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- PDB-5cz2: Crystal structure of a two-domain fragment of MMTV integrase -

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Basic information

Entry
Database: PDB / ID: 5cz2
TitleCrystal structure of a two-domain fragment of MMTV integrase
ComponentsPol polyprotein
KeywordsHYDROLASE / integrase / POL / retrovirus / amino terminal domain / catalytic core domain / zinc binding
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 / structural constituent of virion / aspartic-type endopeptidase activity / 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
Integrase, N-terminal zinc-binding domain / 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 ...Integrase, N-terminal zinc-binding domain / 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 / Retrovirus capsid, C-terminal / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / 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 / Arc Repressor Mutant, subunit A / 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 / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Gag-Pro-Pol polyprotein
Similarity search - Component
Biological speciesMouse mammary tumor virus
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.72 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 / Database references / Category: database_2 / pdbx_audit_support
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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

Downloads & links

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Assembly

Deposited unit
A: Pol polyprotein
B: Pol polyprotein
C: Pol polyprotein
D: Pol polyprotein
E: Pol polyprotein
F: Pol polyprotein
G: Pol polyprotein
H: Pol polyprotein
I: Pol polyprotein
J: Pol polyprotein
K: Pol polyprotein
L: Pol polyprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)286,95624
Polymers286,41712
Non-polymers53812
Water00
1
A: Pol polyprotein
B: Pol polyprotein
G: Pol polyprotein
H: Pol polyprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)95,6528
Polymers95,4724
Non-polymers1794
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
C: Pol polyprotein
D: Pol polyprotein
K: Pol polyprotein
L: Pol polyprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)95,6528
Polymers95,4724
Non-polymers1794
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
E: Pol polyprotein
F: Pol polyprotein
I: Pol polyprotein
J: Pol polyprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)95,6528
Polymers95,4724
Non-polymers1794
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)54.370, 83.150, 141.140
Angle α, β, γ (deg.)90.00, 90.19, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein
Pol polyprotein


Mass: 23868.123 Da / Num. of mol.: 12 / Fragment: UNP residues 1437-1646
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mouse mammary tumor virus (strain BR6) / Strain: BR6 / Gene: pol / Plasmid: pCPH6P / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P03365, RNA-directed DNA polymerase, DNA-directed DNA polymerase, ribonuclease H, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, Hydrolases; Acting on ester bonds
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg
#3: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Zn

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.25 Å3/Da / Density % sol: 45.38 % / Description: needles, rods
Crystal growTemperature: 291.15 K / Method: vapor diffusion, hanging drop / pH: 7.4
Details: 17% (w/v) PEG3350, 0.2M MgCl2, 4% (w/v) 1-Butyl-3-methylimidazolium dicyanamide

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.97726 Å
DetectorType: PSI PILATUS 6M / Detector: PIXEL / Date: Jul 25, 2013
RadiationMonochromator: Si111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97726 Å / Relative weight: 1
Reflection twin
Crystal-IDIDOperatorDomain-IDFraction
11H, K, L10.865
11-h,-k,l20.135
ReflectionResolution: 2.72→70.57 Å / Num. obs: 33784 / % possible obs: 99.3 % / Redundancy: 3.2 % / Rmerge(I) obs: 0.087 / Net I/σ(I): 9.5
Reflection shellResolution: 2.72→2.79 Å / Redundancy: 3.1 % / Rmerge(I) obs: 0.534 / Mean I/σ(I) obs: 2 / % possible all: 99

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155: ???)refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5CZ1 and 3F9K

