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- EMDB-18386: Archaeoglobus fulgidus AfAgo complex with AfAgo-N protein (fAfAgo... -

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Basic information

Entry
Database: EMDB / ID: EMD-18386
TitleArchaeoglobus fulgidus AfAgo complex with AfAgo-N protein (fAfAgo) bound with 17 nt RNA guide and 17 nt DNA target
Map data
Sample
  • Complex: Heterodimeric AfAgo and AfAgo-N complex with 17 nt RNA guide and 17 nt DNA target
    • Complex: AfAgo
      • Protein or peptide: Piwi protein
    • Complex: AfAgo-N
      • Protein or peptide: AfAgo-N protein
    • Complex: DNA target oligonucleotide
      • DNA: DNA target 17 nt
    • Complex: 5'p-RNA guide
      • RNA: RNA guide 17 nt
KeywordsARGONAUTE / PIWI DOMAIN / PROTEIN-DNA COMPLEX / DNA BINDING PROTEIN
Function / homologyPiwi domain / Piwi domain profile. / Piwi domain / Piwi / Ribonuclease H superfamily / nucleic acid binding / Ribonuclease H-like superfamily / Uncharacterized protein / Piwi protein
Function and homology information
Biological speciesArchaeoglobus fulgidus DSM 4304 (archaea) / Escherichia coli (E. coli) / Archaeoglobus fulgidus (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.43 Å
AuthorsManakova EN / Zaremba M / Pocevicuite R / Golovinas E / Zagorskaite E / Silanskas A
Funding supportLithuania, 2 items
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, 2023-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18386.png

Downloads & links

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Map

FileDownload / File: emd_18386.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 8.300000000000001
Minimum - Maximum-35.117545999999997 - 45.093525
Average (Standard dev.)-0.000000000004545 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 211.20001 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_18386_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18386_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Heterodimeric AfAgo and AfAgo-N complex with 17 nt RNA guide and ...

EntireName: Heterodimeric AfAgo and AfAgo-N complex with 17 nt RNA guide and 17 nt DNA target
Components
  • Complex: Heterodimeric AfAgo and AfAgo-N complex with 17 nt RNA guide and 17 nt DNA target
    • Complex: AfAgo
      • Protein or peptide: Piwi protein
    • Complex: AfAgo-N
      • Protein or peptide: AfAgo-N protein
    • Complex: DNA target oligonucleotide
      • DNA: DNA target 17 nt
    • Complex: 5'p-RNA guide
      • RNA: RNA guide 17 nt

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Supramolecule #1: Heterodimeric AfAgo and AfAgo-N complex with 17 nt RNA guide and ...

SupramoleculeName: Heterodimeric AfAgo and AfAgo-N complex with 17 nt RNA guide and 17 nt DNA target
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 100 KDa

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Supramolecule #2: AfAgo

SupramoleculeName: AfAgo / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Archaeoglobus fulgidus DSM 4304 (archaea)

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Supramolecule #3: AfAgo-N

SupramoleculeName: AfAgo-N / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Archaeoglobus fulgidus DSM 4304 (archaea)

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Supramolecule #4: DNA target oligonucleotide

SupramoleculeName: DNA target oligonucleotide / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #5: 5'p-RNA guide

SupramoleculeName: 5'p-RNA guide / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Piwi protein

