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Yorodumi- EMDB-12698: Structure of the foamy viral protease-reverse transcriptase in co... -
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-Basic information
Entry | Database: EMDB / ID: EMD-12698 | |||||||||
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Title | Structure of the foamy viral protease-reverse transcriptase in complex with dsDNA. | |||||||||
Map data | sharpened with b-factor -134.373 | |||||||||
Sample |
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Keywords | reverse trancscriptase / complex with dsDNA / VIRAL PROTEIN | |||||||||
Function / homology | Function and homology information virion component / DNA integration / viral penetration into host nucleus / RNA-directed DNA polymerase activity / host cell / RNA-DNA hybrid ribonuclease activity / DNA recombination / host cell cytoplasm / nucleic acid binding / aspartic-type endopeptidase activity ...virion component / DNA integration / viral penetration into host nucleus / RNA-directed DNA polymerase activity / host cell / RNA-DNA hybrid ribonuclease activity / DNA recombination / host cell cytoplasm / nucleic acid binding / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / proteolysis / metal ion binding / cytoplasm Similarity search - Function | |||||||||
Biological species | White-tufted-ear marmoset simian foamy virus / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.8 Å | |||||||||
Authors | Czarnocki-Cieciura M | |||||||||
Funding support | Poland, 1 items
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Citation | Journal: J Virol / Year: 2021 Title: Structures of Substrate Complexes of Foamy Viral Protease-Reverse Transcriptase. Authors: Marzena Nowacka / Elżbieta Nowak / Mariusz Czarnocki-Cieciura / Justyna Jackiewicz / Krzysztof Skowronek / Roman H Szczepanowski / Birgitta M Wöhrl / Marcin Nowotny / Abstract: Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA (dsDNA), a crucial process for the replication of ...Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA (dsDNA), a crucial process for the replication of retroviruses. Foamy viruses (FVs) possess a unique RT, which is a fusion with the protease (PR) domain. The mechanism of substrate binding by this enzyme has been unknown. Here, we report a crystal structure of monomeric full-length marmoset FV (MFV) PR-RT in complex with an RNA/DNA hybrid substrate. We also describe a structure of MFV PR-RT with an RNase H deletion in complex with a dsDNA substrate in which the enzyme forms an asymmetric homodimer. Cryo-electron microscopy reconstruction of the full-length MFV PR-RT-dsDNA complex confirmed the dimeric architecture. These findings represent the first structural description of nucleic acid binding by a foamy viral RT and demonstrate its ability to change its oligomeric state depending on the type of bound nucleic acid. Reverse transcriptases (RTs) are intriguing enzymes converting single-stranded RNA to dsDNA. Their activity is essential for retroviruses, which are divided into two subfamilies differing significantly in their life cycles: and . The latter family is much more ancient and comprises five genera. A unique feature of foamy viral RTs is that they contain N-terminal protease (PR) domains, which are not present in orthoretroviral enzymes. So far, no structural information for full-length foamy viral PR-RT interacting with nucleic substrates has been reported. Here, we present crystal and cryo-electron microscopy structures of marmoset foamy virus (MFV) PR-RT. These structures revealed the mode of binding of RNA/DNA and dsDNA substrates. Moreover, unexpectedly, the structures and biochemical data showed that foamy viral PR-RT can adopt both a monomeric configuration, which is observed in our structures in the presence of an RNA/DNA hybrid, and an asymmetric dimer arrangement, which we observed in the presence of dsDNA. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_12698.map.gz | 202.4 MB | EMDB map data format | |
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Header (meta data) | emd-12698-v30.xml emd-12698.xml | 18.6 KB 18.6 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_12698_fsc.xml | 13.7 KB | Display | FSC data file |
Images | emd_12698.png | 113.9 KB | ||
Masks | emd_12698_msk_1.map | 216 MB | Mask map | |
Filedesc metadata | emd-12698.cif.gz | 6.5 KB | ||
Others | emd_12698_half_map_1.map.gz emd_12698_half_map_2.map.gz | 171.5 MB 171.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-12698 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-12698 | HTTPS FTP |