5cz1

3f9k


Resolution: 2.72→70.57 Å / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 33.63 / Stereochemistry target values: TWIN_LSQ_F
RfactorNum. reflection% reflectionSelection details
Rfree0.2799 1671 4.95 %RANDOM
Rwork0.2413 ---
obs0.243 33771 99.29 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.72→70.57 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8579 0 12 0 8591
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0028839
X-RAY DIFFRACTIONf_angle_d0.37911949
X-RAY DIFFRACTIONf_dihedral_angle_d10.5795153
X-RAY DIFFRACTIONf_chiral_restr0.0361338
X-RAY DIFFRACTIONf_plane_restr0.0031527
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.7207-2.80070.3981430.33022618X-RAY DIFFRACTION94
2.8007-2.89110.34131250.31942682X-RAY DIFFRACTION95
2.8911-2.99430.32291440.30192682X-RAY DIFFRACTION95
2.9943-3.11420.32851260.27642663X-RAY DIFFRACTION95
3.1142-3.25580.28731440.26352663X-RAY DIFFRACTION95
3.2558-3.42730.31471530.25362676X-RAY DIFFRACTION94
3.4273-3.64190.27041460.23842669X-RAY DIFFRACTION94
3.6419-3.92270.26351360.22662652X-RAY DIFFRACTION94
3.9227-4.31690.24261160.20172666X-RAY DIFFRACTION95
4.3169-4.94010.22491520.19532678X-RAY DIFFRACTION94
4.9401-6.21860.25721260.22672681X-RAY DIFFRACTION95
6.2186-35.6380.30461440.26062746X-RAY DIFFRACTION94
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.1321-0.11490.56483.2445-0.09963.3673-0.1591-0.55130.1521-0.1143-0.0076-0.30970.19060.5530.11020.23190.12490.05050.3892-0.01310.5001-24.043321.9815-12.8282
21.8195-0.17330.04514.22780.21332.5901-0.3525-0.5501-0.05990.24450.17710.56650.10780.0540.16760.23020.13150.02880.4465-0.02670.5128-43.64921.7893-5.7399
31.94760.0560.23672.8422-0.56344.2428-0.28660.3898-0.5581-0.14150.1809-0.15840.08070.68890.08480.2366-0.02740.10830.3809-0.070.5611-5.6194-3.1722-42.5792
41.20480.13920.393.8079-1.05033.5348-0.11970.4148-0.7167-0.44770.41660.42060.5386-0.5218-0.28160.373-0.16210.04680.4797-0.10980.8406-25.2625-9.6051-46.0171
51.58510.2889-0.2373.91040.55153.0189-0.24640.86530.51090.14180.4277-0.4218-0.20130.185-0.15580.3272-0.1057-0.02220.54920.15360.538812.449335.1131-49.4942
61.5066-0.9695-0.71732.26310.55171.896-0.28010.91250.62580.0333-0.15760.0178-0.2478-0.15170.28390.3-0.0861-0.14620.82920.40820.9157-7.512740.4222-51.3011
70.20260.1930.10361.05390.2050.7075-0.0237-0.02790.1519-0.19010.0692-0.2608-0.24370.1014-0.02940.7066-0.1067-0.09690.1414-0.07250.8356-33.076944.768-21.6582
80.350.10990.15270.75261.34212.4670.0592-0.0749-0.7870.1825-0.02290.84171.0276-0.5038-0.01950.8051-0.04940.07340.37110.12941.1852-40.7854-2.3037-14.3004
94.28310.67140.01731.83532.14632.657-0.1779-0.18650.74670.2797-0.05650.5471-0.5106-0.63840.14450.53090.0801-0.07120.489-0.14990.8682-5.196745.8538-28.3558
100.16370.2152-0.25051.5909-0.31591.88510.00290.2411-0.0338-0.33110.0395-0.09840.2250.5548-0.04490.8057-0.38860.10231.2571-0.26890.41733.573316.0161-65.1025
110.1295-0.48250.06432.5526-0.42890.07950.10510.34720.1835-0.58610.00210.119-0.28250.0999-0.05520.8121-0.14050.11251.28030.30330.6443-22.94749.2235-63.634
121.1588-1.09320.04112.4648-0.05011.56410.0071-0.1378-0.130.48630.0409-0.20620.11860.0724-0.0110.65980.19560.14050.27190.19280.5586-14.228-6.7605-18.5444
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1(chain A and resid 51:209)
2X-RAY DIFFRACTION2(chain B and resid 56:209)
3X-RAY DIFFRACTION3(chain C and resid 50:209)
4X-RAY DIFFRACTION4(chain D and resid 55:209)
5X-RAY DIFFRACTION5(chain E and resid 51:209)
6X-RAY DIFFRACTION6(chain F and resid 56:208)
7X-RAY DIFFRACTION7(chain G and resid 1:41)
8X-RAY DIFFRACTION8(chain H and resid 2:41)
9X-RAY DIFFRACTION9(chain I and resid 2:41)
10X-RAY DIFFRACTION10(chain J and resid 1:41)
11X-RAY DIFFRACTION11(chain K and resid 2:41)
12X-RAY DIFFRACTION12(chain L and resid 1:41)

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