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- EMDB-49972: CryoEM structure of M. mazei topoisomerase VI(A-E342Q)-minicircle... -

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Basic information

Entry
Database: EMDB / ID: EMD-49972
TitleCryoEM structure of M. mazei topoisomerase VI(A-E342Q)-minicircle DNA complex in cleavage state
Map dataEMReady2 sharpened map derived from CryoSPARC raw map
Sample
  • Complex: Topoisomerase VI heterotetramer (A2B2) bound to minicircle DNA and ADPNP
    • Complex: Top6 heterotetramer (A2B2)
      • Protein or peptide: Type 2 DNA topoisomerase 6 subunit A
      • Protein or peptide: Type 2 DNA topoisomerase 6 subunit B
    • Complex: 306 bp minicircle DNA
      • DNA: Minicircle DNA sequence
      • DNA: Minicircle DNA sequence
      • DNA: Minicircle DNA sequence
      • DNA: Minicircle DNA sequence
  • Ligand: CALCIUM ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: POTASSIUM ION
KeywordsTOPRIM / 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
Type 2 DNA topoisomerase 6 subunit A / Type 2 DNA topoisomerase 6 subunit B
Similarity search - Component
Biological speciesMethanosarcina mazei Go1 (archaea) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsRichman DE / Wendorff TJ / Rashid F / Beck C / Yan Q / Johnson HR / Eckerty RA / Fogg JM / Baker ML / Zechiedrich L / Berger JM
Funding support United States, 2 items
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 DE / Wendorff TJ / Rashid F / Beck C / Yan Q / Johnson HR / Eckerty RA / Fogg JM / Baker ML / Zechiedrich L / Berger JM
History
DepositionApr 2, 2025-
Header (metadata) releaseFeb 25, 2026-
Map releaseFeb 25, 2026-
UpdateFeb 25, 2026-
Current statusFeb 25, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49972.png

Downloads & links

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Map

FileDownload / File: emd_49972.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEMReady2 sharpened map derived from CryoSPARC raw map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 360 pix.
= 334.8 Å		0.93 Å/pix.
x 360 pix.
= 334.8 Å		0.93 Å/pix.
x 360 pix.
= 334.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.5
Minimum - Maximum-0.12787469 - 27.282429
Average (Standard dev.)-0.013860122 (±0.4856933)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 334.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_49972_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: CryoSPARC auto-sharpened map

Fileemd_49972_additional_1.map
AnnotationCryoSPARC auto-sharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_49972_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_49972_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Topoisomerase VI heterotetramer (A2B2) bound to minicircle DNA an...

EntireName: Topoisomerase VI heterotetramer (A2B2) bound to minicircle DNA and ADPNP
Components
  • Complex: Topoisomerase VI heterotetramer (A2B2) bound to minicircle DNA and ADPNP
    • Complex: Top6 heterotetramer (A2B2)
      • Protein or peptide: Type 2 DNA topoisomerase 6 subunit A
      • Protein or peptide: Type 2 DNA topoisomerase 6 subunit B
    • Complex: 306 bp minicircle DNA
      • DNA: Minicircle DNA sequence
      • DNA: Minicircle DNA sequence
      • DNA: Minicircle DNA sequence
      • DNA: Minicircle DNA sequence
  • Ligand: CALCIUM ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: POTASSIUM ION

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Supramolecule #1: Topoisomerase VI heterotetramer (A2B2) bound to minicircle DNA an...

SupramoleculeName: Topoisomerase VI heterotetramer (A2B2) bound to minicircle DNA and ADPNP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6 / Details: Top6A subunits contain E342Q mutation
Molecular weightTheoretical: 200 KDa

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Supramolecule #2: Top6 heterotetramer (A2B2)

SupramoleculeName: Top6 heterotetramer (A2B2) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2 / Details: Top6A subunits contain E342Q mutation
Source (natural)Organism: Methanosarcina mazei Go1 (archaea)
Strain: ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88

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Supramolecule #3: 306 bp minicircle DNA

SupramoleculeName: 306 bp minicircle DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#6
Details: Approximately 80 bp of the 306 bp minicircle are visible in EM imaging of the Top6-DNA complex
Source (natural)Organism: unidentified (others)

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Macromolecule #1: Type 2 DNA topoisomerase 6 subunit A

