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- EMDB-11926: Cryo-EM structure of human exostosin-like 3 (EXTL3) in complex wi... -

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Basic information

Entry
Database: EMDB / ID: EMD-11926
TitleCryo-EM structure of human exostosin-like 3 (EXTL3) in complex with UDP
Map data
Sample
  • Complex: Homodimer of EXTL3 globular domain
    • Protein or peptide: Exostosin-like 3
  • Ligand: MANGANESE (II) ION
  • Ligand: URIDINE-5'-DIPHOSPHATE
Function / homology
Function and homology information


glucuronosyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / glucuronyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase activity / positive regulation of detection of glucose / protein-hormone receptor activity / heparan sulfate proteoglycan biosynthetic process / negative regulation of keratinocyte differentiation / negative regulation of inflammatory response to wounding / XBP1(S) activates chaperone genes / glycosyltransferase activity / protein glycosylation ...glucuronosyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase / glucuronyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase activity / positive regulation of detection of glucose / protein-hormone receptor activity / heparan sulfate proteoglycan biosynthetic process / negative regulation of keratinocyte differentiation / negative regulation of inflammatory response to wounding / XBP1(S) activates chaperone genes / glycosyltransferase activity / protein glycosylation / negative regulation of cytokine production involved in inflammatory response / negative regulation of inflammatory response / positive regulation of cell growth / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / endoplasmic reticulum membrane / Golgi apparatus / magnesium ion binding / endoplasmic reticulum / nucleus / plasma membrane
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
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsWilson LFL / Dendooven T / Hardwick SW / Chirgadze DY / Luisi BF / Logan DT / Mani K / Dupree P
Funding support United Kingdom, Sweden, 8 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC) United Kingdom
Wellcome Trust200873/Z/16/Z United Kingdom
Swedish Research Council2014-03402 Sweden
Swedish Research Council2016-04855 Sweden
Cancerfonden180710 Sweden
University of Cambridge United Kingdom
OpenPlantBB/L014130/1 United Kingdom
Engineering and Physical Sciences Research Council United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: The structure of EXTL3 helps to explain the different roles of bi-domain exostosins in heparan sulfate synthesis.
Authors: L F L Wilson / T Dendooven / S W Hardwick / A Echevarría-Poza / T Tryfona / K B R M Krogh / D Y Chirgadze / B F Luisi / D T Logan / K Mani / P Dupree /
Abstract: Heparan sulfate is a highly modified O-linked glycan that performs diverse physiological roles in animal tissues. Though quickly modified, it is initially synthesised as a polysaccharide of ...Heparan sulfate is a highly modified O-linked glycan that performs diverse physiological roles in animal tissues. Though quickly modified, it is initially synthesised as a polysaccharide of alternating β-D-glucuronosyl and N-acetyl-α-D-glucosaminyl residues by exostosins. These enzymes generally possess two glycosyltransferase domains (GT47 and GT64)-each thought to add one type of monosaccharide unit to the backbone. Although previous structures of murine exostosin-like 2 (EXTL2) provide insight into the GT64 domain, the rest of the bi-domain architecture is yet to be characterised; hence, how the two domains co-operate is unknown. Here, we report the structure of human exostosin-like 3 (EXTL3) in apo and UDP-bound forms. We explain the ineffectiveness of EXTL3's GT47 domain to transfer β-D-glucuronosyl units, and we observe that, in general, the bi-domain architecture would preclude a processive mechanism of backbone extension. We therefore propose that heparan sulfate backbone polymerisation occurs by a simple dissociative mechanism.
History
DepositionNov 2, 2020-
Header (metadata) releaseMay 18, 2022-
Map releaseMay 18, 2022-
UpdateJun 15, 2022-
Current statusJun 15, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_11926.png

Downloads & links

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Map

FileDownload / File: emd_11926.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 360 pix.
= 383.4 Å		1.07 Å/pix.
x 360 pix.
= 383.4 Å		1.07 Å/pix.
x 360 pix.
= 383.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.065 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.027
Minimum - Maximum-0.06038477 - 0.13254601
Average (Standard dev.)-5.859297e-06 (±0.0024086407)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 383.40002 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Homodimer of EXTL3 globular domain

