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- EMDB-14860: Cryo-EM structure of the MVV CSC intasome at 4.5A resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-14860
TitleCryo-EM structure of the MVV CSC intasome at 4.5A resolution
Map datadeepEMnhancer map
Sample
  • Complex: Maedi-visna virus (MVV) intasome
    • Complex: Intergrase
      • Protein or peptide: Integrase
    • Complex: vDNA
      • DNA: vDNA, non-transferred strand
      • DNA: vDNA, transferred strand
Function / homology
Function and homology information


dUTP diphosphatase / dUTP diphosphatase activity / nucleotide metabolic process / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / exoribonuclease H / exoribonuclease H activity / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA ...dUTP diphosphatase / dUTP diphosphatase activity / nucleotide metabolic process / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / 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 / DNA recombination / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / proteolysis / DNA binding / zinc ion binding
Similarity search - Function
dUTPase-like / dUTPase / dUTPase, trimeric / dUTPase-like superfamily / 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 ...dUTPase-like / dUTPase / dUTPase, trimeric / dUTPase-like superfamily / 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
Biological speciesVisna/maedi virus EV1 KV1772 / Visna-maedi virus / Visna lentivirus (strain 1514)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsBallandras-Colas A / Maskell D / Pye VE / Locke J / Swuec S / Kotecha A / Costa A / Cherepanov P
Funding support United Kingdom, 4 items
OrganizationGrant numberCountry
The Francis Crick InstituteFC001061 United Kingdom
Wellcome TrustFC001061 United Kingdom
Medical Research Council (MRC, United Kingdom)FC001061 United Kingdom
Cancer Research UKFC001061 United Kingdom
CitationJournal: Science / Year: 2017
Title: A supramolecular assembly mediates lentiviral DNA integration.
Authors: Allison Ballandras-Colas / Daniel P Maskell / Erik Serrao / Julia Locke / Paolo Swuec / Stefán R Jónsson / Abhay Kotecha / Nicola J Cook / Valerie E Pye / Ian A Taylor / Valgerdur ...Authors: Allison Ballandras-Colas / Daniel P Maskell / Erik Serrao / Julia Locke / Paolo Swuec / Stefán R Jónsson / Abhay Kotecha / Nicola J Cook / Valerie E Pye / Ian A Taylor / Valgerdur Andrésdóttir / Alan N Engelman / Alessandro Costa / Peter Cherepanov /
Abstract: Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo-electron microscopy, we now visualize the ...Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo-electron microscopy, we now visualize the functional maedi-visna lentivirus intasome at 4.9 angstrom resolution. The intasome comprises a homo-hexadecamer of IN with a tetramer-of-tetramers architecture featuring eight structurally distinct types of IN protomers supporting two catalytically competent subunits. The conserved intasomal core, previously observed in simpler retroviral systems, is formed between two IN tetramers, with a pair of C-terminal domains from flanking tetramers completing the synaptic interface. Our results explain how HIV-1 IN, which self-associates into higher-order multimers, can form a functional intasome, reconcile the bulk of early HIV-1 IN biochemical and structural data, and provide a lentiviral platform for design of HIV-1 IN inhibitors.
History
DepositionApr 28, 2022-
Header (metadata) releaseMay 11, 2022-
Map releaseMay 11, 2022-
SupersessionJun 29, 2022ID: EMD-4138
UpdateOct 12, 2022-
Current statusOct 12, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14860.png

Downloads & links

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Map

FileDownload / File: emd_14860.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationdeepEMnhancer map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.43 Å/pix.
x 300 pix.
= 429. Å		1.43 Å/pix.
x 300 pix.
= 429. Å		1.43 Å/pix.
x 300 pix.
= 429. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.43 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.122
Minimum - Maximum-0.0017862628 - 2.0413592
Average (Standard dev.)0.0012281933 (±0.026059164)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 428.99997 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14860_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: original map - no enhancement

Fileemd_14860_additional_1.map
Annotationoriginal map - no enhancement
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: cryosparc local filter map - to which model...