-Validation report
Summary document | emd_12698_validation.pdf.gz | 1.1 MB | Display | EMDB validaton report |
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Full document | emd_12698_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | emd_12698_validation.xml.gz | 20.7 KB | Display | |
Data in CIF | emd_12698_validation.cif.gz | 27 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12698 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12698 | HTTPS FTP |
-Related structure data
Related structure data | 7o24MC 7o0gC 7o0hC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_12698.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | sharpened with b-factor -134.373 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.86 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_12698_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_12698_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_12698_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : complex of protease-reverse transcriptase with ds DNA
Entire | Name: complex of protease-reverse transcriptase with ds DNA |
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Components |
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-Supramolecule #1: complex of protease-reverse transcriptase with ds DNA
Supramolecule | Name: complex of protease-reverse transcriptase with ds DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Molecular weight | Theoretical: 190 KDa |
-Supramolecule #2: Pr125Pol
Supramolecule | Name: Pr125Pol / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: White-tufted-ear marmoset simian foamy virus |
-Supramolecule #3: DNA
Supramolecule | Name: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3 |
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Source (natural) | Organism: synthetic construct (others) |
-Macromolecule #1: Pr125Pol
Macromolecule | Name: Pr125Pol / type: protein_or_peptide / ID: 1 Details: NCBI Reference Sequence: NC_039030.1 TYG codon placed as LEU 586 Number of copies: 3 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase |
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Source (natural) | Organism: White-tufted-ear marmoset simian foamy virus |
Molecular weight | Theoretical: 85.467289 KDa |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Sequence | String: MTTPPLLQLP VEVKKTELNG FWDTGAQITC IPEAFLKEEI PIGEAQIKTL HGTKLQSVYY LKFKVLGRKV EAEVTTSPFD YVIISPSDI PWYKPQPLEL TVKLPVQDFK KELINKANIN NEEKKQLAKL LDKYDVLWQQ WENQVGHRKI PPHNIATGTV A PRPQRQYH ...String: MTTPPLLQLP VEVKKTELNG FWDTGAQITC IPEAFLKEEI PIGEAQIKTL HGTKLQSVYY LKFKVLGRKV EAEVTTSPFD YVIISPSDI PWYKPQPLEL TVKLPVQDFK KELINKANIN NEEKKQLAKL LDKYDVLWQQ WENQVGHRKI PPHNIATGTV A PRPQRQYH INTKAKPSIQ QVIDDLLKQG VLIKQTSVMN TPIYPVPKPD GKWRMVLDYR AVNKTVPLIG AQNQHSLGIL TN LVRQKYK STIDLSNGFW AHPITKDSQW ITAFTWEGKQ HVWTRLPQGF LNSPALFTAD VVDLLKNIPG ISVYVDDIYF STE TVSEHL KILEKVFKIL LEAGYIVSLK KSALLRYEVT FLGFSITQTG RGLTSEFKDK IQNITSPRTL KELQSILGLF NFAR NFVPN FSEIIKPLYS LISTAEGNNI KWTSEHTRYL EEIVSALNHA GNLEQRDNES PLVVKLNASP KTGYIRYYNK GGQKP IAYA SHVFTNTELK FTPLEKLLVT MHKALIKAID LALGQPIEVY SPIISMQKLQ KTPLPERKAL STRWITWLSY LEDPRI TFY YDKTLPDLKN VPETVTDKKP KMLPIIEYAA VFYTDGSAIR SPDKNKSHSS GMGIVHAVFK PELTIEHQWS IPLGDHT AQ YAEISAVEFA CKKANNISGP VLIVTDSDYV ARSVNEELPF WRSNGFVNNK KKPLKHISKW KNISDSLLLK RDIIIVHE P GHKPSYTSIH TQGNNLADKL ATQGSYTVNN I UniProtKB: Pro-Pol polyprotein |
-Macromolecule #2: DNA (5'-D(*AP*AP*CP*AP*GP*AP*GP*TP*GP*CP*GP*AP*CP*AP*CP*CP*TP*GP*...
Macromolecule | Name: DNA (5'-D(*AP*AP*CP*AP*GP*AP*GP*TP*GP*CP*GP*AP*CP*AP*CP*CP*TP*GP*AP*TP*TP*CP*CP*A)-3') type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 7.34777 KDa |
Sequence | String: (DA)(DA)(DC)(DA)(DG)(DA)(DG)(DT)(DG)(DC) (DG)(DA)(DC)(DA)(DC)(DC)(DT)(DG)(DA)(DT) (DT)(DC)(DC)(DA) |
-Macromolecule #3: DNA (5'-D(*TP*GP*GP*AP*AP*TP*CP*AP*GP*GP*TP*GP*TP*CP*GP*CP*AP*CP*...
Macromolecule | Name: DNA (5'-D(*TP*GP*GP*AP*AP*TP*CP*AP*GP*GP*TP*GP*TP*CP*GP*CP*AP*CP*TP*CP*TP*G)-3') type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 6.783375 KDa |
Sequence | String: (DT)(DG)(DG)(DA)(DA)(DT)(DC)(DA)(DG)(DG) (DT)(DG)(DT)(DC)(DG)(DC)(DA)(DC)(DT)(DC) (DT)(DG) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 7 |
Grid | Model: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Protocol: RIGID BODY FIT |
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Output model | PDB-7o24: |