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- EMDB-26701: Cryo-EM structure of the human Exostosin-1 and Exostosin-2 hetero... -

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Basic information

Entry
Database: EMDB / ID: EMD-26701
TitleCryo-EM structure of the human Exostosin-1 and Exostosin-2 heterodimer in complex with UDP-GlcNAc
Map datasharp by deepemhancer
Sample
  • Complex: hEXT1/2
    • Protein or peptide: Exostosin-1
    • Protein or peptide: Exostosin-2
  • Ligand: URIDINE-5'-DIPHOSPHATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: MANGANESE (II) ION
Function / homology
Function and homology information


glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase / hypersensitivity / heart field specification / lymphocyte adhesion to endothelial cell of high endothelial venule / heparan sulfate N-acetylglucosaminyltransferase activity / glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase activity / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase activity / smoothened signaling pathway involved in lung development / developmental growth involved in morphogenesis ...glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase / hypersensitivity / heart field specification / lymphocyte adhesion to endothelial cell of high endothelial venule / heparan sulfate N-acetylglucosaminyltransferase activity / glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase activity / N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase activity / smoothened signaling pathway involved in lung development / developmental growth involved in morphogenesis / sweat gland development / perichondral bone morphogenesis / mesenchymal cell differentiation involved in bone development / response to leukemia inhibitory factor / UDP-N-acetylglucosamine transferase complex / chondroitin sulfate metabolic process / response to heparin / chondrocyte hypertrophy / embryonic skeletal joint development / hematopoietic stem cell migration to bone marrow / heparan sulfate proteoglycan biosynthetic process, polysaccharide chain biosynthetic process / fluid transport / glucuronosyltransferase activity / limb joint morphogenesis / tight junction organization / heparin biosynthetic process / Defective EXT2 causes exostoses 2 / Defective EXT1 causes exostoses 1, TRPS2 and CHDS / stomach development / sebaceous gland development / glomerular basement membrane development / heparan sulfate proteoglycan biosynthetic process / HS-GAG biosynthesis / glycosaminoglycan biosynthetic process / dendrite self-avoidance / lymphocyte migration into lymphoid organs / chondrocyte proliferation / sulfation / hematopoietic stem cell homeostasis / dendritic cell migration / endochondral bone morphogenesis / glandular epithelial cell differentiation / endochondral bone growth / acetylglucosaminyltransferase activity / sodium ion homeostasis / podocyte differentiation / basement membrane organization / polysaccharide biosynthetic process / cartilage development involved in endochondral bone morphogenesis / vocalization behavior / cranial skeletal system development / stem cell division / vacuole organization / olfactory bulb development / leukocyte tethering or rolling / multicellular organismal-level water homeostasis / endochondral ossification / endoderm development / response to light intensity / fear response / protein N-linked glycosylation / regulation of tumor necrosis factor-mediated signaling pathway / cellular response to fibroblast growth factor stimulus / collagen fibril organization / neural crest cell differentiation / motor behavior / ossification involved in bone maturation / cell adhesion mediated by integrin / optic nerve development / hair follicle morphogenesis / epithelial tube branching involved in lung morphogenesis / heart contraction / glycosyltransferase activity / protein glycosylation / mesoderm development / antigen processing and presentation / social behavior / mesoderm formation / catalytic complex / hematopoietic stem cell differentiation / blood vessel remodeling / fibroblast growth factor receptor signaling pathway / cell fate commitment / canonical Wnt signaling pathway / BMP signaling pathway / chondrocyte differentiation / bone resorption / gastrulation / ossification / axon guidance / synaptic transmission, glutamatergic / wound healing / protein catabolic process / multicellular organism growth / cellular response to virus / regulation of blood pressure / vasodilation / gene expression / protein-containing complex assembly / protein heterodimerization activity
Similarity search - Function
Exostosin-like / Exostosin, GT47 domain / Exostosin family / Glycosyl transferase 64 domain / Glycosyl transferase family 64 domain / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
Exostosin-1 / Exostosin-2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLi H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA231466 United States
CitationJournal: Nat Chem Biol / Year: 2023
Title: Structural basis for heparan sulfate co-polymerase action by the EXT1-2 complex.
Authors: Hua Li / Digantkumar Chapla / Robert A Amos / Annapoorani Ramiah / Kelley W Moremen / Huilin Li /
Abstract: Heparan sulfate (HS) proteoglycans are extended (-GlcAβ1,4GlcNAcα1,4-) co-polymers containing decorations of sulfation and epimerization that are linked to cell surface and extracellular matrix ...Heparan sulfate (HS) proteoglycans are extended (-GlcAβ1,4GlcNAcα1,4-) co-polymers containing decorations of sulfation and epimerization that are linked to cell surface and extracellular matrix proteins. In mammals, HS repeat units are extended by an obligate heterocomplex of two exostosin family members, EXT1 and EXT2, where each protein monomer contains distinct GT47 (GT-B fold) and GT64 (GT-A fold) glycosyltransferase domains. In this study, we generated human EXT1-EXT2 (EXT1-2) as a functional heterocomplex and determined its structure in the presence of bound donor and acceptor substrates. Structural data and enzyme activity of catalytic site mutants demonstrate that only two of the four glycosyltransferase domains are major contributors to co-polymer syntheses: the EXT1 GT-B fold β1,4GlcA transferase domain and the EXT2 GT-A fold α1,4GlcNAc transferase domain. The two catalytic sites are over 90 Å apart, indicating that HS is synthesized by a dissociative process that involves a single catalytic site on each monomer.
History
DepositionApr 20, 2022-
Header (metadata) releaseSep 28, 2022-
Map releaseSep 28, 2022-
UpdateMay 17, 2023-
Current statusMay 17, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26701.png

