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- PDB-7zlg: Cryo-EM structure of C-mannosyltransferase CeDPY19, in complex wi... -

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Basic information

Entry
Database: PDB / ID: 7zlg
TitleCryo-EM structure of C-mannosyltransferase CeDPY19, in complex with acceptor peptide and bound to CMT2-Fab and anti-Fab nanobody
Components
  • Anti-Fab nanobody
  • C-mannosyltransferase dpy-19
  • CMT2-Fab heavy chain
  • CMT2-Fab light chain
  • Synthetic octapeptide WEHI 1886493
KeywordsMEMBRANE PROTEIN / C-mannosyltransferase
Function / homology
Function and homology information


protein C-linked glycosylation via 2'-alpha-mannosyl-L-tryptophan / mannosyltransferase activity / nuclear inner membrane / Transferases; Glycosyltransferases; Hexosyltransferases / nervous system development / cell differentiation / carbohydrate metabolic process / endoplasmic reticulum membrane / perinuclear region of cytoplasm / extracellular region / cytoplasm
Similarity search - Function
Dpy-19/Dpy-19-like / : / Q-cell neuroblast polarisation / Glycoside hydrolase/deacetylase, beta/alpha-barrel / Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin V-set domain / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulin subtype / Immunoglobulins and major histocompatibility complex proteins signature. ...Dpy-19/Dpy-19-like / : / Q-cell neuroblast polarisation / Glycoside hydrolase/deacetylase, beta/alpha-barrel / Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin V-set domain / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulin subtype / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin / Immunoglobulin C-Type / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
C-mannosyltransferase dpy-19 / Ig-like domain-containing protein
Similarity search - Component
Biological speciessynthetic construct (others)
Caenorhabditis elegans (invertebrata)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.72 Å
AuthorsBloch, J.S. / Mukherjee, S. / Mao, R. / Irobalieva, R. / Kossiakoff, A.A. / Goddard-Borger, E.D. / Locher, K.P.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Nat Chem Biol / Year: 2023
Title: Structure, sequon recognition and mechanism of tryptophan C-mannosyltransferase.
Authors: Joël S Bloch / Alan John / Runyu Mao / Somnath Mukherjee / Jérémy Boilevin / Rossitza N Irobalieva / Tamis Darbre / Nichollas E Scott / Jean-Louis Reymond / Anthony A Kossiakoff / Ethan D ...Authors: Joël S Bloch / Alan John / Runyu Mao / Somnath Mukherjee / Jérémy Boilevin / Rossitza N Irobalieva / Tamis Darbre / Nichollas E Scott / Jean-Louis Reymond / Anthony A Kossiakoff / Ethan D Goddard-Borger / Kaspar P Locher /
Abstract: C-linked glycosylation is essential for the trafficking, folding and function of secretory and transmembrane proteins involved in cellular communication processes. The tryptophan C- ...C-linked glycosylation is essential for the trafficking, folding and function of secretory and transmembrane proteins involved in cellular communication processes. The tryptophan C-mannosyltransferase (CMT) enzymes that install the modification attach a mannose to the first tryptophan of WxxW/C sequons in nascent polypeptide chains by an unknown mechanism. Here, we report cryogenic-electron microscopy structures of Caenorhabditis elegans CMT in four key states: apo, acceptor peptide-bound, donor-substrate analog-bound and as a trapped ternary complex with both peptide and a donor-substrate mimic bound. The structures indicate how the C-mannosylation sequon is recognized by this CMT and its paralogs, and how sequon binding triggers conformational activation of the donor substrate: a process relevant to all glycosyltransferase C superfamily enzymes. Our structural data further indicate that the CMTs adopt an unprecedented electrophilic aromatic substitution mechanism to enable the C-glycosylation of proteins. These results afford opportunities for understanding human disease and therapeutic targeting of specific CMT paralogs.
History
DepositionApr 15, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 11, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 18, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2May 10, 2023Group: 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: CMT2-Fab heavy chain
K: Anti-Fab nanobody
L: CMT2-Fab light chain
A: C-mannosyltransferase dpy-19
P: Synthetic octapeptide WEHI 1886493


Theoretical massNumber of molelcules
Total (without water)143,3455
Polymers143,3455
Non-polymers00
Water18010
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Antibody , 3 types, 3 molecules HKL

#1: Antibody CMT2-Fab heavy chain


Mass: 25000.723 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#2: Antibody Anti-Fab nanobody


Mass: 13390.644 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#3: Antibody CMT2-Fab light chain


Mass: 23238.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli) / References: UniProt: Q7Z3Y4

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Protein / Protein/peptide / Non-polymers , 3 types, 12 molecules AP

#4: Protein C-mannosyltransferase dpy-19 / Protein dumpy-19


Mass: 80892.602 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: dpy-19, F22B7.10 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P34413, Transferases; Glycosyltransferases; Hexosyltransferases
#5: Protein/peptide Synthetic octapeptide WEHI 1886493


Mass: 821.900 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: GSWAKWS / Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 10 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: C-mannosyltransferase CeDPY19, in complex with acceptor peptide and bound to CMT2-Fab and anti-Fab nanobody
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 800 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.72 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 324852 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0049854
ELECTRON MICROSCOPYf_angle_d0.71613392
ELECTRON MICROSCOPYf_dihedral_angle_d13.433478
ELECTRON MICROSCOPYf_chiral_restr0.0451501
ELECTRON MICROSCOPYf_plane_restr0.0041687

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