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- PDB-8k52: Cryo-EM structure of chitin synthase -

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Basic information

Entry
Database: PDB / ID: 8k52
TitleCryo-EM structure of chitin synthase
Componentschitin synthase
KeywordsTRANSFERASE / chitin synthase / membrane-integrated glycosyltransferase
Function / homology
Function and homology information


chitin synthase / chitin synthase activity / chitin biosynthetic process / cell wall organization / plasma membrane
Similarity search - Function
Fungal chitin synthase / Chitin synthase / Chitin synthase / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
: / URIDINE-5'-DIPHOSPHATE / Chitin synthase 1
Similarity search - Component
Biological speciesPhytophthora sojae strain P6497 (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsZhang, X. / Niu, S. / Li, P. / Bi, Y.
Funding support China, 1items
OrganizationGrant numberCountry
Other government China
CitationJournal: Int J Mol Sci / Year: 2024
Title: Chitin Translocation Is Functionally Coupled with Synthesis in Chitin Synthase.
Authors: Suhao Niu / Lei Qi / Xiaoyue Zhang / Dongfang He / Pengwei Li / Hao Wang / Yunchen Bi /
Abstract: Chitin, an extracellular polysaccharide, is synthesized by membrane-embedded chitin synthase (CHS) utilizing intracellular substrates. The mechanism of the translocation of synthesized chitin across ...Chitin, an extracellular polysaccharide, is synthesized by membrane-embedded chitin synthase (CHS) utilizing intracellular substrates. The mechanism of the translocation of synthesized chitin across the membrane to extracellular locations remains unresolved. We prove that the chitin synthase from (CHS) is a processive glycosyltransferase, which can rapidly produce and tightly bind with the highly polymerized chitin. We further demonstrate that CHS is a bifunctional enzyme, which is necessary and sufficient to translocate the synthesized chitin. CHS was purified and then reconstituted into proteoliposomes (PLs). The nascent chitin is generated and protected from chitinase degradation unless detergent solubilizes the PLs, showing that CHS translocates the newly produced chitin into the lumen of the PLs. We also attempted to resolve the CHS structure of the synthesized chitin-bound state, although it was not successful; the obtained high-resolution structure of the UDP/Mn-bound state could still assist in describing the characterization of the CHS's transmembrane channel. Consistently, we demonstrate that CHS is indispensable and capable of translocating chitin in a process that is tightly coupled to chitin synthesis.
History
DepositionJul 20, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)210,0916
Polymers209,1732
Non-polymers9184
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein chitin synthase


Mass: 104586.328 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Phytophthora sojae strain P6497 (eukaryote)
Gene: PHYSODRAFT_557500 / Production host: Homo sapiens (human) / References: UniProt: G4Z2L3, chitin synthase
#2: Chemical ChemComp-UDP / URIDINE-5'-DIPHOSPHATE


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


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain
Type: COMPLEX / Entity ID: #1 / Source: MULTIPLE SOURCES
Molecular weightValue: 103 kDa/nm / Experimental value: YES
Source (natural)Organism: Phytophthora sojae (eukaryote)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenConc.: 3 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 FIELD / Nominal defocus max: 5000 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 516823 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312823
ELECTRON MICROSCOPYf_angle_d0.66517400
ELECTRON MICROSCOPYf_dihedral_angle_d5.9331759
ELECTRON MICROSCOPYf_chiral_restr0.0411908
ELECTRON MICROSCOPYf_plane_restr0.0052190

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