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- PDB-9o0g: CryoEM structure of M. mazei topoisomerase VI(A-E342Q)-minicircle... -

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Basic information

Entry
Database: PDB / ID: 9o0g
TitleCryoEM structure of M. mazei topoisomerase VI(A-E342Q)-minicircle DNA complex in cleavage state
Components
  • (Minicircle DNA ...) x 4
  • (Type 2 DNA topoisomerase 6 subunit ...) x 2
KeywordsISOMERASE/DNA / TOPRIM / GHKL / minicircle DNA / Spo11 / ISOMERASE-DNA complex
Function / homology
Function and homology information


DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / DNA topological change / chromosome / magnesium ion binding / DNA binding / ATP binding
Similarity search - Function
DNA topoisomerase VI, subunit B, C-terminal / Type 2 DNA topoisomerase 6 subunit B C-terminal domain / DNA topoisomerase VI, subunit A / : / All-beta domain in DNA topoisomerase VI alpha subunit / Spo11/DNA topoisomerase VI subunit A / Spo11/DNA topoisomerase VI, subunit A, N-terminal / Topoisomerase 6 subunit A/Spo11, TOPRIM domain / Spo11/DNA topoisomerase VI subunit A superfamily / Type IIB DNA topoisomerase ...DNA topoisomerase VI, subunit B, C-terminal / Type 2 DNA topoisomerase 6 subunit B C-terminal domain / DNA topoisomerase VI, subunit A / : / All-beta domain in DNA topoisomerase VI alpha subunit / Spo11/DNA topoisomerase VI subunit A / Spo11/DNA topoisomerase VI, subunit A, N-terminal / Topoisomerase 6 subunit A/Spo11, TOPRIM domain / Spo11/DNA topoisomerase VI subunit A superfamily / Type IIB DNA topoisomerase / Topoisomerase 6 subunit A/Spo11, Toprim domain / Topoisomerase (Topo) IIB-type catalytic domain profile. / DNA topoisomerase VI, subunit B / DNA topoisomerase VI, subunit B, transducer / Topoisomerase VI B subunit, transducer / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase superfamily / Ribosomal protein S13-like, H2TH / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / : / DNA / DNA (> 10) / Type 2 DNA topoisomerase 6 subunit A / Type 2 DNA topoisomerase 6 subunit B
Similarity search - Component
Biological speciesMethanosarcina mazei Go1 (archaea)
unidentified (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsRichman, D.E. / Berger, J.M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)5R35 CA263778 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1F32 GM128269 United States
CitationJournal: To Be Published
Title: Supercoiled DNA recognition and cleavage control in topoisomerase VI
Authors: Richman, D.E. / Wendorff, T.J. / Rashid, F. / Beck, C. / Yan, Q. / Johnson, H.R. / Eckerty, R.A. / Fogg, J.M. / Baker, M.L. / Zechiedrich, L. / Berger, J.M.
History
DepositionApr 2, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 25, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Type 2 DNA topoisomerase 6 subunit A
B: Type 2 DNA topoisomerase 6 subunit B
C: Type 2 DNA topoisomerase 6 subunit A
D: Type 2 DNA topoisomerase 6 subunit B
F: Minicircle DNA sequence
G: Minicircle DNA sequence
H: Minicircle DNA sequence
I: Minicircle DNA sequence
hetero molecules


Theoretical massNumber of molelcules
Total (without water)268,87016
Polymers267,6208
Non-polymers1,2518
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Type 2 DNA topoisomerase 6 subunit ... , 2 types, 4 molecules ACBD

#1: Protein Type 2 DNA topoisomerase 6 subunit A / Type II DNA topoisomerase VI subunit A


Mass: 42178.309 Da / Num. of mol.: 2 / Mutation: E342Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Methanosarcina mazei Go1 (archaea)
Strain: ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88
Gene: top6A, MM_2418 / Plasmid: pST39 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q8PUB7, DNA topoisomerase (ATP-hydrolysing)
#2: Protein Type 2 DNA topoisomerase 6 subunit B / Type II DNA topoisomerase VI subunit B / TopoVI-B


Mass: 68853.367 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Methanosarcina mazei Go1 (archaea)
Strain: ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88
Gene: top6B, MM_2417 / Plasmid: pST39 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q8PUB8, DNA topoisomerase (ATP-hydrolysing)

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Minicircle DNA ... , 4 types, 4 molecules FGHI

#3: DNA chain Minicircle DNA sequence


Mass: 10781.896 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli (E. coli) / Strain (production host): ZYCY10P3S2T
#4: DNA chain Minicircle DNA sequence


Mass: 12011.686 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Plasmid: pUC / Production host: Escherichia coli (E. coli) / Strain (production host): ZYCY10P3S2T
#5: DNA chain Minicircle DNA sequence


Mass: 11360.259 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli (E. coli) / Strain (production host): ZYCY10P3S2T
#6: DNA chain Minicircle DNA sequence


Mass: 11402.311 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli (E. coli) / Strain (production host): ZYCY10P3S2T

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Non-polymers , 3 types, 8 molecules

#7: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
#8: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#9: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K

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Details

Has ligand of interestN
Has protein modificationN

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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
IDNameTypeDetailsEntity IDParent-IDSource
1Topoisomerase VI heterotetramer (A2B2) bound to minicircle DNA and ADPNPCOMPLEXTop6A subunits contain E342Q mutation#1-#60MULTIPLE SOURCES
2Top6 heterotetramer (A2B2)COMPLEXTop6A subunits contain E342Q mutation#1-#21RECOMBINANT
3306 bp minicircle DNACOMPLEXApproximately 80 bp of the 306 bp minicircle are visible in EM imaging of the Top6-DNA complex#3-#61RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.42 MDaNO
210.22 MDaYES
310.2 MDaYES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
32Methanosarcina mazei Go1 (archaea)192952ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88
43unidentified (others)32644
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
32Escherichia coli BL21(DE3) (bacteria)469008pST39
43Escherichia coli (E. coli)562ZYCY10P3S2T
Buffer solutionpH: 7.5 / Details: Etoposide was not observed in the structure
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
250 mMpotassium glutamateC5H8KNO41
310 mMcalcium chlorideCaCl21
41 percent (v/v)glycerolC3H8O31
51 mMTCEPC9H15O6P1
60.25 mMetoposideC29H32O131
71 mMADPNPC10H17N6O12P31
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Summary of order of assembly: minicircle DNA, Top6, then additional buffer components, ending with ADPNP. Sample was soluble and monodisperse.
Specimen supportDetails: Current set to 15 milliamp / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Au-flat 1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K / Details: Vitrification carried out in air

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 1800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 30 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 26728
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1crYOLO1.9.6particle selection
4cryoSPARC4.4.1CTF correction
7Coot0.9.8.91model fitting
8UCSF ChimeraX1.9model fitting
9ISOLDE1.9model fitting
11PHENIX1.21.1_5286model refinement
12cryoSPARC4.4.1initial Euler assignment
13cryoSPARC4.6.0final Euler assignment
15cryoSPARC4.6.03D reconstruction
16EMReady23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2479118
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 329731 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDDetailsSource nameTypeAccession codeInitial refinement model-ID
1ModelAngeloOtherother
22Q2E

2q2e

PDBexperimental model2Q2E2
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 74.27 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003718990
ELECTRON MICROSCOPYf_angle_d0.738326202
ELECTRON MICROSCOPYf_chiral_restr0.04782799
ELECTRON MICROSCOPYf_plane_restr0.00572881
ELECTRON MICROSCOPYf_dihedral_angle_d18.46717526

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