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- PDB-5d7u: Crystal structure of the C-terminal domain of MMTV integrase -

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Basic information

Entry
Database: PDB / ID: 5d7u
TitleCrystal structure of the C-terminal domain of MMTV integrase
ComponentsPr160
KeywordsHYDROLASE / integrase / POL / retrovirus
Function / homology
Function and homology information


dUTP diphosphatase / dUTP diphosphatase activity / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases ...dUTP diphosphatase / dUTP diphosphatase activity / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / RNA-DNA hybrid ribonuclease activity / viral nucleocapsid / DNA recombination / structural constituent of virion / 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 / RNA binding / zinc ion binding
Similarity search - Function
Integrase, C-terminal domain superfamily, retroviral / GAG-polyprotein viral zinc-finger / Beta-retroviral matrix protein / Beta-retroviral matrix superfamily / Retroviral GAG p10 protein / dUTPase-like / dUTPase / dUTPase, trimeric / dUTPase-like superfamily / gag protein p24 N-terminal domain ...Integrase, C-terminal domain superfamily, retroviral / GAG-polyprotein viral zinc-finger / Beta-retroviral matrix protein / Beta-retroviral matrix superfamily / Retroviral GAG p10 protein / dUTPase-like / dUTPase / dUTPase, trimeric / dUTPase-like superfamily / gag protein p24 N-terminal domain / Reverse transcriptase thumb / Reverse transcriptase thumb domain / Integrase Zinc binding domain / Zinc finger integrase-type profile. / Integrase-like, N-terminal / Integrase DNA binding domain / 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. / SH3 type barrels. / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retropepsin-like catalytic 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 / Retroviral matrix protein / 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 / Roll / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily / Mainly Beta
Similarity search - Domain/homology
ISOPROPYL ALCOHOL / Gag-Pro-Pol polyprotein / Gag-Pro-Pol polyprotein
Similarity search - Component
Biological speciesMouse mammary tumor virus
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.5 Å
AuthorsCook, N.J. / Pye, V.E. / Ballandras-Colas, A. / Engelman, A. / Cherepanov, P.
CitationJournal: Nature / Year: 2016
Title: Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function.
Authors: Allison Ballandras-Colas / Monica Brown / Nicola J Cook / Tamaria G Dewdney / Borries Demeler / Peter Cherepanov / Dmitry Lyumkis / Alan N Engelman /
Abstract: Retroviral integrase catalyses the integration of viral DNA into host target DNA, which is an essential step in the life cycle of all retroviruses. Previous structural characterization of integrase- ...Retroviral integrase catalyses the integration of viral DNA into host target DNA, which is an essential step in the life cycle of all retroviruses. Previous structural characterization of integrase-viral DNA complexes, or intasomes, from the spumavirus prototype foamy virus revealed a functional integrase tetramer, and it is generally believed that intasomes derived from other retroviral genera use tetrameric integrase. However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Here, using single-particle cryo-electron microscopy and X-ray crystallography, we determine an unexpected octameric integrase architecture for the intasome of the betaretrovirus mouse mammary tumour virus. The structure is composed of two core integrase dimers, which interact with the viral DNA ends and structurally mimic the integrase tetramer of prototype foamy virus, and two flanking integrase dimers that engage the core structure via their integrase carboxy-terminal domains. Contrary to the belief that tetrameric integrase components are sufficient to catalyse integration, the flanking integrase dimers were necessary for mouse mammary tumour virus integrase activity. The integrase octamer solves a conundrum for betaretroviruses as well as alpharetroviruses by providing critical carboxy-terminal domains to the intasome core that cannot be provided in cis because of evolutionarily restrictive catalytic core domain-carboxy-terminal domain linker regions. The octameric architecture of the intasome of mouse mammary tumour virus provides new insight into the structural basis of retroviral DNA integration.
History
DepositionAug 14, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Feb 17, 2016Provider: repository / Type: Initial release
Revision 1.1Feb 24, 2016Group: Database references
Revision 1.2Mar 2, 2016Group: Database references
Revision 1.3Jan 10, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Pr160
B: Pr160
hetero molecules


Theoretical massNumber of molelcules
Total (without water)12,9434
Polymers12,8232
Non-polymers1202
Water1,42379
1
A: Pr160
hetero molecules


Theoretical massNumber of molelcules
Total (without water)6,4712
Polymers6,4111
Non-polymers601
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Pr160
hetero molecules


Theoretical massNumber of molelcules
Total (without water)6,4712
Polymers6,4111
Non-polymers601
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)35.988, 42.279, 139.087
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221

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Components

#1: Protein Pr160


Mass: 6411.344 Da / Num. of mol.: 2 / Fragment: C-terminal domain, UNP residues 1645-1702
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mouse mammary tumor virus / Gene: gag-pro-pol / Plasmid: pET20b(+) / Details (production host): cleavable His6 tag / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O56220, UniProt: P03365*PLUS
#2: Chemical ChemComp-IPA / ISOPROPYL ALCOHOL / 2-PROPANOL / Isopropyl alcohol


Mass: 60.095 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O / Comment: alkaloid*YM
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 79 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION

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Sample preparation

CrystalDensity Matthews: 2.07 Å3/Da / Density % sol: 40.73 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 20% isopropanol, 0.2M Ammonium Acetate, 0.1M HEPES, pH7.5

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.9763 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Aug 6, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 1.5→46.36 Å / Num. obs: 17494 / % possible obs: 99.8 % / Redundancy: 12.2 % / Rmerge(I) obs: 0.043 / Net I/σ(I): 29.2
Reflection shellResolution: 1.5→1.53 Å / Redundancy: 8.9 % / Rmerge(I) obs: 0.585 / Mean I/σ(I) obs: 3.8 / % possible all: 99.9

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Processing

Software
NameVersionClassification
PHENIX(1.10pre_2091: ???)refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1EX4

1ex4


Resolution: 1.5→34.772 Å / SU ML: 0.13 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 22.9 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2071 870 4.99 %
Rwork0.1589 --
obs0.1613 17448 99.82 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.5→34.772 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms839 0 8 79 926
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.007931
X-RAY DIFFRACTIONf_angle_d1.0481282
X-RAY DIFFRACTIONf_dihedral_angle_d13.534565
X-RAY DIFFRACTIONf_chiral_restr0.068135
X-RAY DIFFRACTIONf_plane_restr0.007162
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.5-1.5940.23841630.15362651X-RAY DIFFRACTION99
1.594-1.71710.22841380.14272740X-RAY DIFFRACTION100
1.7171-1.88980.20331290.12882757X-RAY DIFFRACTION100
1.8898-2.16330.16951280.12972763X-RAY DIFFRACTION100
2.1633-2.72530.20831480.17862776X-RAY DIFFRACTION100
2.7253-34.78110.2111640.16542891X-RAY DIFFRACTION100

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