EntireName: Homodimer of EXTL3 globular domain
Components
  • Complex: Homodimer of EXTL3 globular domain
    • Protein or peptide: Exostosin-like 3
  • Ligand: MANGANESE (II) ION
  • Ligand: URIDINE-5'-DIPHOSPHATE

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Supramolecule #1: Homodimer of EXTL3 globular domain

SupramoleculeName: Homodimer of EXTL3 globular domain / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 170 KDa

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Macromolecule #1: Exostosin-like 3

MacromoleculeName: Exostosin-like 3 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: glucuronosyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 101.705117 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: APQLHHHHHH DLYENLYFQG KLTTLDEADE AGKRIFGPRV GNELCEVKHV LDLCRIRESV SEELLQLEAK RQELNSEIAK LNLKIEACK KSIENAKQDL LQLKNVISQT EHSYKELMAQ NQPKLSLPIR LLPEKDDAGL PPPKATRGCR LHNCFDYSRC P LTSGFPVY ...String:
APQLHHHHHH DLYENLYFQG KLTTLDEADE AGKRIFGPRV GNELCEVKHV LDLCRIRESV SEELLQLEAK RQELNSEIAK LNLKIEACK KSIENAKQDL LQLKNVISQT EHSYKELMAQ NQPKLSLPIR LLPEKDDAGL PPPKATRGCR LHNCFDYSRC P LTSGFPVY VYDSDQFVFG SYLDPLVKQA FQATARANVY VTENADIACL YVILVGEMQE PVVLRPAELE KQLYSLPHWR TD GHNHVII NLSRKSDTQN LLYNVSTGRA MVAQSTFYTV QYRPGFDLVV SPLVHAMSEP NFMEIPPQVP VKRKYLFTFQ GEK IESLRS SLQEARSFEE EMEGDPPADY DDRIIATLKA VQDSKLDQVL VEFTCKNQPK PSLPTEWALC GEREDRLELL KLST FALII TPGDPRLVIS SGCATRLFEA LEVGAVPVVL GEQVQLPYQD MLQWNEAALV VPKPRVTEVH FLLRSLSDSD LLAMR RQGR FLWETYFSTA DSIFNTVLAM IRTRIQIPAA PIREEAAAEI PHRSGKAAGT DPNMADNGDL DLGPVETEPP YASPRY LRN FTLTVTDFYR SWNCAPGPFH LFPHTPFDPV LPSEAKFLGS GTGFRPIGGG AGGSGKEFQA ALGGNVPREQ FTVVMLT YE REEVLMNSLE RLNGLPYLNK VVVVWNSPKL PSEDLLWPDI GVPIMVVRTE KNSLNNRFLP WNEIETEAIL SIDDDAHL R HDEIMFGFRV WREARDRIVG FPGRYHAWDI PHQSWLYNSN YSCELSMVLT GAAFFHKYYA YLYSYVMPQA IRDMVDEYI NCEDIAMNFL VSHITRKPPI KVTSRWTFRC PGCPQALSHD DSHFHERHKC INFFVKVYGY MPLLYTQFRV DSVLFKTRLP HDKTKCFKF I

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Macromolecule #4: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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Macromolecule #5: URIDINE-5'-DIPHOSPHATE

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

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

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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: 6.8
GridModel: Quantifoil / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: INTEGRATING / Average electron dose: 48.46 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1133069
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: OTHER
Details: initial model generated with SGD-algorithm, The structure was determined ab initio using Buccaneer, which attempts to thread the polypeptide chain into the density map. Alignment with PDB ...Details: initial model generated with SGD-algorithm, The structure was determined ab initio using Buccaneer, which attempts to thread the polypeptide chain into the density map. Alignment with PDB entry 1OMX was used to calibrate pixel size.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 238379
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7aua:
Cryo-EM structure of human exostosin-like 3 (EXTL3) in complex with UDP

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