Downloads & links

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Map

FileDownload / File: emd_26701.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharp by deepemhancer
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 211.968 Å		0.83 Å/pix.
x 256 pix.
= 211.968 Å		0.83 Å/pix.
x 256 pix.
= 211.968 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0144
Minimum - Maximum-0.0017306556 - 2.0266192
Average (Standard dev.)0.0022433659 (±0.033530224)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.968 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26701_msk_1.map
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Additional map: sharp by postprocess in Relion

Fileemd_26701_additional_1.map
Annotationsharp by postprocess in Relion
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Half map: #2

Fileemd_26701_half_map_1.map
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Half map: #1

Fileemd_26701_half_map_2.map
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Sample components

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Entire : hEXT1/2

EntireName: hEXT1/2
Components
  • Complex: hEXT1/2
    • Protein or peptide: Exostosin-1
    • Protein or peptide: Exostosin-2
  • Ligand: URIDINE-5'-DIPHOSPHATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: hEXT1/2

SupramoleculeName: hEXT1/2 / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Exostosin-1

MacromoleculeName: Exostosin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 83.404023 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GFRASRSHSR REEHSGRNGL HHPSPDHFWP RFPDALRPFV PWDQLENEDS SVHISPRQKR DANSSIYKGK KCRMESCFDF TLCKKNGFK VYVYPQQKGE KIAESYQNIL AAIEGSRFYT SDPSQACLFV LSLDTLDRDQ LSPQYVHNLR SKVQSLHLWN N GRNHLIFN ...String:
GFRASRSHSR REEHSGRNGL HHPSPDHFWP RFPDALRPFV PWDQLENEDS SVHISPRQKR DANSSIYKGK KCRMESCFDF TLCKKNGFK VYVYPQQKGE KIAESYQNIL AAIEGSRFYT SDPSQACLFV LSLDTLDRDQ LSPQYVHNLR SKVQSLHLWN N GRNHLIFN LYSGTWPDYT EDVGFDIGQA MLAKASISTE NFRPNFDVSI PLFSKDHPRT GGERGFLKFN TIPPLRKYML VF KGKRYLT GIGSDTRNAL YHVHNGEDVV LLTTCKHGKD WQKHKDSRCD RDNTEYEKYD YREMLHNATF CLVPRGRRLG SFR FLEALQ AACVPVMLSN GWELPFSEVI NWNQAAVIGD ERLLLQIPST IRSIHQDKIL ALRQQTQFLW EAYFSSVEKI VLTT LEIIQ DRIFKHISRN SLIWNKHPGG LFVLPQYSSY LGDFPYYYAN LGLKPPSKFT AVIHAVTPLV SQSQPVLKLL VAAAK SQYC AQIIVLWNCD KPLPAKHRWP ATAVPVVVIE GESKVMSSRF LPYDNIITDA VLSLDEDTVL STTEVDFAFT VWQSFP ERI VGYPARSHFW DNSKERWGYT SKWTNDYSMV LTGAAIYHKY YHYLYSHYLP ASLKNMVDQL ANCEDILMNF LVSAVTK LP PIKVTQKKQY KETMMGQTSR ASRWADPDHF AQRQSCMNTF ASWFGYMPLI HSQMRLDPVL FKDQVSILRK KYRDIERL

