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- PDB-8ok9: Heterodimeric complex of Archaeoglobus fulgidus Argonaute protein... -

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Basic information

Entry
Database: PDB / ID: 8ok9
TitleHeterodimeric complex of Archaeoglobus fulgidus Argonaute protein Af1318 (AfAgo) with DNA and AfAgo-N protein containing N-L1-L2 domains
Components
  • Archaeoglobus fulgidus AfAgo-N protein
  • DNA (5'-D(P*AP*TP*CP*GP*AP*CP*CP*AP*GP*GP*CP*TP*AP*CP*G)-3')
  • Piwi protein AF_1318
KeywordsRNA BINDING PROTEIN / Protein-nucleic acid interactions / Argonaute / pAgo / guide and target specificity
Function / homology
Function and homology information


DNA binding / RNA binding / metal ion binding
Similarity search - Function
Piwi domain / Piwi domain profile. / Piwi domain / Piwi / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
ACETIC ACID / DI(HYDROXYETHYL)ETHER / DNA / DNA (> 10) / Uncharacterized protein / Piwi protein
Similarity search - Component
Biological speciesArchaeoglobus fulgidus DSM 4304 (archaea)
Escherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.5 Å
AuthorsManakova, E.N. / Zaremba, M.
Funding supportLithuania, European Union, 2items
OrganizationGrant numberCountry
Research Council of LithuaniaS-MIP-20-37Lithuania
iNEXTH2020 Grant # 653706European Union
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
DepositionMar 27, 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 AF_1318
C: Archaeoglobus fulgidus AfAgo-N protein
R: DNA (5'-D(P*AP*TP*CP*GP*AP*CP*CP*AP*GP*GP*CP*TP*AP*CP*G)-3')
S: DNA (5'-D(P*AP*TP*CP*GP*AP*CP*CP*AP*GP*GP*CP*TP*AP*CP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)91,63820
Polymers90,6554
Non-polymers98316
Water3,603200
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS, SEC-SAXS experiment, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9330 Å2
ΔGint-27 kcal/mol
Surface area30230 Å2
Unit cell
Length a, b, c (Å)81.714, 105.893, 144.347
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 2 types, 2 molecules AC

#1: Protein Piwi protein AF_1318 / / AfAgo / AfPiwi / PIWI/MID domain protein


Mass: 50921.121 Da / Num. of mol.: 1 / Fragment: Arhaeoglobus fulgidus Argonaute protein / Mutation: N-terminal His tag
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Archaeoglobus fulgidus DSM 4304 (archaea)
Strain: DSM 4304 / Gene: AF_1318 / Plasmid: pBAD / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O28951
#2: Protein Archaeoglobus fulgidus AfAgo-N protein


Mass: 30575.756 Da / Num. of mol.: 1 / Mutation: N-terminal His tag
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Archaeoglobus fulgidus DSM 4304 (archaea)
Strain: DSM 8774 / Gene: AFULGI_00014290 / Plasmid: pBAD / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A075WKW4

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DNA chain , 1 types, 2 molecules RS

#3: DNA chain DNA (5'-D(P*AP*TP*CP*GP*AP*CP*CP*AP*GP*GP*CP*TP*AP*CP*G)-3')


Mass: 4578.987 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 5 types, 216 molecules

#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-ACY / ACETIC ACID / Acetic acid


Mass: 60.052 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C2H4O2
#6: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C2H6O2
#7: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER / Diethylene glycol


Mass: 106.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O3
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 200 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.9 Å3/Da / Density % sol: 68.45 %
Crystal growTemperature: 280 K / Method: vapor diffusion, sitting drop / pH: 5.5
Details: BisTris pH 5.5 0.1 M;Ammonium acetate 0.1 M; PEG10k 8%; cryo-cooling with addition of ethylene glycol to 30 %

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 1.01 Å
DetectorType: DECTRIS PILATUS3 X 6M / Detector: PIXEL / Date: Jun 25, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.01 Å / Relative weight: 1
ReflectionResolution: 2.5→144.35 Å / Num. obs: 44100 / % possible obs: 98.1 % / Redundancy: 13.3 % / Biso Wilson estimate: 64.922 Å2 / CC1/2: 0.999 / R split: 0.023 / Rmerge(I) obs: 0.054 / Rpim(I) all: 0.022 / Rrim(I) all: 0.058 / Χ2: 1.02 / Net I/av σ(I): 9.3 / Net I/σ(I): 30.2
Reflection shellResolution: 2.5→2.55 Å / Redundancy: 13.7 % / Rmerge(I) obs: 0.413 / Mean I/σ(I) obs: 6.6 / Num. unique obs: 4576 / CC1/2: 0.98 / Rpim(I) all: 0.167 / Rrim(I) all: 0.446 / Χ2: 1.01 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(1.18.2_3874: ???)refinement
XDSVERSION Jun 17, 2015data reduction
Aimlessversion 0.7.2 : 27/05/18data scaling
MOLREPversion 6.1 : 23/01/06phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.5→64.69 Å / SU ML: 0.29 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 24.29 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2269 4216 10.08 %Random selection
Rwork0.1981 ---
obs0.201 41834 99.92 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 57.56 Å2
Displacement parametersBiso mean: 60.13 Å2
Refinement stepCycle: LAST / Resolution: 2.5→64.69 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5456 471 64 200 6191
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0056185
X-RAY DIFFRACTIONf_angle_d0.8768458
X-RAY DIFFRACTIONf_dihedral_angle_d21.6462338
X-RAY DIFFRACTIONf_chiral_restr0.059924
X-RAY DIFFRACTIONf_plane_restr0.005994
LS refinement shell

Refine-ID: X-RAY DIFFRACTION

Resolution (Å)Rfactor RfreeNum. reflection Rfree% reflection Rfree (%)Rfactor RworkNum. reflection Rwork% reflection obs (%)
2.5-2.530.267829011.480.27462524100
2.53-2.560.32992890.27342511100
2.56-2.590.29842730.26642517100
2.59-2.620.33013280.26262462100
2.62-2.660.30982600.26672508100
2.66-2.690.28042750.24862521100
2.69-2.730.30172810.25112548100
2.73-2.770.30072910.24552467100
2.77-2.820.26872930.24662506100
2.82-2.860.29632520.24522515100
2.86-2.910.29042690.24012535100
2.91-2.960.27523010.24512451100
2.96-3.020.28222570.23492556100
3.02-3.080.27882840.25422490100
3.08-3.150.29732900.23772508100
3.15-3.220.30992650.23592516100
3.22-3.30.24882610.22432555100
3.3-3.390.2622600.22142501100
3.39-3.490.25743130.21432460100
3.49-3.610.25232490.20072569100
3.61-3.730.24922940.20212502100
3.73-3.880.22452530.18592538100
3.88-4.060.1782850.17752517100
4.06-4.270.18573060.16312471100
4.27-4.540.17922870.14792499100
4.54-4.890.16813260.15172450100
4.89-5.390.17962710.16862523100
5.39-6.160.21782770.19692505100
6.16-7.760.2032840.18882499100
7.76-64.690.1822680.1693251299

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