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- PDB-8qg0: Archaeoglobus fulgidus AfAgo complex with AfAgo-N protein (fAfAgo... -

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Basic information

Entry
Database: PDB / ID: 8qg0
TitleArchaeoglobus fulgidus AfAgo complex with AfAgo-N protein (fAfAgo) bound with 17 nt RNA guide and 17 nt DNA target
Components
  • AfAgo-N protein
  • DNA target 17 nt
  • Piwi protein
  • RNA guide 17 nt
KeywordsDNA BINDING PROTEIN / ARGONAUTE / PIWI DOMAIN / PROTEIN-DNA COMPLEX
Function / homology
Function and homology information


nucleic acid binding
Similarity search - Function
Piwi domain / Piwi domain profile. / Piwi domain / Piwi / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / Uncharacterized protein / Piwi protein
Similarity search - Component
Biological speciesArchaeoglobus fulgidus (archaea)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.43 Å
AuthorsManakova, E.N. / Zaremba, M. / Pocevicuite, R. / Golovinas, E. / Zagorskaite, E. / Silanskas, A.
Funding supportLithuania, 2items
OrganizationGrant numberCountry
Research Council of LithuaniaS-MIP-23-131Lithuania
Research Council of LithuaniaS-MIP-20-37Lithuania
CitationJournal: Nucleic Acids Res / Year: 2024
Title: The missing part: the Archaeoglobus fulgidus Argonaute forms a functional heterodimer with an N-L1-L2 domain protein.
Authors: Elena Manakova / Edvardas Golovinas / Reda Pocevičiūtė / Giedrius Sasnauskas / Arunas Silanskas / Danielis Rutkauskas / Marija Jankunec / Evelina Zagorskaitė / Edvinas Jurgelaitis / ...Authors: Elena Manakova / Edvardas Golovinas / Reda Pocevičiūtė / Giedrius Sasnauskas / Arunas Silanskas / Danielis Rutkauskas / Marija Jankunec / Evelina Zagorskaitė / Edvinas Jurgelaitis / Algirdas Grybauskas / Česlovas Venclovas / Mindaugas Zaremba
Abstract: Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind complementary nucleic acid ...Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind complementary nucleic acid targets and are responsible for gene expression regulation, mobile genome element silencing, and defence against viruses or plasmids. According to their domain organization, Agos are divided into long and short Agos. Long Agos found in prokaryotes (long-A and long-B pAgos) and eukaryotes (eAgos) comprise four major functional domains (N, PAZ, MID and PIWI) and two structural linker domains L1 and L2. The majority (∼60%) of pAgos are short pAgos, containing only the MID and inactive PIWI domains. Here we focus on the prokaryotic Argonaute AfAgo from Archaeoglobus fulgidus DSM4304. Although phylogenetically classified as a long-B pAgo, AfAgo contains only MID and catalytically inactive PIWI domains, akin to short pAgos. We show that AfAgo forms a heterodimeric complex with a protein encoded upstream in the same operon, which is a structural equivalent of the N-L1-L2 domains of long pAgos. This complex, structurally equivalent to a long PAZ-less pAgo, outperforms standalone AfAgo in guide RNA-mediated target DNA binding. Our findings provide a missing piece to one of the first and the most studied pAgos.
History
DepositionSep 5, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 24, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Piwi protein
B: AfAgo-N protein
P: DNA target 17 nt
Q: RNA guide 17 nt


Theoretical massNumber of molelcules
Total (without water)91,2894
Polymers91,2894
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, SAXS
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11770 Å2
ΔGint-103 kcal/mol
Surface area27670 Å2
MethodPISA

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Components

#1: Protein Piwi protein /


Mass: 49302.434 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Archaeoglobus fulgidus (archaea) / Strain: DSM4304 / Gene: XD48_2091 / Plasmid: pBAD / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A101DYI0
#2: Protein AfAgo-N protein


Mass: 31356.576 Da / Num. of mol.: 1 / Mutation: N-terminal His-tag
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Archaeoglobus fulgidus (archaea) / Strain: DSM8774 / Gene: AFULGI_00014290 / Plasmid: pBAD / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A075WKW4
#3: DNA chain DNA target 17 nt


Mass: 5202.384 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#4: RNA chain RNA guide 17 nt


Mass: 5427.286 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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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

Component
IDNameTypeEntity IDParent-IDSource
1Heterodimeric AfAgo and AfAgo-N complex with 17 nt RNA guide and 17 nt DNA targetCOMPLEXall0RECOMBINANT
2AfAgoCOMPLEX#11RECOMBINANT
3AfAgo-NCOMPLEX#21RECOMBINANT
4DNA target oligonucleotideCOMPLEX#31RECOMBINANT
55'p-RNA guideCOMPLEX#41RECOMBINANT
Molecular weightValue: 0.1 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Archaeoglobus fulgidus DSM 4304 (archaea)224325
33Archaeoglobus fulgidus DSM 4304 (archaea)224325
44Escherichia coli (E. coli)562
55Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
22Escherichia coli BL21(DE3) (bacteria)469008pBAD
33Escherichia coli BL21(DE3) (bacteria)469008pBAD
44synthetic construct (others)32630
55synthetic construct (others)32630
Buffer solutionpH: 8.5
Buffer component
IDConc.NameFormulaBuffer-ID
1125 mMKClKCl1
220 mMTris hydrochloride pH8.5C4H11NO31
32 mMMgCl2MgCl21
42 mMDTTC4H10O2S21
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: fAfAgo complex with 17/17 guide-target heteroduplexes was mixed and applied on grid
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 92000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingAverage exposure time: 46.33 sec. / Electron dose: 31 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2152
Image scansWidth: 4000 / Height: 4000

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv.4.2.1particle selection
2EPU3image acquisition
3UCSF Chimera1.17.1masking
4cryoSPARCv.4.2.1CTF correction
5UCSF Chimera1.17.1model fitting
6Coot0.9other
9cryoSPARCv.4.2.1initial Euler assignment
10cryoSPARCv.4.2.1final Euler assignment
11cryoSPARCv.4.2.1classification
12cryoSPARCv.4.2.13D reconstruction
13PHENIX1.18.2_3874:model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1480423 / Details: Blob particle picking
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 364005 / Symmetry type: POINT
Atomic model buildingB value: 48 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Accession code: 8ok9 / Details: fAfAgo complex was used as initial model / Initial refinement model-ID: 1 / PDB-ID: 8ok9

8ok9

/ Source name: PDB / Type: experimental model

IDPdb chain-IDChain-IDChain residue rangePdb chain residue range
1AA1-4271-427
2CC20-24520-245
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046295
ELECTRON MICROSCOPYf_angle_d0.6218684
ELECTRON MICROSCOPYf_dihedral_angle_d18.1841084
ELECTRON MICROSCOPYf_chiral_restr0.044972
ELECTRON MICROSCOPYf_plane_restr0.005974

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