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Macromolecule #2: Exostosin-2

MacromoleculeName: Exostosin-2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 77.238031 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GWPHSIESSN DWNVEKRSIR DVPVVRLPAD SPIPERGDLS CRMHTCFDVY RCGFNPKNKI KVYIYALKKY VDDFGVSVSN TISREYNEL LMAISDSDYY TDDINRACLF VPSIDVLNQN TLRIKETAQA MAQLSRWDRG TNHLLFNMLP GGPPDYNTAL D VPRDRALL ...String:
GWPHSIESSN DWNVEKRSIR DVPVVRLPAD SPIPERGDLS CRMHTCFDVY RCGFNPKNKI KVYIYALKKY VDDFGVSVSN TISREYNEL LMAISDSDYY TDDINRACLF VPSIDVLNQN TLRIKETAQA MAQLSRWDRG TNHLLFNMLP GGPPDYNTAL D VPRDRALL AGGGFSTWTY RQGYDVSIPV YSPLSAEVDL PEKGPGPRQY FLLSSQVGLH PEYREDLEAL QVKHGESVLV LD KCTNLSE GVLSVRKRCH KHQVFDYPQV LQEATFCVVL RGARLGQAVL SDVLQAGCVP VVIADSYILP FSEVLDWKRA SVV VPEEKM SDVYSILQSI PQRQIEEMQR QARWFWEAYF QSIKAIALAT LQIINDRIYP YAAISYEEWN DPPAVKWGSV SNPL FLPLI PPQSQGFTAI VLTYDRVESL FRVITEVSKV PSLSKLLVVW NNQNKNPPED SLWPKIRVPL KVVRTAENKL SNRFF PYDE IETEAVLAID DDIIMLTSDE LQFGYEVWRE FPDRLVGYPG RLHLWDHEMN KWKYESEWTN EVSMVLTGAA FYHKYF NYL YTYKMPGDIK NWVDAHMNCE DIAMNFLVAN VTGKAVIKVT PRKKFKCPEC TAIDGLSLDQ THMVERSECI NKFASVF GT MPLKVVEHRA DPVLYKDDFP EKLKSFPNIG SL

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Macromolecule #4: URIDINE-5'-DIPHOSPHATE

MacromoleculeName: URIDINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 2 / Formula: UDP
Molecular weightTheoretical: 404.161 Da
Chemical component information

ChemComp-UDP:
URIDINE-5'-DIPHOSPHATE / UDP*YM

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

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

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Macromolecule #6: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MN
Molecular weightTheoretical: 54.938 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

Concentration0.3 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMHEPES
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 299 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 193.0 K / Max: 193.0 K
Alignment procedureComa free - Residual tilt: 0.05 mrad
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 5460 / Average exposure time: 1.5 sec. / Average electron dose: 62.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4783612
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 161338
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7uqx:
Cryo-EM structure of the human Exostosin-1 and Exostosin-2 heterodimer in complex with UDP-GlcNAc

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