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- EMDB-14453: MVV strand transfer complex (STC) intasome in complex with LEDGF/... -

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Basic information

Entry
Database: EMDB / ID: EMD-14453
TitleMVV strand transfer complex (STC) intasome in complex with LEDGF/p75 at 3.5 A resolution
Map datadeepEMhancer tight target map
Sample
  • Complex: MVV STC intasome in complex with LEDGF
    • Protein or peptide: Pol polyprotein
    • Protein or peptide: PC4 and SFRS1-interacting protein
    • DNA: 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: DNA (37-MER)
    • DNA: DNA (5'-D(P*TP*TP*GP*AP*TP*TP*AP*GP*GP*GP*TP*G)-3')
  • Ligand: ZINC ION
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. / Ribonuclease H superfamily / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
PC4 and SFRS1-interacting protein / Gag-Pol polyprotein
Similarity search - Component
Biological speciesVisna/maedi virus EV1 KV1772 / Homo sapiens (human) / DNA molecule (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsBallandras-Colas A / Nans A / Cherepanov P
Funding support United Kingdom, United States, 2 items
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 DT / Shan Z / Singh PK / Pye VE / McLean RK / Bedwell GJ / Li W / Nans A / Cook NJ / Fadel HJ / Poeschla EM / Griffiths DJ / Vargas J / Taylor IA / ...Authors: Ballandras-Colas A / Chivukula V / Gruszka DT / Shan Z / Singh PK / Pye VE / McLean RK / Bedwell GJ / Li W / Nans A / Cook NJ / Fadel HJ / Poeschla EM / Griffiths DJ / Vargas J / Taylor IA / Lyumkis D / Yardimci H / Engelman AN / Cherepanov P
History
DepositionFeb 25, 2022-
Header (metadata) releaseMay 11, 2022-
Map releaseMay 11, 2022-
UpdateDec 21, 2022-
Current statusDec 21, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14453.png

Downloads & links

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Map

FileDownload / File: emd_14453.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationdeepEMhancer tight target map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.38 Å/pix.
x 320 pix.
= 441.6 Å		1.38 Å/pix.
x 320 pix.
= 441.6 Å		1.38 Å/pix.
x 320 pix.
= 441.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.38 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.0018081084 - 2.1215732
Average (Standard dev.)0.0007304996 (±0.018725066)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 441.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14453_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: original volume map - no enhancement

Fileemd_14453_additional_1.map
Annotationoriginal volume map - no enhancement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Phenix local sharpen map - this map was...

Fileemd_14453_additional_2.map
AnnotationPhenix local sharpen map - this map was used for model real space refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_14453_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_14453_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : MVV STC intasome in complex with LEDGF

EntireName: MVV STC intasome in complex with LEDGF
Components
  • Complex: MVV STC intasome in complex with LEDGF
    • Protein or peptide: Pol polyprotein
    • Protein or peptide: PC4 and SFRS1-interacting protein
    • DNA: 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: DNA (37-MER)
    • DNA: DNA (5'-D(P*TP*TP*GP*AP*TP*TP*AP*GP*GP*GP*TP*G)-3')
  • Ligand: ZINC ION

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Supramolecule #1: MVV STC intasome in complex with LEDGF

SupramoleculeName: MVV STC intasome in complex with LEDGF / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Visna/maedi virus EV1 KV1772
Molecular weightTheoretical: 480 KDa

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Macromolecule #1: Pol polyprotein

MacromoleculeName: Pol polyprotein / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases
Source (natural)Organism: Visna/maedi virus EV1 KV1772 / Strain: KV1772
Molecular weightTheoretical: 32.368826 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: WIENIPLAEE EHNKWHQDAV SLHLEFGIPR TAAEDIVQQC DVCQENKMPS TLRGSNKRGI DHWQVDYTHY EDKIILVWVE TNSGLIYAE RVKGETGQEF RVQTMKWYAM FAPKSLQSDN GPAFVAESTQ LLMKYLGIEH TTGIPWNPQS QALVERTHQT L KNTLEKLI ...String:
WIENIPLAEE EHNKWHQDAV SLHLEFGIPR TAAEDIVQQC DVCQENKMPS TLRGSNKRGI DHWQVDYTHY EDKIILVWVE TNSGLIYAE RVKGETGQEF RVQTMKWYAM FAPKSLQSDN GPAFVAESTQ LLMKYLGIEH TTGIPWNPQS QALVERTHQT L KNTLEKLI PMFNAFESAL AGTLITLNIK RKGGLGTSPM DIFIFNKEQQ RIQQQSKSKQ EKIRFCYYRT RKRGHPGEWQ GP TQVLWGG DGAIVVKDRG TDRYLVIANK DVKFIPPPKE IQKE

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Macromolecule #2: PC4 and SFRS1-interacting protein

MacromoleculeName: PC4 and SFRS1-interacting protein / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.07597 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SMDSRLQRIH AEIKNSLKID NLDVNRCIEA LDELASLQVT MQQAQKHTEM ITTLKKIRRF KVSQVIMEKS TMLYNKFKNM FLVGEGDSV LEVLFQ

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

MacromoleculeName: 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')
type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 7.064532 KDa
SequenceString:
(DG)(DC)(DT)(DG)(DC)(DG)(DA)(DG)(DA)(DT) (DC)(DC)(DG)(DC)(DT)(DC)(DC)(DG)(DG)(DT) (DG)(DT)(DT)

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Macromolecule #4: DNA (37-MER)

MacromoleculeName: DNA (37-MER) / type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 15.387863 KDa
SequenceString:
(DA)(DA)(DC)(DA)(DC)(DC)(DG)(DG)(DA)(DG) (DC)(DG)(DG)(DA)(DT)(DC)(DT)(DC)(DG)(DC) (DA)(DG)(DT)(DC)(DG)(DA)(DC)(DC)(DA) (DC)(DC)(DC)(DT)(DA)(DA)(DT)(DC)(DA)(DA) (DG) (DT)(DT)(DT)(DT)(DT)(DT)(DG)(DG) (DG)(DG)

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Macromolecule #5: DNA (5'-D(P*TP*TP*GP*AP*TP*TP*AP*GP*GP*GP*TP*G)-3')

MacromoleculeName: DNA (5'-D(P*TP*TP*GP*AP*TP*TP*AP*GP*GP*GP*TP*G)-3') / type: dna / ID: 5 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 7.0736 KDa
SequenceString:
(DC)(DC)(DC)(DC)(DA)(DA)(DA)(DA)(DA)(DA) (DC)(DT)(DT)(DG)(DA)(DT)(DT)(DA)(DG)(DG) (DG)(DT)(DG)

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 12 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration0.15 mg/mL
BufferpH: 6.5
Details: 310 mM NaCl, 3 mM CaCl2, 25 mM BisTris-HCl, pH 6.5.
GridModel: EMS Lacey Carbon / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
DetailsThe 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.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 36232 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-50 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 121619
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

5m0r

,

3hph

)
Detailsinteractive fitting/rebuilding in coot and real space refinement in Phenix.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7z1z:
MVV strand transfer complex (STC) intasome in complex with LEDGF/p75 at 3.5 A resolution

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