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- EMDB-32546: CryoEM structure of chitin synthase 1 mutant E495A from Phytophth... -

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Basic information

Entry
Database: EMDB / ID: EMD-32546
TitleCryoEM structure of chitin synthase 1 mutant E495A from Phytophthora sojae complexed with UDP-GlcNAc
Map dataCryoEM map of chitin synthase 1 mutant E495A with UDP-GlcNAc
Sample
  • Complex: Chitin synthase 1
    • Protein or peptide: Chitin synthase
  • Ligand: URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE
  • Ligand: MANGANESE (II) ION
Keywordscarbohydate / biosynthetic protein / membrane protein / transferase
Function / homology
Function and homology information


chitin synthase / chitin synthase activity / : / cell septum / cell periphery / membrane
Similarity search - Function
Fungal chitin synthase / Chitin synthase / Chitin synthase / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
Biological speciesPhytophthora sojae strain P6497 (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsChen W / Cao P
Funding support China, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32161133010 China
National Natural Science Foundation of China (NSFC)31830076 China
CitationJournal: Nature / Year: 2022
Title: Structural basis for directional chitin biosynthesis.
Authors: Wei Chen / Peng Cao / Yuansheng Liu / Ailing Yu / Dong Wang / Lei Chen / Rajamanikandan Sundarraj / Zhiguang Yuchi / Yong Gong / Hans Merzendorfer / Qing Yang /
Abstract: Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of N-acetylglucosamine (GlcNAc) units. The key reactions of chitin biosynthesis are catalysed by chitin ...Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of N-acetylglucosamine (GlcNAc) units. The key reactions of chitin biosynthesis are catalysed by chitin synthase, a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain. However, the precise mechanism of this process has yet to be elucidated. Here we report five cryo-electron microscopy structures of a chitin synthase from the devastating soybean root rot pathogenic oomycete Phytophthora sojae (PsChs1). They represent the apo, GlcNAc-bound, nascent chitin oligomer-bound, UDP-bound (post-synthesis) and chitin synthase inhibitor nikkomycin Z-bound states of the enzyme, providing detailed views into the multiple steps of chitin biosynthesis and its competitive inhibition. The structures reveal the chitin synthesis reaction chamber that has the substrate-binding site, the catalytic centre and the entrance to the polymer-translocating channel that allows the product polymer to be discharged. This arrangement reflects consecutive key events in chitin biosynthesis from UDP-GlcNAc binding and polymer elongation to the release of the product. We identified a swinging loop within the chitin-translocating channel, which acts as a 'gate lock' that prevents the substrate from leaving while directing the product polymer into the translocating channel for discharge to the extracellular side of the cell membrane. This work reveals the directional multistep mechanism of chitin biosynthesis and provides a structural basis for inhibition of chitin synthesis.
History
DepositionJan 7, 2022-
Header (metadata) releaseSep 28, 2022-
Map releaseSep 28, 2022-
UpdateJun 26, 2024-
Current statusJun 26, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32546.png

Downloads & links

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Map

FileDownload / File: emd_32546.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM map of chitin synthase 1 mutant E495A with UDP-GlcNAc
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.09211784 - 0.17026772
Average (Standard dev.)0.0001855089 (±0.0031348576)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-150-150-150
Dimensions300300300
Spacing300300300
CellA=B=C: 321.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Chitin synthase 1

EntireName: Chitin synthase 1
Components
  • Complex: Chitin synthase 1
    • Protein or peptide: Chitin synthase
  • Ligand: URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: Chitin synthase 1

SupramoleculeName: Chitin synthase 1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Phytophthora sojae strain P6497 (eukaryote)

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Macromolecule #1: Chitin synthase

MacromoleculeName: Chitin synthase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: chitin synthase
Source (natural)Organism: Phytophthora sojae strain P6497 (eukaryote) / Strain: P6497
Molecular weightTheoretical: 103.088664 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSGAPPPSSG FAPRSYGQQP LSHAPRSSMM SVEYDGIPLP PPSIRSCGSQ QYVTSYIPTG AAFPPSSVQD MISSMKSYAS ATDLVRTYS EIPSVEEALS TLDRAAAALN ARRYRDALKL YLEGGYAMAN VAERQANPKI CNLLTSKGFE TLNWCARLCD W IEGRIKEK ...String:
MSGAPPPSSG FAPRSYGQQP LSHAPRSSMM SVEYDGIPLP PPSIRSCGSQ QYVTSYIPTG AAFPPSSVQD MISSMKSYAS ATDLVRTYS EIPSVEEALS TLDRAAAALN ARRYRDALKL YLEGGYAMAN VAERQANPKI CNLLTSKGFE TLNWCARLCD W IEGRIKEK HPRPGVHKVG IPVSNWDEDW VGPFMDEEEA RRMWYTPVYC PHPIDFSNLG YRLRCVETGR RPRLMICITM YN EGPQQLK ATLKKLANNL AYLKEQMPGD EKSLTGAFAG DDVWQNVLVC IVADGREQVH PKTLDYLEAI GLYDEDLLTI NSA GIGAQC HLFEHTLQLS VNGKCLLPIQ TVFALKENKA SKLDSHHWYF NAFAEQIQPE YTAVMDVGTM LTKSALYHLL FAFE RNHQI GGACGQLTVD NPFENLSNWV ISAQHFEYKI SNILDKSLES CFGFISVLPG AFSAYRYEAI RGAPLDAYFQ TLNIE LDVL GPFIGNMYLA ADRILSFEVV ARKNCNWTMH YVKDAVARTD VPHDLVGLIS QRKRWLNGAF FATLFSIWNW GRIYSE SKH TFVRKMAFLV FYVYHLLYTA FGFFLPANLY LALFFIVFQG FQQNRLEFID TSEYSQTVLD CAVYIYNFSY LFGLLML II IGLGNNPKHM KLTYYFVGAV FGLMMMLSSL VGAGIFFSTP ATVHSIVVSI LTVGVYFIAS ALHGEVHHIF MTFTHYTA L IPSFVNIFTI YSFCNLQDLS WGTKGLHDDP LLAASLDETE KGDFKDVIAK RRALEELRRE EKERVENRKK NFEAFRTNV LLTWAFSNLI FALFVVYFAS SSTYMPVLYI FVASLNTCRL LGSIGHWVYI HTEGLRGRVI DKSECGNGTG RYPQNSYVQL EEHYAALAE DQRTYASGRT NASVRTVNDV SSAA

UniProtKB: chitin synthase

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Macromolecule #2: URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE

MacromoleculeName: URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE / type: ligand / ID: 2 / Number of copies: 2 / Formula: UD1
Molecular weightTheoretical: 607.354 Da
Chemical component information

ChemComp-UD1:
URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE

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Macromolecule #3: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 3 / Number of copies: 2 / 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

Concentration5 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 350132
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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