MacromoleculeName: Piwi protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Archaeoglobus fulgidus (archaea) / Strain: DSM4304
Molecular weightTheoretical: 49.302434 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MMEYKIVENG LTYRIGNGAS VPISNTGELI KGLRNYGPYE VPSLKYNQIA LIHNNQFSSL INQLKSQISS KIDEVWHIHN INISEFIYD SPHFDSIKSQ VDNAIDTGVD GIMLVLPEYN TPLYYKLKSY LINSIPSQFM RYDILSNRNL TFYVDNLLVQ F VSKLGGKP ...String:
MMEYKIVENG LTYRIGNGAS VPISNTGELI KGLRNYGPYE VPSLKYNQIA LIHNNQFSSL INQLKSQISS KIDEVWHIHN INISEFIYD SPHFDSIKSQ VDNAIDTGVD GIMLVLPEYN TPLYYKLKSY LINSIPSQFM RYDILSNRNL TFYVDNLLVQ F VSKLGGKP WILNVDPEKG SDIIIGTGAT RIDNVNLFCF AMVFKKDGTM LWNEISPIVT SSEYLTYLKS TIKKVVYGFK KS NPDWDVE KLTLHVSGKR PKMKDGETKI LKETVEELKK QEMVSRDVKY AILHLNETHP FWVMGDPNNR FHPYEGTKVK LSS KRYLLT LLQPYLKRNG LEMVTPIKPL SVEIVSDNWT SEEYYHNVHE ILDEIYYLSK MNWRGFRSRN LPVTVNYPKL VAGI IANVN RYGGYPINPE GNRSLQTNPW FL

UniProtKB: Piwi protein

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Macromolecule #2: AfAgo-N protein

MacromoleculeName: AfAgo-N protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Archaeoglobus fulgidus (archaea) / Strain: DSM8774
Molecular weightTheoretical: 31.356576 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGGSHHHHHH GMASENLYFQ GGGGEIPLSS GNVNTPDVRS SGILYINIYP IVNYPETIKV SAIPYYEEFL PGKWKKRIGD LIYLYGYGI ENEFDEIDNS NALFGKIFRK YLLDILSENI ATPWQLKELG STLRLVKEIT ENYEFSNIIK LQYELIINVH H WQNTNFGI ...String:
MGGSHHHHHH GMASENLYFQ GGGGEIPLSS GNVNTPDVRS SGILYINIYP IVNYPETIKV SAIPYYEEFL PGKWKKRIGD LIYLYGYGI ENEFDEIDNS NALFGKIFRK YLLDILSENI ATPWQLKELG STLRLVKEIT ENYEFSNIIK LQYELIINVH H WQNTNFGI IVDLKINILD RENNQRISYT KIKDKYGESV KKKIWVSVQA FHRHLTPEGK KYATAMRDKF NLLTGLLKEA FG SSEDEKT FSTPDGEIKI VFKPLEIVEV SNNDGI

UniProtKB: Uncharacterized protein

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Macromolecule #3: DNA target 17 nt

MacromoleculeName: DNA target 17 nt / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 5.202384 KDa
SequenceString:
(DA)(DT)(DT)(DC)(DG)(DG)(DC)(DC)(DG)(DT) (DG)(DT)(DA)(DC)(DA)(DA)(DT)

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Macromolecule #4: RNA guide 17 nt

MacromoleculeName: RNA guide 17 nt / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 5.427286 KDa
SequenceString:
AUUGUACACG GCCGAAU

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 8.5
Component:
ConcentrationFormulaName
125.0 mMKClKCl
20.0 mMC4H11NO3Tris hydrochloride pH8.5
2.0 mMMgCl2MgCl2
2.0 mMC4H10O2S2DTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsfAfAgo complex with 17/17 guide-target heteroduplexes was mixed and applied on grid

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 92000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number grids imaged: 1 / Number real images: 2152 / Average exposure time: 46.33 sec. / Average electron dose: 31.0 e/Å2

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Image processing

Particle selectionNumber selected: 1480423 / Details: Blob particle picking
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8ok9


Details: Protein only chains
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v.4.2.1)
Final 3D classificationNumber classes: 105 / Avg.num./class: 14300 / Software - Name: cryoSPARC (ver. v.4.2.1)
Details: Particles of selected classes were further sorted by heterogenous refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v.4.2.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.43 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v.4.2.1) / Number images used: 364005
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

8ok9

chain_id: A, residue_range: 1-427, source_name: PDB, initial_model_type: experimental modelfAfAgo complex was used as initial model

8ok9

chain_id: C, residue_range: 20-245, source_name: PDB, initial_model_type: experimental modelfAfAgo complex was used as initial model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 48
Output model

PDB-8qg0:
Archaeoglobus fulgidus AfAgo complex with AfAgo-N protein (fAfAgo) bound with 17 nt RNA guide and 17 nt DNA target

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