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- EMDB-60365: Cryo-EM structure of the yeast Htm1/Pdi1 complex at a resolution ... -

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Basic information

Entry
Database: EMDB / ID: EMD-60365
TitleCryo-EM structure of the yeast Htm1/Pdi1 complex at a resolution of 3.0 angstrom
Map datasharpened map
Sample
  • Complex: A complex of yeast Htm1/Pdi1
    • Protein or peptide: ER degradation-enhancing alpha-mannosidase-like protein 1
    • Protein or peptide: Protein disulfide-isomerase
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM ION
Keywordscomplex / carbohydrate cleavage / peptide binding / PEPTIDE BINDING PROTEIN
Function / homology
Function and homology information


mannosyl-oligosaccharide 1,2-alpha-mannosidase complex / Calnexin/calreticulin cycle / : / ubiquitin-dependent glycoprotein ERAD pathway / alpha-mannosidase / mannosyl-oligosaccharide 1,2-alpha-mannosidase activity / endoplasmic reticulum mannose trimming / protein disulfide-isomerase / endoplasmic reticulum quality control compartment / positive regulation of endoplasmic reticulum unfolded protein response ...mannosyl-oligosaccharide 1,2-alpha-mannosidase complex / Calnexin/calreticulin cycle / : / ubiquitin-dependent glycoprotein ERAD pathway / alpha-mannosidase / mannosyl-oligosaccharide 1,2-alpha-mannosidase activity / endoplasmic reticulum mannose trimming / protein disulfide-isomerase / endoplasmic reticulum quality control compartment / positive regulation of endoplasmic reticulum unfolded protein response / protein glycosylation / protein disulfide isomerase activity / protein-disulfide reductase activity / endoplasmic reticulum unfolded protein response / response to endoplasmic reticulum stress / unfolded protein binding / protein folding / carbohydrate binding / carbohydrate metabolic process / endoplasmic reticulum lumen / calcium ion binding / endoplasmic reticulum / membrane
Similarity search - Function
ER degradation-enhancing alpha-mannosidase-like protein 1/2/3 / Glycoside hydrolase family 47 / Seven-hairpin glycosidases / Glycosyl hydrolase family 47 / Protein disulphide isomerase / Thioredoxin-like domain / Endoplasmic reticulum targeting sequence. / Thioredoxin / Six-hairpin glycosidase-like superfamily / Thioredoxin, conserved site ...ER degradation-enhancing alpha-mannosidase-like protein 1/2/3 / Glycoside hydrolase family 47 / Seven-hairpin glycosidases / Glycosyl hydrolase family 47 / Protein disulphide isomerase / Thioredoxin-like domain / Endoplasmic reticulum targeting sequence. / Thioredoxin / Six-hairpin glycosidase-like superfamily / Thioredoxin, conserved site / Thioredoxin family active site. / Thioredoxin domain profile. / Thioredoxin domain / Thioredoxin-like superfamily
Similarity search - Domain/homology
Protein disulfide-isomerase / ER degradation-enhancing alpha-mannosidase-like protein 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsWu XW / Zhao D / Rapoport TA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM052586 United States
CitationJournal: bioRxiv / Year: 2024
Title: Initiation of ERAD by the bifunctional complex of Mnl1 mannosidase and protein disulfide isomerase.
Authors: Dan Zhao / Xudong Wu / Tom A Rapoport /
Abstract: Misfolded glycoproteins in the endoplasmic reticulum (ER) lumen are translocated into the cytosol and degraded by the proteasome, a conserved process called ER-associated protein degradation (ERAD). ...Misfolded glycoproteins in the endoplasmic reticulum (ER) lumen are translocated into the cytosol and degraded by the proteasome, a conserved process called ER-associated protein degradation (ERAD). In , the glycan of these proteins is trimmed by the luminal mannosidase Mnl1 (Htm1) to generate a signal that triggers degradation. Curiously, Mnl1 is permanently associated with protein disulfide isomerase (Pdi1). Here, we have used cryo-electron microscopy, biochemical, and experiments to clarify how this complex initiates ERAD. The Mnl1-Pdi1 complex first de-mannosylates misfolded, globular proteins that are recognized through a C-terminal domain (CTD) of Mnl1; Pdi1 causes the CTD to ignore completely unfolded polypeptides. The disulfides of these globular proteins are then reduced by the Pdi1 component of the complex, generating unfolded polypeptides that can be translocated across the membrane. Mnl1 blocks the canonical oxidative function of Pdi1, but allows it to function as the elusive disulfide reductase in ERAD.
History
DepositionMay 31, 2024-
Header (metadata) releaseNov 13, 2024-
Map releaseNov 13, 2024-
UpdateNov 27, 2024-
Current statusNov 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_60365.png

Downloads & links

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Map

FileDownload / File: emd_60365.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 260 pix.
= 214.5 Å		0.83 Å/pix.
x 260 pix.
= 214.5 Å		0.83 Å/pix.
x 260 pix.
= 214.5 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.045093752 - 0.08517909
Average (Standard dev.)0.00007025792 (±0.0025725395)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 214.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: unsharpened map

Fileemd_60365_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

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Histogram

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Half map: halfmap B

Fileemd_60365_half_map_1.map
Annotationhalfmap_B
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: halfmap A

