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- EMDB-18386: Archaeoglobus fulgidus AfAgo complex with AfAgo-N protein (fAfAgo... -
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Open data
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Basic information
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Title | Archaeoglobus fulgidus AfAgo complex with AfAgo-N protein (fAfAgo) bound with 17 nt RNA guide and 17 nt DNA target | |||||||||
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![]() | ARGONAUTE / PIWI DOMAIN / PROTEIN-DNA COMPLEX / DNA BINDING PROTEIN | |||||||||
Function / homology | Piwi domain / Piwi domain profile. / Piwi domain / Piwi / nucleic acid binding / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Uncharacterized protein / Piwi protein![]() | |||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.43 Å | |||||||||
![]() | Manakova EN / Zaremba M / Pocevicuite R / Golovinas E / Zagorskaite E / Silanskas A | |||||||||
Funding support | Lithuania, 2 items
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![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 24.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 26.2 KB 26.2 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 6.3 KB | Display | ![]() |
Images | ![]() | 164.5 KB | ||
Filedesc metadata | ![]() | 7.4 KB | ||
Others | ![]() ![]() | 25.1 MB 25.1 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 714.5 KB | Display | ![]() |
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Full document | ![]() | 714 KB | Display | |
Data in XML | ![]() | 13.9 KB | Display | |
Data in CIF | ![]() | 17.5 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8qg0MC ![]() 8ok9C ![]() 8oldC ![]() 8oljC ![]() 8pvvC C: citing same article ( M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 1.1 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_18386_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_18386_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Heterodimeric AfAgo and AfAgo-N complex with 17 nt RNA guide and ...
Entire | Name: Heterodimeric AfAgo and AfAgo-N complex with 17 nt RNA guide and 17 nt DNA target |
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Components |
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-Supramolecule #1: Heterodimeric AfAgo and AfAgo-N complex with 17 nt RNA guide and ...
Supramolecule | Name: 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 |
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Molecular weight | Theoretical: 100 KDa |
-Supramolecule #2: AfAgo
Supramolecule | Name: AfAgo / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: ![]() ![]() |
-Supramolecule #3: AfAgo-N
Supramolecule | Name: AfAgo-N / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
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Source (natural) | Organism: ![]() ![]() |
-Supramolecule #4: DNA target oligonucleotide
Supramolecule | Name: DNA target oligonucleotide / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3 |
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Source (natural) | Organism: ![]() ![]() |
-Supramolecule #5: 5'p-RNA guide
Supramolecule | Name: 5'p-RNA guide / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #4 |
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Source (natural) | Organism: ![]() ![]() |
-Macromolecule #1: Piwi protein
Macromolecule | Name: Piwi protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 49.302434 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #2: AfAgo-N protein
Macromolecule | Name: AfAgo-N protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 31.356576 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #3: DNA target 17 nt
Macromolecule | Name: DNA target 17 nt / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 5.202384 KDa |
Sequence | String: (DA)(DT)(DT)(DC)(DG)(DG)(DC)(DC)(DG)(DT) (DG)(DT)(DA)(DC)(DA)(DA)(DT) |
-Macromolecule #4: RNA guide 17 nt
Macromolecule | Name: RNA guide 17 nt / type: rna / ID: 4 / Number of copies: 1 |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 5.427286 KDa |
Sequence | String: AUUGUACACG GCCGAAU |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 1 mg/mL | |||||||||||||||
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Buffer | pH: 8.5 Component:
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Grid | Model: 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. | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV | |||||||||||||||
Details | fAfAgo complex with 17/17 guide-target heteroduplexes was mixed and applied on grid |
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Electron microscopy
Microscope | TFS GLACIOS |
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Image recording | Film 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 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 92000 |
Sample stage | Specimen holder model: OTHER / Cooling holder cryogen: NITROGEN |
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Image processing
-Atomic model buiding 1
Initial model |
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Refinement | Space: REAL / Protocol: FLEXIBLE FIT / Overall B value: 48 | |||||||||
Output model | ![]() PDB-8qg0: |