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- PDB-9e1t: CryoEM structure of LARGE1 bound to UDP-GlcA -

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Basic information

Entry
Database: PDB / ID: 9e1t
TitleCryoEM structure of LARGE1 bound to UDP-GlcA
ComponentsXylosyl- and glucuronyltransferase LARGE1
KeywordsTRANSFERASE / Glycosyltransferase
Function / homology
Function and homology information


post-embryonic hindlimb morphogenesis / Defective LARGE causes MDDGA6 and MDDGB6 / principal sensory nucleus of trigeminal nerve development / xylosyltransferase activity / walking behavior / connective tissue development / Transferases; Glycosyltransferases / glycosphingolipid biosynthetic process / O-linked glycosylation / skeletal muscle organ development ...post-embryonic hindlimb morphogenesis / Defective LARGE causes MDDGA6 and MDDGB6 / principal sensory nucleus of trigeminal nerve development / xylosyltransferase activity / walking behavior / connective tissue development / Transferases; Glycosyltransferases / glycosphingolipid biosynthetic process / O-linked glycosylation / skeletal muscle organ development / glucuronosyltransferase activity / UDP-xylosyltransferase activity / synaptic assembly at neuromuscular junction / localization of cell / protein O-linked glycosylation via mannose / reactive gliosis / N-acetylglucosamine metabolic process / glycoprotein biosynthetic process / neuromuscular process controlling posture / acetylglucosaminyltransferase activity / water transport / plasma membrane organization / retina layer formation / retina vasculature development in camera-type eye / skeletal muscle fiber differentiation / nerve development / basement membrane organization / hexosyltransferase activity / neuromuscular synaptic transmission / dentate gyrus development / skeletal muscle tissue regeneration / protein O-linked glycosylation / astrocyte differentiation / cardiac muscle cell development / acetylcholine receptor signaling pathway / protein targeting to membrane / Transferases; Glycosyltransferases; Hexosyltransferases / muscle cell cellular homeostasis / : / blood vessel development / glycosyltransferase activity / response to light stimulus / macrophage differentiation / Transferases; Glycosyltransferases; Pentosyltransferases / response to mechanical stimulus / behavioral fear response / striated muscle contraction / skeletal muscle fiber development / myelination / cytoskeleton organization / potassium ion transmembrane transport / post-translational protein modification / determination of adult lifespan / protein localization to plasma membrane / neuromuscular junction / intracellular protein transport / sensory perception of sound / bone development / multicellular organism growth / memory / long-term synaptic potentiation / neuron migration / manganese ion binding / protein-containing complex assembly / gene expression / Golgi membrane / Golgi apparatus / protein-containing complex / plasma membrane
Similarity search - Function
: / Glycosyl-transferase for dystroglycan / Glycosyl transferase, family 8 / Glycosyl transferase family 8 / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
: / URIDINE-5'-DIPHOSPHATE-GLUCURONIC ACID / Xylosyl- and glucuronyltransferase LARGE1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsJoseph, S. / Spellmon, N. / Campbell, K.P.
Funding support United States, 3items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)U54 NS053672 United States
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)U24 GM129547 United States
CitationJournal: Nat Commun / Year: 2025
Title: LARGE1 processively polymerizes length-controlled matriglycan on prodystroglycan.
Authors: Soumya Joseph / Nicholas J Schnicker / Nicholas Spellmon / Zhen Xu / Rui Yan / Zhiheng Yu / Omar Davulcu / Tiandi Yang / Jesse Hopkins / Mary E Anderson / David Venzke / Kevin P Campbell /
Abstract: Matriglycan is a linear glycan (xylose-β1,3-glucuronate), which binds proteins in the extracellular matrix that contain laminin-globular domains and Lassa Fever Virus. It is indispensable for ...Matriglycan is a linear glycan (xylose-β1,3-glucuronate), which binds proteins in the extracellular matrix that contain laminin-globular domains and Lassa Fever Virus. It is indispensable for neuromuscular function. Matriglycan of insufficient length can cause muscular dystrophy with abnormal brain and eye development. LARGE1 (Like-acetylglucosaminyltransferase-1) uniquely synthesizes matriglycan on dystroglycan. The mechanism of matriglycan synthesis is not obvious from cryo-EM reconstructions of LARGE1. However, by reconstituting activity in vitro on recombinant prodystroglycan we show that the presence of the dystroglycan N-terminal domain (DGN), phosphorylated core M3, and a xylose-glucuronate primer are necessary for matriglycan polymerization by LARGE1. By introducing active site mutations, we demonstrate that LARGE1 processively polymerizes matriglycan on prodystroglycan, with its length regulated by the dystroglycan prodomain, DGN. Our enzymatic analysis of LARGE1 uncovers the mechanism of matriglycan synthesis on dystroglycan, which can form the basis for therapeutic strategies to treat matriglycan-deficient neuromuscular disorders and arenaviral infections.
History
DepositionOct 21, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release
Revision 1.0Feb 12, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 12, 2025Data content type: Additional map / Data content type: Additional map / Provider: repository / Type: Initial release
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Revision 1.1May 21, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 21, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name
Revision 2.0Oct 22, 2025Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Database references / Derived calculations / Non-polymer description / Refinement description / Structure summary
Category: atom_site / audit_author ...atom_site / audit_author / chem_comp / chem_comp_atom / chem_comp_bond / citation / citation_author / em_admin / em_author_list / em_image_recording / em_software / entity / pdbx_contact_author / pdbx_entity_instance_feature / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_conn_angle / pdbx_validate_close_contact / pdbx_validate_torsion / refine_ls_restr / struct / struct_conn
Item: _audit_author.name / _chem_comp.formula ..._audit_author.name / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.name / _chem_comp.pdbx_synonyms / _chem_comp.type / _citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update / _em_author_list.author / _em_image_recording.avg_electron_dose_per_image / _entity.formula_weight / _entity.pdbx_description / _pdbx_entity_nonpoly.comp_id / _pdbx_entity_nonpoly.name / _pdbx_nonpoly_scheme.auth_mon_id / _pdbx_nonpoly_scheme.auth_seq_num / _pdbx_nonpoly_scheme.mon_id / _pdbx_nonpoly_scheme.pdb_mon_id / _pdbx_nonpoly_scheme.pdb_seq_num / _struct.title
Description: Ligand geometry
Details: Coordination distances between LARGE, MN and UGA required additional remodeling.
Provider: author / Type: Coordinate replacement

