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- PDB-8pn9: Structure of human oligosaccharyltransferase OST-A complex bound ... -

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Basic information

Entry
Database: PDB / ID: 8pn9
TitleStructure of human oligosaccharyltransferase OST-A complex bound to NGI-1
Components
  • (Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit ...) x 5
  • Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit
  • Oligosaccharyltransferase complex subunit OSTCOligosaccharyltransferase
  • Transmembrane protein 258Transmembrane protein
KeywordsTRANSFERASE / N-glycosylation / OST-A complex / NGI-1 inhibitor
Function / homology
Function and homology information


oligosaccharyltransferase complex binding / oligosaccharyltransferase I complex / oligosaccharyltransferase III complex / Asparagine N-linked glycosylation / membrane-bounded organelle / co-translational protein modification / oligosaccharyltransferase complex / dolichyl-diphosphooligosaccharide-protein glycotransferase / dolichyl-diphosphooligosaccharide-protein glycotransferase activity / protein N-linked glycosylation via asparagine ...oligosaccharyltransferase complex binding / oligosaccharyltransferase I complex / oligosaccharyltransferase III complex / Asparagine N-linked glycosylation / membrane-bounded organelle / co-translational protein modification / oligosaccharyltransferase complex / dolichyl-diphosphooligosaccharide-protein glycotransferase / dolichyl-diphosphooligosaccharide-protein glycotransferase activity / protein N-linked glycosylation via asparagine / protein N-linked glycosylation / epithelial cell apoptotic process / azurophil granule membrane / protein glycosylation / blastocyst development / Advanced glycosylation endproduct receptor signaling / SRP-dependent cotranslational protein targeting to membrane / enzyme activator activity / rough endoplasmic reticulum / response to endoplasmic reticulum stress / T cell activation / post-translational protein modification / response to cytokine / regulation of protein stability / protein modification process / melanosome / Maturation of spike protein / nuclear body / inflammatory response / intracellular membrane-bounded organelle / apoptotic process / Neutrophil degranulation / endoplasmic reticulum membrane / negative regulation of apoptotic process / endoplasmic reticulum / protein-containing complex / RNA binding / membrane / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
DAD/Ost2 / Oligosaccharyltransferase complex subunit / DAD family / Oligosaccharyltransferase subunit 5 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48kDa subunit / Oligosaccharyltransferase complex subunit OSTC / Oligosaccharyltransferase 48 kDa subunit beta / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit Swp1 / Oligosaccharyltransferase subunit Ribophorin II / Oligosaccaryltransferase ...DAD/Ost2 / Oligosaccharyltransferase complex subunit / DAD family / Oligosaccharyltransferase subunit 5 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48kDa subunit / Oligosaccharyltransferase complex subunit OSTC / Oligosaccharyltransferase 48 kDa subunit beta / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit Swp1 / Oligosaccharyltransferase subunit Ribophorin II / Oligosaccaryltransferase / Oligosaccharyltransferase subunit ost4p superfamily / Oligosaccaryltransferase / Ribophorin I / Ribophorin I / Oligosaccharyl transferase complex, subunit OST3/OST6 / OST3 / OST6 family, transporter family / : / STT3/PglB/AglB core domain / : / Oligosaccharyl transferase, STT3 subunit / Oligosaccharyl transferase STT3, N-terminal
Similarity search - Domain/homology
Chem-EGY / Chem-KZB / : / Chem-OTP / : / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 4 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A ...Chem-EGY / Chem-KZB / : / Chem-OTP / : / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 4 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A / Transmembrane protein 258 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit DAD1 / Oligosaccharyltransferase complex subunit OSTC
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.61 Å
AuthorsRamirez, A.S. / Kowal, J. / Locher, K.P.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_196862 Switzerland
CitationJournal: Cell / Year: 2024
Title: Positive selection CRISPR screens reveal a druggable pocket in an oligosaccharyltransferase required for inflammatory signaling to NF-κB.
Authors: Benjamin L Lampson / Ana S Ramίrez / Marta Baro / Lixia He / Mudra Hegde / Vidyasagar Koduri / Jamie L Pfaff / Ruth E Hanna / Julia Kowal / Nitin H Shirole / Yanfeng He / John G Doench / ...Authors: Benjamin L Lampson / Ana S Ramίrez / Marta Baro / Lixia He / Mudra Hegde / Vidyasagar Koduri / Jamie L Pfaff / Ruth E Hanna / Julia Kowal / Nitin H Shirole / Yanfeng He / John G Doench / Joseph N Contessa / Kaspar P Locher / William G Kaelin /
Abstract: Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome ...Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc-Man-Glc, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.
History
DepositionJun 30, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 8, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A
B: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 4
C: Transmembrane protein 258
D: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit DAD1
E: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1
F: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2
H: Oligosaccharyltransferase complex subunit OSTC
G: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)321,36221
Polymers311,5338
Non-polymers9,83013
Water181
1


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

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Components

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Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit ... , 5 types, 5 molecules ABDEF

#1: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A / STT3-A / B5 / Integral membrane protein 1 / Transmembrane protein TMC


Mass: 80607.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: STT3A, ITM1, TMC / Production host: Homo sapiens (human)
References: UniProt: P46977, dolichyl-diphosphooligosaccharide-protein glycotransferase
#2: Protein/peptide Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 4


Mass: 4196.004 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: OST4 / Production host: Homo sapiens (human) / References: UniProt: P0C6T2
#4: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit DAD1 / Oligosaccharyl transferase subunit DAD1 / Defender against cell death 1 / DAD-1