Fileemd_60365_half_map_2.map
Annotationhalfmap_A
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Sample components

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Entire : A complex of yeast Htm1/Pdi1

EntireName: A complex of yeast Htm1/Pdi1
Components
  • Complex: A complex of yeast Htm1/Pdi1
    • Protein or peptide: ER degradation-enhancing alpha-mannosidase-like protein 1
    • Protein or peptide: Protein disulfide-isomerase
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM ION

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Supramolecule #1: A complex of yeast Htm1/Pdi1

SupramoleculeName: A complex of yeast Htm1/Pdi1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 150 KDa

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Macromolecule #1: ER degradation-enhancing alpha-mannosidase-like protein 1

MacromoleculeName: ER degradation-enhancing alpha-mannosidase-like protein 1
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: alpha-mannosidase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 91.336508 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MVCCLWVLLA LLLHLDHVAC EDDAYSFTSK ELKAYKQEVK ELFYFGFDNY LEHGYPYDEV KPISCVPKKR NFEDPTDQGT NDILGNFTI TLIDSLTTIA ILEDRPQFLK AVRLVERTFP DGNFDIDSTI QVFEITIRVI GSLLSSHLYA TDPTKAVYLG D DYDGSLLR ...String:
MVCCLWVLLA LLLHLDHVAC EDDAYSFTSK ELKAYKQEVK ELFYFGFDNY LEHGYPYDEV KPISCVPKKR NFEDPTDQGT NDILGNFTI TLIDSLTTIA ILEDRPQFLK AVRLVERTFP DGNFDIDSTI QVFEITIRVI GSLLSSHLYA TDPTKAVYLG D DYDGSLLR LAQNMADRLL PAYLTSTGLP MPRRNIKRKW DVSEFPEFLE TENNVAAMAS PMFEFTILSY LTGDPKYEKV TR YAFDKTW SLRTGLDLLP MSFHPEKLTP YTPMTGIGAS IDSLFEYALK GAILFDDSEL MEVWNVAYEA LKTNCKNDWF FAN VMADTG HLFVPWIDSL SAFFSGLQVL AGDLDDAIAN HLMFLKMWNT FGGIPERWNF SPPEFPPLSP LERSGAVALD NILP LEWYP LRPEFFESTY FLYRATKDPF YLNIGVHLLK DLKQRFKSNC GFAGFQNVIT GELQDRMETF VLSETLKYLY LLFDE ENEL HNSASDVIFS TEAHPMWLPQ EVRSNYKRNA KFNNSVYSSH LEICQKKDRE QAGENTLSQR IVGFAKSIFH KGPPDE EAT DPIIDYTIDT ELPGTCSIKP HHVIGDEFWY SPMLSNFDRL FEIDSRFAAT LIKPSHMHNY NAIELEPGFY NRWSNPQ FS TCLIPPTTEI FELLFDLPGY HQLNPLMLEN KTITFETFGG RSRLKIEKLQ IYQIDYYGDL ITASTFQDVS RKDIFSNA C DAVASLYSPT YLYRVVAING RILPRHGSVQ IKKHSPVLTS NGTREEDEFK MDGIGINDHS QLMLECTPII NLFIV

UniProtKB: ER degradation-enhancing alpha-mannosidase-like protein 1

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Macromolecule #2: Protein disulfide-isomerase

MacromoleculeName: Protein disulfide-isomerase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: protein disulfide-isomerase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 58.279816 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MKFSAGAVLS WSSLLLASSV FAQQEAVAPE DSAVVKLATD SFNEYIQSHD LVLAEFFAPW CGHCKNMAPE YVKAAETLVE KNITLAQID CTENQDLCME HNIPGFPSLK IFKNSDVNNS IDYEGPRTAE AIVQFMIKQS QPAVAVVADL PAYLANETFV T PVIVQSGK ...String:
MKFSAGAVLS WSSLLLASSV FAQQEAVAPE DSAVVKLATD SFNEYIQSHD LVLAEFFAPW CGHCKNMAPE YVKAAETLVE KNITLAQID CTENQDLCME HNIPGFPSLK IFKNSDVNNS IDYEGPRTAE AIVQFMIKQS QPAVAVVADL PAYLANETFV T PVIVQSGK IDADFNATFY SMANKHFNDY DFVSAENADD DFKLSIYLPS AMDEPVVYNG KKADIADADV FEKWLQVEAL PY FGEIDGS VFAQYVESGL PLGYLFYNDE EELEEYKPLF TELAKKNRGL MNFVSIDARK FGRHAGNLNM KEQFPLFAIH DMT EDLKYG LPQLSEEAFD ELSDKIVLES KAIESLVKDF LKGDASPIVK SQEIFENQDS SVFQLVGKNH DEIVNDPKKD VLVL YYAPW CGHCKRLAPT YQELADTYAN ATSDVLIAKL DHTENDVRGV VIEGYPTIVL YPGGKKSESV VYQGSRSLDS LFDFI KENG HFDVDGKALY EEAQEKAAEE ADADAELADE EDAIHDEL

UniProtKB: Protein disulfide-isomerase

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 313324
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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