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Xylosyl- and glucuronyltransferase LARGE1
B: Xylosyl- and glucuronyltransferase LARGE1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)180,8836
Polymers179,6122
Non-polymers1,2704
Water00
1


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

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Components

#1: Protein Xylosyl- and glucuronyltransferase LARGE1 / Acetylglucosaminyltransferase-like 1A / Glycosyltransferase-like protein / LARGE xylosyl- and ...Acetylglucosaminyltransferase-like 1A / Glycosyltransferase-like protein / LARGE xylosyl- and glucuronyltransferase 1


Mass: 89806.242 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LARGE1, KIAA0609, LARGE / Production host: Homo sapiens (human)
References: UniProt: O95461, Transferases; Glycosyltransferases, Transferases; Glycosyltransferases; Pentosyltransferases, Transferases; Glycosyltransferases; Hexosyltransferases
#2: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
#3: Chemical ChemComp-UGA / URIDINE-5'-DIPHOSPHATE-GLUCURONIC ACID / UDP-GLUCURONIC ACID


Mass: 580.285 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C15H22N2O18P2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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: LARGE1 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 6.6
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
10cryoSPARCinitial Euler assignment
11RELIONfinal Euler assignment
13RELION3D reconstruction
14PHENIX3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 347589 / Symmetry type: POINT

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