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- PDB-7z1z: MVV strand transfer complex (STC) intasome in complex with LEDGF/... -

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Basic information

Entry
Database: PDB / ID: 7z1z
TitleMVV strand transfer complex (STC) intasome in complex with LEDGF/p75 at 3.5 A resolution
Components
  • DNA (37-MER)
  • DNA (5'-D(*GP*CP*TP*GP*CP*GP*AP*GP*AP*TP*CP*CP*GP*CP*TP*CP*CP*GP*GP*TP*G)-3')
  • DNA (5'-D(P*TP*TP*GP*AP*TP*TP*AP*GP*GP*GP*TP*G)-3')
  • PC4 and SFRS1-interacting protein
  • Pol polyprotein
KeywordsVIRAL PROTEIN / Intasome / Integrase / MVV / Lentivirus / HIV / strand transfer complex / LEDGF / IBD / DNA / integration
Function / homology
Function and homology information


dUTP diphosphatase / dUTP diphosphatase activity / nucleotide metabolic process / supercoiled DNA binding / Integration of viral DNA into host genomic DNA / Autointegration results in viral DNA circles / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / Formation of WDR5-containing histone-modifying complexes / 2-LTR circle formation ...dUTP diphosphatase / dUTP diphosphatase activity / nucleotide metabolic process / supercoiled DNA binding / Integration of viral DNA into host genomic DNA / Autointegration results in viral DNA circles / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / Formation of WDR5-containing histone-modifying complexes / 2-LTR circle formation / Vpr-mediated nuclear import of PICs / Integration of provirus / APOBEC3G mediated resistance to HIV-1 infection / mRNA 5'-splice site recognition / heterochromatin / nuclear periphery / euchromatin / exoribonuclease H / exoribonuclease H activity / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA / establishment of integrated proviral latency / viral capsid / RNA-directed DNA polymerase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / RNA-DNA hybrid ribonuclease activity / response to heat / DNA-binding transcription factor binding / DNA recombination / response to oxidative stress / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase / transcription coactivator activity / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / chromatin remodeling / symbiont entry into host cell / chromatin binding / positive regulation of transcription by RNA polymerase II / proteolysis / DNA binding / RNA binding / zinc ion binding / nucleoplasm / nucleus / cytosol
Similarity search - Function
Lens epithelium-derived growth factor, integrase-binding domain / HIV integrase-binding domain superfamily / Lens epithelium-derived growth factor (LEDGF) / TFIIS/LEDGF domain superfamily / dUTPase-like / dUTPase / dUTPase, trimeric / dUTPase-like superfamily / domain with conserved PWWP motif / PWWP domain ...Lens epithelium-derived growth factor, integrase-binding domain / HIV integrase-binding domain superfamily / Lens epithelium-derived growth factor (LEDGF) / TFIIS/LEDGF domain superfamily / dUTPase-like / dUTPase / dUTPase, trimeric / dUTPase-like superfamily / domain with conserved PWWP motif / PWWP domain / PWWP domain profile. / PWWP domain / gag protein p24 N-terminal domain / Integrase Zinc binding domain / Zinc finger integrase-type profile. / Integrase-like, N-terminal / Integrase, C-terminal domain superfamily, retroviral / Integrase, N-terminal zinc-binding domain / Integrase, C-terminal, retroviral / Integrase DNA binding domain profile. / RNase H / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Ribonuclease H domain / RNase H type-1 domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Retrovirus capsid, C-terminal / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / PC4 and SFRS1-interacting protein / Gag-Pol polyprotein
Similarity search - Component
Biological speciesVisna/maedi virus EV1 KV1772
Homo sapiens (human)
DNA molecule (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsPye, V.E. / Ballandras-Colas, A. / Cherepanov, P.
Funding support United Kingdom, United States, 2items
OrganizationGrant numberCountry
The Francis Crick InstituteFC10061 United Kingdom
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P50AI150481 United States
CitationJournal: Nat Commun / Year: 2022
Title: Multivalent interactions essential for lentiviral integrase function.
Authors: Ballandras-Colas, A. / Chivukula, V. / Gruszka, D.T. / Shan, Z. / Singh, P.K. / Pye, V.E. / McLean, R.K. / Bedwell, G.J. / Li, W. / Nans, A. / Cook, N.J. / Fadel, H.J. / Poeschla, E.M. / ...Authors: Ballandras-Colas, A. / Chivukula, V. / Gruszka, D.T. / Shan, Z. / Singh, P.K. / Pye, V.E. / McLean, R.K. / Bedwell, G.J. / Li, W. / Nans, A. / Cook, N.J. / Fadel, H.J. / Poeschla, E.M. / Griffiths, D.J. / Vargas, J. / Taylor, I.A. / Lyumkis, D. / Yardimci, H. / Engelman, A.N. / Cherepanov, P.
History
DepositionFeb 25, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 11, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 21, 2022Group: Data collection / Database references / Category: citation / pdbx_initial_refinement_model / Item: _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Pol polyprotein
B: Pol polyprotein
C: Pol polyprotein
D: Pol polyprotein
E: Pol polyprotein
F: Pol polyprotein
G: Pol polyprotein
H: Pol polyprotein
R: PC4 and SFRS1-interacting protein
Q: PC4 and SFRS1-interacting protein
X: DNA (5'-D(*GP*CP*TP*GP*CP*GP*AP*GP*AP*TP*CP*CP*GP*CP*TP*CP*CP*GP*GP*TP*G)-3')
W: DNA (37-MER)
U: DNA (5'-D(P*TP*TP*GP*AP*TP*TP*AP*GP*GP*GP*TP*G)-3')
I: Pol polyprotein
J: Pol polyprotein
K: Pol polyprotein
L: Pol polyprotein
M: Pol polyprotein
N: Pol polyprotein
O: Pol polyprotein
P: Pol polyprotein
Y: DNA (5'-D(*GP*CP*TP*GP*CP*GP*AP*GP*AP*TP*CP*CP*GP*CP*TP*CP*CP*GP*GP*TP*G)-3')
Z: DNA (37-MER)
a: DNA (5'-D(P*TP*TP*GP*AP*TP*TP*AP*GP*GP*GP*TP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)599,89036
Polymers599,10524
Non-polymers78512
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 18 molecules ABCDEFGHIJKLMNOPRQ

