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- PDB-8olj: Crystal structure of Archaeoglobus fulgidus AfAgo-N protein repre... -

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Basic information

Entry
Database: PDB / ID: 8olj
TitleCrystal structure of Archaeoglobus fulgidus AfAgo-N protein representing N-L1-L2 domains
ComponentsArchaeoglobus fulgidus AfAgo-N protein representing N-L1-L2 domains
KeywordsRNA BINDING PROTEIN / Protein-nucleic acid interactions / Argonaute / pAgo / guide and target specificity / PROTEIN BINDING
Function / homologyACETIC ACID / ISOPROPYL ALCOHOL / : / Uncharacterized protein
Function and homology information
Biological speciesArchaeoglobus fulgidus DSM 8774 (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.4 Å
AuthorsManakova, E.N. / Zaremba, M. / Grazulis, S.
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 30, 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

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Assembly

Deposited unit
B: Archaeoglobus fulgidus AfAgo-N protein representing N-L1-L2 domains
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,87720
Polymers30,5761
Non-polymers1,30119
Water4,774265
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4120 Å2
ΔGint33 kcal/mol
Surface area13010 Å2
Unit cell
Length a, b, c (Å)75.264, 75.264, 94.722
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number154
Space group name H-MP3221
Components on special symmetry positions
IDModelComponents
11B-317-

K

21B-402-

HOH

31B-661-

HOH

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Components

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Protein , 1 types, 1 molecules B

#1: Protein Archaeoglobus fulgidus AfAgo-N protein representing N-L1-L2 domains


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 8774 (archaea)
Gene: AFULGI_00014290 / Plasmid: pBAD / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A075WKW4

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Non-polymers , 6 types, 284 molecules

#2: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C3H8O3
#3: Chemical
ChemComp-IPA / ISOPROPYL ALCOHOL / 2-PROPANOL


Mass: 60.095 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C3H8O / Comment: alkaloid*YM
#4: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: K
#5: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#6: Chemical ChemComp-ACY / ACETIC ACID


Mass: 60.052 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H4O2
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 265 / 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: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.47 Å3/Da / Density % sol: 50.12 %
Crystal growTemperature: 280 K / Method: vapor diffusion, sitting drop
Details: Protein concentration 4.1 mg/ml. Reservoir: TrisHCl pH 8.0 0.05 M; iPrOH 27%; ammonium acetate 0.11 M; Bicine pH 9.0 0.05 M. Cryo-protection: short wash in 0.16 M Ammonium acetate; 0.08 M ...Details: Protein concentration 4.1 mg/ml. Reservoir: TrisHCl pH 8.0 0.05 M; iPrOH 27%; ammonium acetate 0.11 M; Bicine pH 9.0 0.05 M. Cryo-protection: short wash in 0.16 M Ammonium acetate; 0.08 M TrisHCl pH 8.5; 24% iPrOH; Glycerol 20%
PH range: 8-9

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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: P13 (MX1) / Wavelength: 0.9797 Å
DetectorType: DECTRIS PILATUS3 X 6M / Detector: PIXEL / Date: Nov 23, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9797 Å / Relative weight: 1
ReflectionResolution: 1.4→94.72 Å / Num. obs: 61624 / % possible obs: 100 % / Redundancy: 9.7 % / Biso Wilson estimate: 19.676 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.047 / Rpim(I) all: 0.016 / Rrim(I) all: 0.049 / Χ2: 0.97 / Net I/av σ(I): 6.6 / Net I/σ(I): 23.8
Reflection shellResolution: 1.4→1.42 Å / Redundancy: 8.4 % / Rmerge(I) obs: 0.908 / Mean I/σ(I) obs: 2.3 / Num. unique obs: 3045 / CC1/2: 0.81 / Rpim(I) all: 0.331 / Rrim(I) all: 0.967 / Χ2: 0.9 / % possible all: 100

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
XDSVERSION Jan 26, 2018 BUILT=20180126data reduction
Aimlessversion 0.7.2data scaling
MOLREPVers 11.6.04phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.4→53.7 Å / Cor.coef. Fo:Fc: 0.977 / Cor.coef. Fo:Fc free: 0.968 / Cross valid method: THROUGHOUT / ESU R: 0.056 / ESU R Free: 0.054 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflectionSelection details
Rfree0.17772 6208 10.1 %RANDOM
Rwork0.14458 ---
obs0.1479 55371 99.99 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 25.532 Å2
Baniso -1Baniso -2Baniso -3
1--0.04 Å2-0.02 Å2-0 Å2
2---0.04 Å20 Å2
3---0.14 Å2
Refinement stepCycle: 1 / Resolution: 1.4→53.7 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1926 0 81 265 2272
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0122199
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.611.642984
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.3525270
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.35823.889108
X-RAY DIFFRACTIONr_dihedral_angle_3_deg11.85915397
X-RAY DIFFRACTIONr_dihedral_angle_4_deg12.05158
X-RAY DIFFRACTIONr_chiral_restr0.1080.2282
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.021661
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.3131.9351028
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it2.9972.911314
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it4.8482.5061171
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined6.03130.6193447
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr5.10232199
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.4→1.436 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.28 475 -
Rwork0.247 4031 -
obs--99.96 %

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