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- PDB-5cz1: Crystal structure of the catalytic core domain of MMTV integrase -

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Basic information

Entry
Database: PDB / ID: 5cz1
TitleCrystal structure of the catalytic core domain of MMTV integrase
Componentsintegrase
KeywordsHYDROLASE / integrase / POL / retrovirus / catalytic core domain
Function / homology
Function and homology information


dUTP diphosphatase / dUTP diphosphatase activity / ribonuclease H / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / RNA stem-loop binding / RNA-directed DNA polymerase activity ...dUTP diphosphatase / dUTP diphosphatase activity / ribonuclease H / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / RNA stem-loop binding / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / viral nucleocapsid / DNA recombination / DNA-directed DNA polymerase / aspartic-type endopeptidase activity / structural constituent of virion / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase activity / viral translational frameshifting / symbiont entry into host cell / proteolysis / DNA binding / zinc ion binding
Similarity search - Function
Beta-retroviral matrix protein / Beta-retroviral matrix superfamily / Retroviral GAG p10 protein / GAG-polyprotein viral zinc-finger / dUTPase-like / dUTPase / dUTPase, trimeric / dUTPase-like superfamily / gag protein p24 N-terminal domain / Ribonuclease H-like superfamily/Ribonuclease H ...Beta-retroviral matrix protein / Beta-retroviral matrix superfamily / Retroviral GAG p10 protein / GAG-polyprotein viral zinc-finger / dUTPase-like / dUTPase / dUTPase, trimeric / dUTPase-like superfamily / gag protein p24 N-terminal domain / Ribonuclease H-like superfamily/Ribonuclease H / 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. / Retropepsin-like catalytic domain / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / RNase H type-1 domain profile. / Ribonuclease H domain / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / 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. / Nucleotidyltransferase; domain 5 / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Gag-Pro-Pol polyprotein / Gag-Pro-Pol polyprotein
Similarity search - Component
Biological speciesMouse mammary tumor virus
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.7 Å
AuthorsCook, N. / Ballandras-Colas, A. / Engelman, A. / Cherepanov, P.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI070042 United States
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
DepositionJul 31, 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 1.3Mar 30, 2022Group: Author supporting evidence / Data collection / Database references
Category: database_2 / diffrn_radiation_wavelength / pdbx_audit_support
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _diffrn_radiation_wavelength.wavelength / _pdbx_audit_support.funding_organization
Revision 1.4Jan 10, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: integrase
B: integrase
C: integrase
D: integrase


Theoretical massNumber of molelcules
Total (without water)77,2404
Polymers77,2404
Non-polymers00
Water7,873437
1
A: integrase


Theoretical massNumber of molelcules
Total (without water)19,3101
Polymers19,3101
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: integrase


Theoretical massNumber of molelcules
Total (without water)19,3101
Polymers19,3101
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: integrase


Theoretical massNumber of molelcules
Total (without water)19,3101
Polymers19,3101
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: integrase


Theoretical massNumber of molelcules
Total (without water)19,3101
Polymers19,3101
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)51.886, 53.707, 69.647
Angle α, β, γ (deg.)69.69, 82.08, 63.97
Int Tables number1
Space group name H-MP1

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Components

#1: Protein
integrase


Mass: 19310.104 Da / Num. of mol.: 4 / Fragment: UNP residues 1487-1648
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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 437 / 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: 2.14 Å3/Da / Density % sol: 42.49 %
Crystal growTemperature: 291.15 K / Method: vapor diffusion, hanging drop
Details: 12.5% PEG3350, 0.15 M Ammonium Citrate Tribasic, pH6.5.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: BM14 / Wavelength: 0.95372 / Wavelength: 1 Å
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Jun 27, 2015
RadiationMonochromator: Si111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.953721
211
ReflectionResolution: 1.7→46.62 Å / Num. obs: 69082 / % possible obs: 99.1 % / Redundancy: 5.2 % / Rmerge(I) obs: 0.06 / Net I/σ(I): 21
Reflection shellResolution: 1.7→1.73 Å / Redundancy: 2.8 % / Rmerge(I) obs: 0.571 / Mean I/σ(I) obs: 2 / % possible all: 95.6

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Processing

Software
NameVersionClassification
PHENIX(1.10pre_2084: ???)refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1ASV

1asv


Resolution: 1.7→32.801 Å / SU ML: 0.2 / Cross valid method: FREE R-VALUE / σ(F): 1.96 / Phase error: 23.27 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2219 3319 4.8 %
Rwork0.1869 --
obs0.1886 69075 99.1 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.7→32.801 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4978 0 0 437 5415
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0075123
X-RAY DIFFRACTIONf_angle_d0.9546953
X-RAY DIFFRACTIONf_dihedral_angle_d10.8833001
X-RAY DIFFRACTIONf_chiral_restr0.045762
X-RAY DIFFRACTIONf_plane_restr0.005878
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.7-1.72430.33061350.28662636X-RAY DIFFRACTION95
1.7243-1.750.31581330.26732702X-RAY DIFFRACTION97
1.75-1.77740.27361570.24742666X-RAY DIFFRACTION99
1.7774-1.80650.28831480.23472739X-RAY DIFFRACTION99
1.8065-1.83770.28441460.22642711X-RAY DIFFRACTION99
1.8377-1.87110.27211420.2162709X-RAY DIFFRACTION99
1.8711-1.90710.22251270.212759X-RAY DIFFRACTION99
1.9071-1.9460.28991330.2012749X-RAY DIFFRACTION99
1.946-1.98830.26731480.20522766X-RAY DIFFRACTION99
1.9883-2.03450.2285960.1952731X-RAY DIFFRACTION99
2.0345-2.08540.22271170.1952789X-RAY DIFFRACTION99
2.0854-2.14180.23551280.18822760X-RAY DIFFRACTION99
2.1418-2.20480.19521260.18572734X-RAY DIFFRACTION99
2.2048-2.27590.2171270.17872748X-RAY DIFFRACTION99
2.2759-2.35730.22771400.18992792X-RAY DIFFRACTION100
2.3573-2.45160.22571290.20162757X-RAY DIFFRACTION100
2.4516-2.56320.2281500.19042776X-RAY DIFFRACTION100
2.5632-2.69820.23781600.19722702X-RAY DIFFRACTION100
2.6982-2.86720.21671760.18592721X-RAY DIFFRACTION100
2.8672-3.08840.2091710.18942752X-RAY DIFFRACTION100
3.0884-3.39890.25111510.18262729X-RAY DIFFRACTION100
3.3989-3.89010.18871170.16642795X-RAY DIFFRACTION100
3.8901-4.89850.17711440.15132752X-RAY DIFFRACTION100
4.8985-32.80710.21231180.18452781X-RAY DIFFRACTION100

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