound to dolichol-phosphate and dolichol-phosphate-mannose
Components
  • Complex: Chaetomium thermophilum Pmt4 homodimer bound to dolichol-phosphate and dolichol-phosphate-mannose
    • Protein or peptide: Dolichyl-phosphate-mannose--protein mannosyltransferase
  • Ligand: dolichol-phosphate-mannose (n=2)
  • Ligand: dolichol-phosphate (n=4)

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Supramolecule #1: Chaetomium thermophilum Pmt4 homodimer bound to dolichol-phosphat...

SupramoleculeName: Chaetomium thermophilum Pmt4 homodimer bound to dolichol-phosphate and dolichol-phosphate-mannose
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Thermochaetoides thermophila (fungus)
Molecular weightTheoretical: 180 KDa

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Macromolecule #1: Dolichyl-phosphate-mannose--protein mannosyltransferase

MacromoleculeName: Dolichyl-phosphate-mannose--protein mannosyltransferase
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: dolichyl-phosphate-mannose-protein mannosyltransferase
Source (natural)Organism: Thermochaetoides thermophila (fungus)
Molecular weightTheoretical: 92.046484 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSGSPAPQGA LRQRIVASTP KKTKESKDGA ASDAELDKLV KEAAAAKAPA GSERDYKIAF AIITALAFLT RFWGISHPDQ VVFDEVHFG KFASYYLQRT YFFDVHPPLG KLLFAFMGWL VGYDGHFHFD NIGDPYVINK VPYVAFRALP AILGSLTVSV V FLIMWESG ...String:
MSGSPAPQGA LRQRIVASTP KKTKESKDGA ASDAELDKLV KEAAAAKAPA GSERDYKIAF AIITALAFLT RFWGISHPDQ VVFDEVHFG KFASYYLQRT YFFDVHPPLG KLLFAFMGWL VGYDGHFHFD NIGDPYVINK VPYVAFRALP AILGSLTVSV V FLIMWESG YSLPACILAS GLVLLDNAHI GQTRLILLDA TLVFAMACSL LCYIKFYKLR HEPFSRKWWK WLILTGFALS CD ISTKYVG LFAFITIGSA VCIDLWDLLN INRPKGALTL PQFGKHFAAR AFGLIFMPFM FYLFWFQVHF SILTRSGPGD DFM TPEFQE TLSDNIMLAN AVTIDYWDTI TIKHKETKAY LHSHPDRYPL RYDDGRVSSQ GQQVTGYPFN DTNNWWQILP AGPF EEPKL GRHVKHRDLV RLRHVGTDTY LLSHDVASPY YPTNQEFTTV SFNEAYGDRA ADTLFEVRIE HGKPGQEFKS ISSHF KLIH NPSKVAMWTH PTPLPDWGHR QQEINGNKQI APSSNVWLVE DIVSLPADHK RREKPERKVK TLPFLRKWFE LQRSMF WHN NQLTASHPYA SLPYQWPFLL RGVSFWTNSE TRQQIYFLGN PVGWWIASSV LAIYVGIVLA DQFSLRRGID ALDHRTR SR LYNSTGFFFL AWATHYFPFF VMGRQLFLHH YLPAHLASTL VTGALVEFIF SPEAPEHEIA AQQAKSSSSG KRGGVAMK R HITARERFAG QSLMGSWIAT AVILALVAWS WWFFLPLTYG YPGMSVQQVL RRKWLGYDLH FAKLEVLFQG PGSHHHHHH HHHH

UniProtKB: Dolichyl-phosphate-mannose--protein mannosyltransferase

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Macromolecule #2: dolichol-phosphate-mannose (n=2)

MacromoleculeName: dolichol-phosphate-mannose (n=2) / type: ligand / ID: 2 / Number of copies: 2 / Formula: A1I0N
Molecular weightTheoretical: 684.88 Da

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Macromolecule #3: dolichol-phosphate (n=4)

MacromoleculeName: dolichol-phosphate (n=4) / type: ligand / ID: 3 / Number of copies: 2 / Formula: A1I0M
Molecular weightTheoretical: 658.974 Da

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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

Concentration1.7 mg/mL
BufferpH: 7.5
Details: 20 mM HEPES pH 7.5 200 mM NaCl 0.01% (w/v) LMNG 0.001% (w/v) cholesterol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 1 / Number real images: 9879 / Average electron dose: 42.7 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2199173
CTF correctionSoftware - Name: RELION (ver. 3.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 347202
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cisTEM
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

9fd1

chain_id: A, source_name: PDB, initial_model_type: experimental modelMIR domain crystal structure

9i5k

source_name: PDB, initial_model_type: experimental modelcryoEM structure of TMDs from C2 symmeterised map
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9i5l:
Structure of the Chaetomium thermophilum Pmt4 homodimer (C1 symmetry)

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