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- PDB-6p28: Crystal structure of the MIR domain (aa 337-532) of the S. cerevi... -

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Basic information

Entry
Database: PDB / ID: 6p28
TitleCrystal structure of the MIR domain (aa 337-532) of the S. cerevisiae mannosyltransferase Pmt2
ComponentsDolichyl-phosphate-mannose--protein mannosyltransferase 2
KeywordsTRANSFERASE / complex / glycosylation
Function / homology
Function and homology information


dolichyl-phosphate-mannose-protein mannosyltransferase Pmt5p-Pmt2p dimer complex / dolichyl-phosphate-mannose-protein mannosyltransferase / dolichyl-phosphate-mannose-protein mannosyltransferase activity / dolichyl-phosphate-mannose-protein mannosyltransferase Pmt1p-Pmt2p dimer complex / regulation of endoplasmic reticulum unfolded protein response / fungal-type cell wall biogenesis / protein O-linked mannosylation / protein exit from endoplasmic reticulum / protein O-linked glycosylation / : ...dolichyl-phosphate-mannose-protein mannosyltransferase Pmt5p-Pmt2p dimer complex / dolichyl-phosphate-mannose-protein mannosyltransferase / dolichyl-phosphate-mannose-protein mannosyltransferase activity / dolichyl-phosphate-mannose-protein mannosyltransferase Pmt1p-Pmt2p dimer complex / regulation of endoplasmic reticulum unfolded protein response / fungal-type cell wall biogenesis / protein O-linked mannosylation / protein exit from endoplasmic reticulum / protein O-linked glycosylation / : / endoplasmic reticulum membrane / endoplasmic reticulum
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 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.35 Å
AuthorsBai, L. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01-CA231466 United States
CitationJournal: Nat Struct Mol Biol / Year: 2019
Title: Structure of the eukaryotic protein O-mannosyltransferase Pmt1-Pmt2 complex.
Authors: Lin Bai / Amanda Kovach / Qinglong You / Alanna Kenny / Huilin Li /
Abstract: In eukaryotes, a nascent peptide entering the endoplasmic reticulum (ER) is scanned by two Sec61 translocon-associated large membrane machines for protein N-glycosylation and protein O-mannosylation, ...In eukaryotes, a nascent peptide entering the endoplasmic reticulum (ER) is scanned by two Sec61 translocon-associated large membrane machines for protein N-glycosylation and protein O-mannosylation, respectively. While the structure of the eight-protein oligosaccharyltransferase complex has been determined recently, the structures of mannosyltransferases of the PMT family, which are an integral part of ER protein homeostasis, are still unknown. Here we report cryo-EM structures of the Saccharomyces cerevisiae Pmt1-Pmt2 complex bound to a donor and an acceptor peptide at 3.2-Å resolution, showing that each subunit contains 11 transmembrane helices and a lumenal β-trefoil fold termed the MIR domain. The structures reveal the substrate recognition model and confirm an inverting mannosyl-transferring reaction mechanism by the enzyme complex. Furthermore, we found that the transmembrane domains of Pmt1 and Pmt2 share a structural fold with the catalytic subunits of oligosaccharyltransferases, confirming a previously proposed evolutionary relationship between protein O-mannosylation and protein N-glycosylation.
History
DepositionMay 21, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 10, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Aug 21, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Dolichyl-phosphate-mannose--protein mannosyltransferase 2


Theoretical massNumber of molelcules
Total (without water)22,4991
Polymers22,4991
Non-polymers00
Water3,441191
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area9080 Å2
MethodPISA
Unit cell
Length a, b, c (Å)34.289, 71.403, 37.886
Angle α, β, γ (deg.)90.00, 96.37, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Dolichyl-phosphate-mannose--protein mannosyltransferase 2 / Pmt2


Mass: 22498.826 Da / Num. of mol.: 1 / Fragment: MIR domain (UNP residues 337-532)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PMT2, FUN25, YAL023C / Production host: Escherichia coli (E. coli)
References: UniProt: P31382, dolichyl-phosphate-mannose-protein mannosyltransferase
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 191 / Source method: isolated from a natural source / Formula: H2O

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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: 2.09 Å3/Da / Density % sol: 41.06 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / Details: 25% PEG20000 MME

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-D / Wavelength: 1.078 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Oct 8, 2018
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.078 Å / Relative weight: 1
ReflectionResolution: 1.35→37.65 Å / Num. obs: 40003 / % possible obs: 96.7 % / Redundancy: 3.5 % / Rmerge(I) obs: 0.134 / Net I/σ(I): 13.4
Reflection shellResolution: 1.35→1.38 Å / Rmerge(I) obs: 0.023 / Num. unique obs: 17096

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Processing

Software
NameVersionClassification
REFMAC5.8.0155refinement
HKL-2000data reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 6P25

6p25


Resolution: 1.35→37.65 Å / Cor.coef. Fo:Fc: 0.972 / Cor.coef. Fo:Fc free: 0.962 / SU B: 0.924 / SU ML: 0.038 / Cross valid method: THROUGHOUT / ESU R: 0.057 / ESU R Free: 0.06 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.213 1965 5.2 %RANDOM
Rwork0.187 ---
obs0.187 36188 95.38 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 19.885 Å2
Baniso -1Baniso -2Baniso -3
1-0 Å20 Å20 Å2
2--0 Å20 Å2
3---0 Å2
Refinement stepCycle: 1 / Resolution: 1.35→37.65 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1555 0 0 191 1746
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0250.0191596
X-RAY DIFFRACTIONr_bond_other_d0.0020.021443
X-RAY DIFFRACTIONr_angle_refined_deg2.3591.9162166
X-RAY DIFFRACTIONr_angle_other_deg1.23933321
X-RAY DIFFRACTIONr_dihedral_angle_1_deg8.1015191
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.96924.65186
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.15915267
X-RAY DIFFRACTIONr_dihedral_angle_4_deg22.1671510
X-RAY DIFFRACTIONr_chiral_restr0.1840.2223
X-RAY DIFFRACTIONr_gen_planes_refined0.0120.0211848
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02396
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.4071.67767
X-RAY DIFFRACTIONr_mcbond_other2.3981.669766
X-RAY DIFFRACTIONr_mcangle_it3.7682.508957
X-RAY DIFFRACTIONr_mcangle_other3.7692.508958
X-RAY DIFFRACTIONr_scbond_it3.1682.029829
X-RAY DIFFRACTIONr_scbond_other3.1672.029830
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other4.8542.91210
X-RAY DIFFRACTIONr_long_range_B_refined7.02433.3736997
X-RAY DIFFRACTIONr_long_range_B_other6.87632.7756822
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.348→1.383 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.254 128 -
Rwork0.234 2274 -
obs--81.81 %

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