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- EMDB-12698: Structure of the foamy viral protease-reverse transcriptase in co... -

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Basic information

Entry
Database: EMDB / ID: EMD-12698
TitleStructure of the foamy viral protease-reverse transcriptase in complex with dsDNA.
Map datasharpened with b-factor -134.373
Sample
  • Complex: complex of protease-reverse transcriptase with ds DNA
    • Complex: Pr125Pol
      • Protein or peptide: Pr125Pol
    • Complex: DNA
      • DNA: 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')
      • DNA: 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')
Function / homology
Function and homology information


virion component / DNA integration / viral penetration into host nucleus / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / DNA recombination / host cell cytoplasm / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / proteolysis ...virion component / DNA integration / viral penetration into host nucleus / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / DNA recombination / host cell cytoplasm / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / proteolysis / RNA binding / metal ion binding / cytoplasm
Similarity search - Function
Spumavirus aspartic protease A9 / Retroviral integrase, C-terminal SH3 domain / Spumavirus aspartic protease (A9) / Retroviral integrase C-terminal SH3 domain / Foamy virus protease (FV PR) domain profile. / Integrase zinc-binding domain / Integrase zinc binding domain / Reverse transcriptase/retrotransposon-derived protein, RNase H-like domain / RNase H-like domain found in reverse transcriptase / RNase H ...Spumavirus aspartic protease A9 / Retroviral integrase, C-terminal SH3 domain / Spumavirus aspartic protease (A9) / Retroviral integrase C-terminal SH3 domain / Foamy virus protease (FV PR) domain profile. / Integrase zinc-binding domain / Integrase zinc binding domain / Reverse transcriptase/retrotransposon-derived protein, RNase H-like domain / RNase H-like domain found in reverse transcriptase / RNase H / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Ribonuclease H domain / RNase H type-1 domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Biological speciesWhite-tufted-ear marmoset simian foamy virus / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsCzarnocki-Cieciura M
Funding support Poland, 1 items
OrganizationGrant numberCountry
Polish National Science CentreUMO-2016/21/B/NZ1/02757 Poland
CitationJournal: 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
DepositionMar 30, 2021-
Header (metadata) releaseJun 30, 2021-
Map releaseJun 30, 2021-
UpdateJan 19, 2022-
Current statusJan 19, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7o24
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12698.png

Downloads & links

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Map

FileDownload / File: emd_12698.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened with b-factor -134.373
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 384 pix.
= 330.24 Å		0.86 Å/pix.
x 384 pix.
= 330.24 Å		0.86 Å/pix.
x 384 pix.
= 330.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.012 / Movie #1: 0.012
Minimum - Maximum-0.01831851 - 0.039614014
Average (Standard dev.)2.8691395e-05 (±0.0015421817)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 330.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.860.860.86
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z330.240330.240330.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0180.0400.000

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Supplemental data

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Mask #1

Fileemd_12698_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_12698_half_map_1.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_12698_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : complex of protease-reverse transcriptase with ds DNA

EntireName: complex of protease-reverse transcriptase with ds DNA
Components
  • Complex: complex of protease-reverse transcriptase with ds DNA
    • Complex: Pr125Pol
      • Protein or peptide: Pr125Pol
    • Complex: DNA
      • DNA: 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')
      • DNA: 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')

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Supramolecule #1: complex of protease-reverse transcriptase with ds DNA

SupramoleculeName: complex of protease-reverse transcriptase with ds DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightExperimental: 190 KDa

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Supramolecule #2: Pr125Pol

SupramoleculeName: Pr125Pol / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: White-tufted-ear marmoset simian foamy virus
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: Pr125Pol

MacromoleculeName: 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
Source (natural)Organism: White-tufted-ear marmoset simian foamy virus
Molecular weightTheoretical: 85.467289 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: 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

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Macromolecule #2: DNA (5'-D(*AP*AP*CP*AP*GP*AP*GP*TP*GP*CP*GP*AP*CP*AP*CP*CP*TP*GP*...

MacromoleculeName: 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
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.34777 KDa
SequenceString:
(DA)(DA)(DC)(DA)(DG)(DA)(DG)(DT)(DG)(DC) (DG)(DA)(DC)(DA)(DC)(DC)(DT)(DG)(DA)(DT) (DT)(DC)(DC)(DA)

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Macromolecule #3: DNA (5'-D(*TP*GP*GP*AP*AP*TP*CP*AP*GP*GP*TP*GP*TP*CP*GP*CP*AP*CP*...

MacromoleculeName: 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
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.783375 KDa
SequenceString:
(DT)(DG)(DG)(DA)(DA)(DT)(DC)(DA)(DG)(DG) (DT)(DG)(DT)(DC)(DG)(DC)(DA)(DC)(DT)(DC) (DT)(DG)

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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: 7
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 344421
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 20071
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-7o24:
Structure of the foamy viral protease-reverse transcriptase in complex with dsDNA.

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