Fileemd_14860_additional_2.map
Annotationcryosparc local filter map - to which model was fitted and refined against
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_14860_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_14860_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Maedi-visna virus (MVV) intasome

EntireName: Maedi-visna virus (MVV) intasome
Components
  • Complex: Maedi-visna virus (MVV) intasome
    • Complex: Intergrase
      • Protein or peptide: Integrase
    • Complex: vDNA
      • DNA: vDNA, non-transferred strand
      • DNA: vDNA, transferred strand

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Supramolecule #1: Maedi-visna virus (MVV) intasome

SupramoleculeName: Maedi-visna virus (MVV) intasome / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 542.45 KDa

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Supramolecule #2: Intergrase

SupramoleculeName: Intergrase / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Visna/maedi virus EV1 KV1772
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: vDNA

SupramoleculeName: vDNA / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Visna-maedi virus

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Macromolecule #1: Integrase

MacromoleculeName: Integrase / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
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: vDNA, non-transferred strand

MacromoleculeName: vDNA, non-transferred strand / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Visna lentivirus (strain 1514)
Molecular weightTheoretical: 6.456146 KDa
SequenceString:
(DG)(DC)(DT)(DG)(DC)(DG)(DA)(DG)(DA)(DT) (DC)(DC)(DG)(DC)(DT)(DC)(DC)(DG)(DG)(DT) (DG)

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Macromolecule #3: vDNA, transferred strand

MacromoleculeName: vDNA, transferred strand / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Visna-maedi virus
Molecular weightTheoretical: 5.815762 KDa
SequenceString:
(DC)(DA)(DC)(DC)(DG)(DG)(DA)(DG)(DC)(DG) (DG)(DA)(DT)(DC)(DT)(DC)(DG)(DC)(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

Concentration0.3 mg/mL
BufferpH: 6.5
Component:
ConcentrationFormulaName
1.0 MNaClSodium chlorideSodium Chloride
3.0 mMCaCl2Calcium Chloride
25.0 mMBisTris-HCl

Details: 1 M NaCl, 3 mM CaCl2 and 25 mM BisTris-HCl, pH 6.5
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293.15 K / Instrument: FEI VITROBOT MARK IV
Details: To lower salt concentration before plunge-freezing, the grids were blotted for 0.5 s, immediately hydrated with a 4-ul drop of 200 mM NaCl, 3 mM CaCl2 and 25 mM BisTris-HCl pH 6.5 and ...Details: To lower salt concentration before plunge-freezing, the grids were blotted for 0.5 s, immediately hydrated with a 4-ul drop of 200 mM NaCl, 3 mM CaCl2 and 25 mM BisTris-HCl pH 6.5 and blotted again for 2.5 s followed by plunging into liquid ethane..
DetailsA 4 ul drop of freshly prepared intasome in 1 M NaCl, 3 mM CaCl2 and 25 mM BisTris-HCl pH 6.5 was applied onto glow-discharged lacey carbon grids coated with ultrathin carbon (product 01824, Ted Pella). The grids were incubated for 30 s under 100% humidity in a Vitrobot Mark IV (FEI) at 20 oC. To lower salt concentration before plunge-freezing, the grids were blotted for 0.5 s, immediately hydrated with a 4 ul drop of 200 mM NaCl, 3 mM CaCl2, and 25 mM BisTris-HCl pH 6.5 and blotted again for 2.5 s, followed by plunging into liquid ethane.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.5 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 253785
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: NONE / Details: ab-initio in cryoSPARC
Initial angle assignmentType: PROJECTION MATCHING
Final 3D classificationSoftware - Name: cryoSPARC (ver. 2)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Details: Non Uniform Refinement in cryoSPARC-2 / Number images used: 128974
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5m0q
PDB Unreleased entry

Details5M0Q model was docked to new map (updated relion version and pixel size corrected) in Chimera and refined using phenix.real_space refine and interactively adjusted in coot.
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 100 / Target criteria: correlation coefficient
Output model

PDB-7zpp:
Cryo-EM structure of the MVV CSC intasome at 4.5A resolution

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