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- EMDB-40591: Xenopus laevis hyaluronan synthase 1 -

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Basic information

Entry
Database: EMDB / ID: EMD-40591
TitleXenopus laevis hyaluronan synthase 1
Map dataXenopus laevis hyaluronan synthase 1
Sample
  • Complex: Xenopus laevis hyaluronan synthase 1 in complex with a Fab molecule
    • Protein or peptide: Hyaluronan synthase 1
    • Protein or peptide: Fab15 heavy chain
    • Protein or peptide: Fab15 light chain
Keywordshyaluronic acid / hyaluronan / HA / HAS / glycosyltransferase / GT / membrane protein / Fab / Transferase-Immune System complex
Function / homologyhyaluronan synthase / hyaluronan synthase activity / Glycosyltransferase like family 2 / extracellular matrix assembly / hyaluronan biosynthetic process / : / Nucleotide-diphospho-sugar transferases / membrane / Hyaluronan synthase 1
Function and homology information
Biological speciesXenopus laevis (African clawed frog) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsGorniak I / Zimmer J
Funding support United States, Germany, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM144130 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM117372 United States
Boehringer Ingelheim Fonds (BIF) Germany
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structural insights into translocation and tailored synthesis of hyaluronan.
Authors: Ireneusz Górniak / Zachery Stephens / Satchal K Erramilli / Tomasz Gawda / Anthony A Kossiakoff / Jochen Zimmer /
Abstract: Hyaluronan (HA) is an essential component of the vertebrate extracellular matrix. It is a heteropolysaccharide of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) reaching several megadaltons ...Hyaluronan (HA) is an essential component of the vertebrate extracellular matrix. It is a heteropolysaccharide of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) reaching several megadaltons in healthy tissues. HA is synthesized and translocated in a coupled reaction by HA synthase (HAS). Here, structural snapshots of HAS provide insights into HA biosynthesis, from substrate recognition to HA elongation and translocation. We monitor the extension of a GlcNAc primer with GlcA, reveal the coordination of the uridine diphosphate product by a conserved gating loop and capture the opening of a translocation channel to coordinate a translocating HA polymer. Furthermore, we identify channel-lining residues that modulate HA product lengths. Integrating structural and biochemical analyses suggests an avenue for polysaccharide engineering based on finely tuned enzymatic activity and HA coordination.
History
DepositionApr 26, 2023-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40591.png

Downloads & links

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Map

FileDownload / File: emd_40591.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationXenopus laevis hyaluronan synthase 1
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 320 pix.
= 345.6 Å		1.08 Å/pix.
x 320 pix.
= 345.6 Å		1.08 Å/pix.
x 320 pix.
= 345.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.238
Minimum - Maximum-1.9089223 - 2.9916883
Average (Standard dev.)-0.00042717895 (±0.038553465)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_40591_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_40591_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Xenopus laevis hyaluronan synthase 1 in complex with a Fab molecule

EntireName: Xenopus laevis hyaluronan synthase 1 in complex with a Fab molecule
Components
  • Complex: Xenopus laevis hyaluronan synthase 1 in complex with a Fab molecule
    • Protein or peptide: Hyaluronan synthase 1
    • Protein or peptide: Fab15 heavy chain
    • Protein or peptide: Fab15 light chain

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Supramolecule #1: Xenopus laevis hyaluronan synthase 1 in complex with a Fab molecule

SupramoleculeName: Xenopus laevis hyaluronan synthase 1 in complex with a Fab molecule
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 118 KDa

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Macromolecule #1: Hyaluronan synthase 1

MacromoleculeName: Hyaluronan synthase 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: hyaluronan synthase
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 70.385047 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MHHHHHHHHH HHHMKEKAAE TMEIPEGIPK DLEPKHPTLW RIIYYSFGVV LLATITAAYV AEFQVLKHEA ILFSLGLYGL AMLLHLMMQ SLFAFLEIRR VNKSELPCSF KKTVALTIAG YQENPEYLIK CLESCKYVKY PKDKLKIILV IDGNTEDDAY M MEMFKDVF ...String:
MHHHHHHHHH HHHMKEKAAE TMEIPEGIPK DLEPKHPTLW RIIYYSFGVV LLATITAAYV AEFQVLKHEA ILFSLGLYGL AMLLHLMMQ SLFAFLEIRR VNKSELPCSF KKTVALTIAG YQENPEYLIK CLESCKYVKY PKDKLKIILV IDGNTEDDAY M MEMFKDVF HGEDVGTYVW KGNYHTVKKP EETNKGSCPE VSKPLNEDEG INMVEELVRN KRCVCIMQQW GGKREVMYTA FQ AIGTSVD YVQVCDSDTK LDELATVEMV KVLESNDMYG AVGGDVRILN PYDSFISFMS SLRYWMAFNV ERACQSYFDC VSC ISGPLG MYRNNILQVF LEAWYRQKFL GTYCTLGDDR HLTNRVLSMG YRTKYTHKSR AFSETPSLYL RWLNQQTRWT KSYF REWLY NAQWWHKHHI WMTYESVVSF IFPFFITATV IRLIYAGTIW NVVWLLLCIQ IMSLFKSIYA CWLRGNFIML LMSLY SMLY MTGLLPSKYF ALLTLNKTGW GTSGRKKIVG NYMPILPLSI WAAVLCGGVG YSIYMDCQND WSTPEKQKEM YHLLYG CVG YVMYWVIMAV MYWVWVKRCC RKRSQTVTLV HDIPDMCV

UniProtKB: Hyaluronan synthase 1

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Macromolecule #2: Fab15 heavy chain

MacromoleculeName: Fab15 heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.674379 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVSSYYIHWV RQAPGKGLEW VASISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARSGYYWGPY FGGFDYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF P EPVTVSWN ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NVSSYYIHWV RQAPGKGLEW VASISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARSGYYWGPY FGGFDYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF P EPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHT

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Macromolecule #3: Fab15 light chain

MacromoleculeName: Fab15 light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.258783 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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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

Concentration8 mg/mL
BufferpH: 7.8
Component:
ConcentrationNameFormula
20.0 mMtris(hydroxymethyl)aminomethane
150.0 mMsodium chlorideNaCl
0.01 %glyco-diosgeninGDN
GridModel: C-flat-1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
Details: Glow discharged in the presence of 1 drop of amylamine
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 20.0 µm / Nominal defocus min: 10.0 µm / Nominal magnification: 81000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6543217
Startup modelType of model: INSILICO MODEL
In silico model: Alphafold2 prediction of Xenopus laevis hyaluronan synthase 1
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 305106
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-8smm:
Xenopus laevis hyaluronan synthase 1

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