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- PDB-8xk4: Structure of the Argonaute protein from Kurthia massiliensis in c... -

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Basic information

Entry
Database: PDB / ID: 8xk4
TitleStructure of the Argonaute protein from Kurthia massiliensis in complex with guide DNA and 19-mer target DNA in target-released state
Components
  • KmAgo
  • guide DNA
  • target DNA
KeywordsDNA BINDING PROTEIN/DNA / Mesophilic prokaryotic Argonaute / DNA BINDING PROTEIN-DNA COMPLEX
Function / homology2'-DEOXYCYTIDINE-5'-MONOPHOSPHATE / : / DNA / DNA (> 10)
Function and homology information
Biological speciesKurthia massiliensis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsTao, X. / Ding, H. / Wu, S.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2021YFC2100100 China
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Structural and mechanistic insights into a mesophilic prokaryotic Argonaute.
Authors: Xin Tao / Hui Ding / Shaowen Wu / Fei Wang / Hu Xu / Jie Li / Chao Zhai / Shunshun Li / Kai Chen / Shan Wu / Yang Liu / Lixin Ma /
Abstract: Argonaute (Ago) proteins are programmable nucleases found in all domains of life, playing a crucial role in biological processes like DNA/RNA interference and gene regulation. Mesophilic prokaryotic ...Argonaute (Ago) proteins are programmable nucleases found in all domains of life, playing a crucial role in biological processes like DNA/RNA interference and gene regulation. Mesophilic prokaryotic Agos (pAgos) have gained increasing research interest due to their broad range of potential applications, yet their molecular mechanisms remain poorly understood. Here, we present seven cryo-electron microscopy structures of Kurthia massiliensis Ago (KmAgo) in various states. These structures encompass the steps of apo-form, guide binding, target recognition, cleavage, and release, revealing that KmAgo employs a unique DDD catalytic triad, instead of a DEDD tetrad, for DNA target cleavage under 5'P-DNA guide conditions. Notably, the last catalytic residue, D713, is positioned outside the catalytic pocket in the absence of guide. After guide binding, D713 enters the catalytic pocket. In contrast, the corresponding catalytic residue in other Agos has been consistently located in the catalytic pocket. Moreover, we identified several sites exhibiting enhanced catalytic activity through alanine mutagenesis. These sites have the potential to serve as engineering targets for augmenting the catalytic efficiency of KmAgo. This structural analysis of KmAgo advances the understanding of the diversity of molecular mechanisms by Agos, offering insights for developing and optimizing mesophilic pAgos-based programmable DNA and RNA manipulation tools.
History
DepositionDec 22, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 1, 2025Provider: repository / Type: Initial release
Revision 1.1Jan 8, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: KmAgo
B: guide DNA
C: target DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)94,5637
Polymers94,0913
Non-polymers4724
Water362
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

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Protein , 1 types, 1 molecules A

#1: Protein KmAgo


Mass: 85485.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Kurthia massiliensis (bacteria) / Production host: Escherichia coli (E. coli)

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DNA chain , 2 types, 2 molecules BC

#2: DNA chain guide DNA


Mass: 5641.660 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Kurthia massiliensis (bacteria)
#3: DNA chain target DNA


Mass: 2963.969 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Kurthia massiliensis (bacteria)

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Non-polymers , 3 types, 6 molecules

#4: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-DC / 2'-DEOXYCYTIDINE-5'-MONOPHOSPHATE


Type: DNA linking / Mass: 307.197 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H14N3O7P / Feature type: SUBJECT OF INVESTIGATION / Comment: dCMP*YM
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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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: KmAgo / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Kurthia massiliensis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding 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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (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: 259549 / Symmetry type: POINT
RefinementHighest resolution: 2.94 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036220
ELECTRON MICROSCOPYf_angle_d0.5288499
ELECTRON MICROSCOPYf_dihedral_angle_d11.623958
ELECTRON MICROSCOPYf_chiral_restr0.042947
ELECTRON MICROSCOPYf_plane_restr0.0051002

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