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

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Basic information

Entry
Database: PDB / ID: 7zpp
TitleCryo-EM structure of the MVV CSC intasome at 4.5A resolution
Components
  • Integrase
  • vDNA, non-transferred strand
  • vDNA, transferred strand
KeywordsVIRAL PROTEIN / Integrase / intasome / MVV / nucleoprotein complex / retrovirus
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
: / DNA / DNA (> 10) / Gag-Pol polyprotein
Similarity search - Component
Biological speciesVisna/maedi virus EV1 KV1772
Visna lentivirus
Visna-maedi virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsBallandras-Colas, A. / Maskell, D. / Pye, V.E. / Locke, J. / Swuec, S. / Kotecha, A. / Costa, A. / Cherepanov, P.
Funding support United Kingdom, 4items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 11, 2022Provider: repository / Type: Initial release
SupersessionJun 29, 2022ID: 5M0Q
Revision 1.1Jun 29, 2022Group: Advisory / Database references ...Advisory / Database references / Source and taxonomy / Structure summary
Category: em_entity_assembly / em_entity_assembly_naturalsource ...em_entity_assembly / em_entity_assembly_naturalsource / em_entity_assembly_recombinant / entity / entity_name_com / pdbx_database_PDB_obs_spr / pdbx_entity_src_syn / struct_ref / struct_ref_seq
Item: _entity.pdbx_description / _entity.pdbx_ec ..._entity.pdbx_description / _entity.pdbx_ec / _pdbx_entity_src_syn.ncbi_taxonomy_id / _pdbx_entity_src_syn.organism_scientific / _struct_ref.db_code / _struct_ref.db_name / _struct_ref.pdbx_align_begin / _struct_ref.pdbx_db_accession / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.db_align_beg / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_db_accession
Revision 1.2Oct 12, 2022Group: Structure summary / Category: struct / Item: _struct.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Integrase
B: Integrase
C: Integrase
D: Integrase
E: Integrase
F: Integrase
G: Integrase
H: Integrase
I: Integrase
J: Integrase
K: Integrase
L: Integrase
M: Integrase
N: Integrase
O: Integrase
P: Integrase
Q: vDNA, non-transferred strand
R: vDNA, transferred strand
S: vDNA, non-transferred strand
T: vDNA, transferred strand


Theoretical massNumber of molelcules
Total (without water)542,44520
Polymers542,44520
Non-polymers00
Water0
1


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

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Components

#1: Protein
Integrase / / IN


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, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, Hydrolases; Acting on ester bonds
#2: DNA chain vDNA, non-transferred strand


Mass: 6456.146 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Visna lentivirus (strain 1514)
#3: DNA chain vDNA, transferred strand


Mass: 5815.762 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Visna-maedi virus / References: GenBank: J04359.1

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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

Component
IDNameTypeEntity IDParent-IDSource
1Maedi-visna virus (MVV) intasomeCOMPLEXall0MULTIPLE SOURCES
2IntergraseCOMPLEX#11RECOMBINANT
3vDNACOMPLEX#2-#31MULTIPLE SOURCES
Molecular weightValue: 0.54245 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Visna/maedi virus EV1 KV177236374
33Visna-maedi virus2169971
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
Buffer solutionpH: 6.5 / Details: 1 M NaCl, 3 mM CaCl2 and 25 mM BisTris-HCl, pH 6.5
Buffer component
IDConc.NameFormulaBuffer-ID
11 MSodium ChlorideNaClSodium chloride1
23 mMCalcium ChlorideCaCl21
325 mMBisTris-HCl1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: A 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, ...Details: A 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.
Specimen supportGrid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293.15 K
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.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5000 nm / Nominal defocus min: 3500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: dev_4213: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10Cootmodel refinement
12RELION3.1final Euler assignment
13cryoSPARC2classification
14cryoSPARC23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 253785
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 128974 / Algorithm: FOURIER SPACE / Details: Non Uniform Refinement in cryoSPARC-2 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 100 / Protocol: OTHER / Space: REAL / Target criteria: correlation coefficient
Details: 5M0Q 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.
Atomic model buildingPDB-ID: 5M0Q

5m0q
PDB Unreleased entry

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00234119
ELECTRON MICROSCOPYf_angle_d0.61846515
ELECTRON MICROSCOPYf_dihedral_angle_d13.9674959
ELECTRON MICROSCOPYf_chiral_restr0.0445006
ELECTRON MICROSCOPYf_plane_restr0.0045714

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