Mass: 12503.631 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DAD1 / Production host: Homo sapiens (human) / References: UniProt: P61803
#5: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 67 kDa subunit / Ribophorin I / RPN- ...Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 67 kDa subunit / Ribophorin I / RPN-I / Ribophorin-1


Mass: 68656.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RPN1 / Production host: Homo sapiens (human) / References: UniProt: P04843
#6: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 63 kDa subunit / RIBIIR / Ribophorin ...Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 63 kDa subunit / RIBIIR / Ribophorin II / RPN-II / Ribophorin-2


Mass: 69347.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RPN2 / Production host: Homo sapiens (human) / References: UniProt: P04844

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Protein , 3 types, 3 molecules CHG

#3: Protein Transmembrane protein 258 / Transmembrane protein / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit TMEM258 / Oligosaccharyl ...Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit TMEM258 / Oligosaccharyl transferase subunit TMEM258


Mass: 9083.804 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TMEM258, C11orf10, HSPC005 / Production host: Homo sapiens (human) / References: UniProt: P61165
#7: Protein Oligosaccharyltransferase complex subunit OSTC / Oligosaccharyltransferase / Hydrophobic protein HSF-28


Mass: 16844.215 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: OSTC, DC2, HDCMD45P, HSPC307 / Production host: Homo sapiens (human) / References: UniProt: Q9NRP0
#8: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit / DDOST 48 kDa subunit / Oligosaccharyl transferase 48 kDa subunit


Mass: 50293.977 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DDOST, KIAA0115, OST48, OK/SW-cl.45 / Production host: Homo sapiens (human) / References: UniProt: P39656

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Sugars , 4 types, 4 molecules

#9: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#10: Polysaccharide alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D- ...alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)-alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1397.245 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-2DManpa1-2DManpa1-3[DManpa1-6DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,8,7/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3-3-3/a4-b1_b4-c1_c3-d1_c6-g1_d2-e1_e2-f1_g6-h1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(2+1)][a-D-Manp]{[(2+1)][a-D-Manp]{}}}[(6+1)][a-D-Manp]{[(6+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#11: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]alpha-D-mannopyranose-(1-6)-[alpha-D- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]alpha-D-mannopyranose-(1-6)-[alpha-D-mannopyranose-(1-3)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1235.105 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpa1-6[DManpa1-3]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,7,6/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3-3/a4-b1_b4-c1_c3-d1_c6-e1_e3-f1_e6-g1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#12: Polysaccharide alpha-D-glucopyranose-(1-2)-alpha-D-glucopyranose-(1-3)-alpha-D-glucopyranose-(1-3)-alpha-D- ...alpha-D-glucopyranose-(1-2)-alpha-D-glucopyranose-(1-3)-alpha-D-glucopyranose-(1-3)-alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)-alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-alpha-D-glucopyranose


Type: oligosaccharide / Mass: 1883.668 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpa1-2DGlcpa1-3DGlcpa1-3DManpa1-2DManpa1-2DManpa1-3[DManpa1-6DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAca1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/5,11,10/[a2122h-1a_1-5_2*NCC/3=O][a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5][a2122h-1a_1-5]/1-2-3-4-4-4-5-5-5-4-4/a4-b1_b4-c1_c3-d1_c6-j1_d2-e1_e2-f1_f3-g1_g3-h1_h2-i1_j6-k1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(2+1)][a-D-Manp]{[(2+1)][a-D-Manp]{[(3+1)][a-D-Glcp]{[(3+1)][a-D-Glcp]{[(2+1)][a-D-Glcp]{}}}}}}[(6+1)][a-D-Manp]{[(6+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE

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Non-polymers , 6 types, 10 molecules

#13: Chemical
ChemComp-KZB / (2~{S},3~{R},4~{R},5~{S},6~{S})-2-(hydroxymethyl)-6-[(1~{S},2~{R},3~{R},4~{R},5'~{S},6~{S},7~{R},8~{S},9~{R},12~{R},13~{R},15~{S},16~{S},18~{R})-5',7,9,13-tetramethyl-3,15-bis(oxidanyl)spiro[5-oxapentacyclo[10.8.0.0^{2,9}.0^{4,8}.0^{13,18}]icosane-6,2'-oxane]-16-yl]oxy-oxane-3,4,5-triol


Mass: 610.776 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Formula: C33H54O10
#14: Chemical ChemComp-EGY / (4R,7R)-4-hydroxy-N,N,N-trimethyl-4,9-dioxo-7-[(undecanoyloxy)methyl]-3,5,8-trioxa-4lambda~5~-phosphadocosan-1-aminium


Mass: 636.861 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C33H67NO8P / Comment: phospholipid*YM
#15: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn
#16: Chemical ChemComp-OTP / (2E,6E,10E,14E,18E,22E,26E)-3,7,11,15,19,23,27,31-OCTAMETHYLDOTRIACONTA-2,6,10,14,18,22,26,30-OCTAENYL TRIHYDROGEN DIPHOSPHATE / OCTAPRENYL PYROPHOSPHATE


Mass: 722.911 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H68O7P2
#17: Chemical ChemComp-ZXT / 5-(dimethylsulfamoyl)-~{N}-(5-methyl-1,3-thiazol-2-yl)-2-pyrrolidin-1-yl-benzamide


Mass: 394.512 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H22N4O3S2 / Feature type: SUBJECT OF INVESTIGATION
#18: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: OLIGOSACCHARYLTRANSFERASE COMPLEX OST-A WITH BOUND NGI-1 INHIBITOR
Type: COMPLEX / Entity ID: #2-#5, #7 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2400 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 54.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 101699 / Symmetry type: POINT

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