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- PDB-7x06: CryoEM structure of chitin synthase 1 from Phytophthora sojae com... -

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Basic information

Entry
Database: PDB / ID: 7x06
TitleCryoEM structure of chitin synthase 1 from Phytophthora sojae complexed with UDP
ComponentsChitin synthase
KeywordsTRANSFERASE / carbohydate / biosynthetic protein / membrane protein
Function / homology
Function and homology information


chitin biosynthetic process / chitin synthase / chitin synthase activity / membrane
Similarity search - Function
Fungal chitin synthase / Chitin synthase / Chitin synthase / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
URIDINE-5'-DIPHOSPHATE / chitin synthase
Similarity search - Component
Biological speciesPhytophthora sojae strain P6497 (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsChen, W. / Cao, P. / Gong, Y. / Yang, Q.
Funding support China, 3items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32161133010 China
National Natural Science Foundation of China (NSFC)31830076 China
National Natural Science Foundation of China (NSFC)31901916 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
DepositionFeb 21, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 28, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 5, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Oct 12, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Oct 26, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Chitin synthase
B: Chitin synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)207,1506
Polymers206,2932
Non-polymers8574
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13140 Å2
ΔGint-101 kcal/mol
Surface area71420 Å2

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Components

#1: Protein Chitin synthase /


Mass: 103146.703 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Phytophthora sojae strain P6497 (eukaryote)
Strain: P6497 / Gene: PHYSODRAFT_557500 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: G4Z2L3, chitin synthase
#2: Chemical ChemComp-UDP / URIDINE-5'-DIPHOSPHATE / Uridine diphosphate


Type: RNA linking / Mass: 404.161 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C9H14N2O12P2 / Comment: UDP*YM
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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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: Chitin synthase 1 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Phytophthora sojae strain P6497 (eukaryote)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 8
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 224465 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313312
ELECTRON MICROSCOPYf_angle_d0.48618082
ELECTRON MICROSCOPYf_dihedral_angle_d10.3434730
ELECTRON MICROSCOPYf_chiral_restr0.0381984
ELECTRON MICROSCOPYf_plane_restr0.0042282

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