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

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Basic information

Entry
Database: PDB / ID: 8pvv
TitleArchaeoglobus fulgidus AfAgo complex with AfAgo-N protein (fAfAgo) bound with 30 nt RNA guide and 51 nt DNA target
Components
  • Archaeoglobus fulgidus AfAgo-N protein
  • DNA (51-MER)
  • Piwi protein
  • RNA (30-MER)
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)
Archaeoglobus fulgidus DSM 8774 (archaea)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.81 Å
AuthorsManakova, E.N. / Zaremba, M. / Pocevicuite, R. / Golovinas, E. / Sasnauskas, G. / 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
DepositionJul 18, 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
C: Archaeoglobus fulgidus AfAgo-N protein
R: RNA (30-MER)
S: DNA (51-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)106,0565
Polymers106,0324
Non-polymers241
Water64936
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AC

#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 Archaeoglobus fulgidus AfAgo-N protein


Mass: 31356.576 Da / Num. of mol.: 1 / Mutation: N-terminal His tag
Source method: isolated from a genetically manipulated source
Details: N-terminal His tag for purification
Source: (gene. exp.) Archaeoglobus fulgidus DSM 8774 (archaea)
Gene: AFULGI_00014290 / Plasmid: plasmid / Details (production host): pBAD / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A075WKW4

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RNA chain / DNA chain , 2 types, 2 molecules RS

#3: RNA chain RNA (30-MER)


Mass: 9792.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#4: DNA chain DNA (51-MER)


Mass: 15579.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 2 types, 37 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 36 / Source method: isolated from a natural source / Formula: H2O

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Details

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

Component
IDNameTypeEntity IDParent-IDSource
1AfAgo and AfAgo-N complex with 30 nt RNA guide and 51 nt DNA targetCOMPLEX#1-#40MULTIPLE SOURCES
2Piwi proteinCOMPLEX#11RECOMBINANT
3Archaeoglobus fulgidus AfAgo-N proteinCOMPLEX#21RECOMBINANT
4RNA (30-MER)COMPLEX#31RECOMBINANT
5DNA (51-MER)COMPLEX#41RECOMBINANT
Molecular weightValue: 0.09 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Archaeoglobus fulgidus (archaea)2234
33Archaeoglobus fulgidus DSM 8774 (archaea)1344584
44Escherichia coli (E. coli)562
55Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
22Escherichia coli BL21(DE3) (bacteria)469008BL21(DE3)pBAD
33Escherichia coli BL21(DE3) (bacteria)469008
44synthetic construct (others)32630
55synthetic construct (others)32630
Buffer solutionpH: 8.5
Details: fAfAgo complex with 30/51 guide-target heteroduplexes was mixed and applied on grid
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 30/51 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: SPOT SCAN
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
2PHENIX1.18.2_3874:model refinement
3EPUimage acquisition
4UCSF Chimera1.17.1masking
5cryoSPARCv.4.2.1CTF correction
6UCSF Chimera1.17.1model fitting
7Coot0.9other
10cryoSPARCv.4.2.1initial Euler assignment
11cryoSPARCv.4.2.1final Euler assignment
12cryoSPARCv.4.2.1classification
13cryoSPARCv.4.2.13D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 2326555 / Details: Blob particle picking
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.81 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 498038 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY 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_d0.0036698
ELECTRON MICROSCOPYf_angle_d0.5659308
ELECTRON MICROSCOPYf_dihedral_angle_d19.0321265
ELECTRON MICROSCOPYf_chiral_restr0.0431047
ELECTRON MICROSCOPYf_plane_restr0.005995

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