#1: Protein
Pol polyprotein


Mass: 32368.826 Da / Num. of mol.: 16 / Fragment: UNP residues 821-1101
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Visna/maedi virus EV1 KV1772 / Strain: KV1772 / Gene: pol / Production host: Escherichia coli (E. coli)
References: UniProt: P35956, Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases, RNA-directed DNA polymerase, retroviral ribonuclease H, exoribonuclease H, dUTP diphosphatase, ...References: UniProt: P35956, Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases, RNA-directed DNA polymerase, retroviral ribonuclease H, exoribonuclease H, dUTP diphosphatase, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, Hydrolases; Acting on ester bonds
#2: Protein PC4 and SFRS1-interacting protein / CLL-associated antigen KW-7 / Dense fine speckles 70 kDa protein / DFS 70 / Lens epithelium-derived ...CLL-associated antigen KW-7 / Dense fine speckles 70 kDa protein / DFS 70 / Lens epithelium-derived growth factor / Transcriptional coactivator p75/p52


Mass: 11075.970 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PSIP1, DFS70, LEDGF, PSIP2 / Production host: Escherichia coli (E. coli) / References: UniProt: O75475

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DNA chain , 3 types, 6 molecules XYWZUa

#3: DNA chain DNA (5'-D(*GP*CP*TP*GP*CP*GP*AP*GP*AP*TP*CP*CP*GP*CP*TP*CP*CP*GP*GP*TP*G)-3')


Mass: 7064.532 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#4: DNA chain DNA (37-MER)


Mass: 15387.863 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#5: DNA chain DNA (5'-D(P*TP*TP*GP*AP*TP*TP*AP*GP*GP*GP*TP*G)-3')


Mass: 7073.600 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)

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Non-polymers , 1 types, 12 molecules

#6: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: MVV STC intasome in complex with LEDGF / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.48 MDa / Experimental value: NO
Source (natural)Organism: Visna/maedi virus EV1 KV1772
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.5
Details: 310 mM NaCl, 3 mM CaCl2, 25 mM BisTris-HCl, pH 6.5.
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The intasomes were typically assembled by incubating 7 uM MVV IN, 8 uM LEDGF/p75, and 3.75 uM annealed vDNA (or the strand transfer product mimic) in 80 mM NaCl, 40 mM potassium acetate, 3 ...Details: The intasomes were typically assembled by incubating 7 uM MVV IN, 8 uM LEDGF/p75, and 3.75 uM annealed vDNA (or the strand transfer product mimic) in 80 mM NaCl, 40 mM potassium acetate, 3 mM CaCl2 10 uM ZnCl2, 1 mM dithiothreitol (DTT) and 25 mM BisTris-HCl, pH 6.0 in a total volume of 200 ul at 37 deg C for 10 min (to prepare samples for cryo-EM, the reaction was upscaled to a total volume of 1 ml). The opalescent mixture was supplemented with 50 mM BisTris-HCl, pH 6.5 and 190 mM NaCl and incubated on ice for 5 min to clear. If starting volume exceeded 200 ul, the mixture was concentrated by ultrafiltration in a VivaSpin device to a final volume of 200 ul. Intasomes were purified by size exclusion chromatography through a Superdex-200 10/30 column (GE Healthcare) pre-equilibrated in 310 mM NaCl, 3 mM CaCl2, 25 mM BisTris-HCl, pH 6.5.
Specimen supportGrid type: EMS Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 36232 X / Nominal defocus max: 4500 nm / Nominal defocus min: 1000 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: dev_4213: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
9RELION2.1initial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
13PHENIXdev-4213-000model refinement
14Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 121619 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: interactive fitting/rebuilding in coot and real space refinement in Phenix.
Atomic model building
IDPDB-ID 3D fitting-ID
15M0R

5m0r

1
23HPH

3hph

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00236711
ELECTRON MICROSCOPYf_angle_d0.49150260
ELECTRON MICROSCOPYf_dihedral_angle_d15.5855685
ELECTRON MICROSCOPYf_chiral_restr0.045432
ELECTRON MICROSCOPYf_plane_restr0.0045975

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