MacromoleculeName: Type 2 DNA topoisomerase 6 subunit A / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA topoisomerase (ATP-hydrolysing)
Source (natural)Organism: Methanosarcina mazei Go1 (archaea)
Strain: ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88
Molecular weightTheoretical: 42.178309 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MEGEKGSKTR KGDALAREKL LEIAEKIYNQ FEEEVVPSVS LPSRTKANLE YSDESDVWVY GDRESERSAK TVKGAFQLLK TTYATDFLI NEHLARNRGS TLRELYYISE GWDYAKFKEQ GESDRLIEDL EILTSLQREY FHMRPEEDGA TMFGPIEITE Q TKRGERNI ...String:
MEGEKGSKTR KGDALAREKL LEIAEKIYNQ FEEEVVPSVS LPSRTKANLE YSDESDVWVY GDRESERSAK TVKGAFQLLK TTYATDFLI NEHLARNRGS TLRELYYISE GWDYAKFKEQ GESDRLIEDL EILTSLQREY FHMRPEEDGA TMFGPIEITE Q TKRGERNI HCQKDVGEGG YQIPFNVENI EFQKHDASMI IAIETGGMYA RLMENGFDEA YNAILVHLKG QPARSTRRII KR MNEELGI PVAVFTDGDP WSYRIYASVA YGAIKSAHLS EFMATPAAKF LGLQPSDIVE YELSTDKLTE QDVSALRSEL SDP RFESDY WKEQIQLQLD IGKKAQQQAF AGKGLDFVTE VYLPNRLKEM GMI

UniProtKB: Type 2 DNA topoisomerase 6 subunit A

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Macromolecule #2: Type 2 DNA topoisomerase 6 subunit B

MacromoleculeName: Type 2 DNA topoisomerase 6 subunit B / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA topoisomerase (ATP-hydrolysing)
Source (natural)Organism: Methanosarcina mazei Go1 (archaea)
Strain: ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88
Molecular weightTheoretical: 68.853367 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: METPIAEELA KKQKSISVAE FFEKNRQILG FDSAPRSLIT TVKEAVDNAL DACEEAGILP DILVQVERTG PDYVTVIIED NGPGIVREQ IPKVFAKLLY GSRFHALKQS RGQQGIGISA AVLYAQMTAG RHTKILSKTS PTAPAHYYEL MINTSTNEPD I LVDEVRDW ...String:
METPIAEELA KKQKSISVAE FFEKNRQILG FDSAPRSLIT TVKEAVDNAL DACEEAGILP DILVQVERTG PDYVTVIIED NGPGIVREQ IPKVFAKLLY GSRFHALKQS RGQQGIGISA AVLYAQMTAG RHTKILSKTS PTAPAHYYEL MINTSTNEPD I LVDEVRDW FRPHGTQIEL EMRAAYVKGR RQSIYEYLKA TAIVNPHARI TLIDPDGNEE VFERATDKMP EPAEEILPHP EG IELGTLM KMLHYTERQK LAPFLRYSFC KIGLLTAEEI CKAAGLDPEI DPHALGRHEA RKLIEAFEKV KIMAPPTDCL SPI GEDLIY RGLEKETTVD FIATSTRKPA VYSGNPFVVE VGMAYGGNLP KEEKISIMRF ANRVPLLYQQ GGCVTTHAVE DIKW KQYGL NQPGGGIPVG PVILLIHVAS INVPFTSESK DAIADIPVIK EEIDLAIKEV ARKLKHYLSK QSNLKKRREK EIIIT KVLP KLAAKVAHVL EKDVPDINPV VAKIMGNLLV HRVIKNNGDG TVDVAIKVKN FGTSAYSFRV HEMLPCKVSG AKPEPK VVT MGNDYDYVWD ISASAGSSKV LSYKIESASE EELQKLPQLI VEGIEEELVT GAKAFKGV

UniProtKB: Type 2 DNA topoisomerase 6 subunit B

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Macromolecule #3: Minicircle DNA sequence

MacromoleculeName: Minicircle DNA sequence / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 10.781896 KDa
SequenceString:
(DA)(DG)(DG)(DT)(DT)(DA)(DC)(DC)(DC)(DC) (DA)(DG)(DT)(DT)(DG)(DG)(DG)(DG)(DC)(DG) (DG)(DG)(DC)(DC)(DC)(DC)(DT)(DT)(DG) (DT)(DA)(DC)(DA)(DG)(DC)

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Macromolecule #4: Minicircle DNA sequence

MacromoleculeName: Minicircle DNA sequence / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 12.011686 KDa
SequenceString:
(DT)(DC)(DG)(DT)(DC)(DC)(DA)(DT)(DG)(DC) (DC)(DG)(DA)(DG)(DA)(DG)(DT)(DG)(DA)(DT) (DC)(DC)(DC)(DG)(DG)(DC)(DG)(DG)(DC) (DG)(DG)(DT)(DC)(DA)(DC)(DG)(DA)(DA)(DC)

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Macromolecule #5: Minicircle DNA sequence

MacromoleculeName: Minicircle DNA sequence / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 11.360259 KDa
SequenceString:
(DG)(DT)(DT)(DC)(DG)(DT)(DG)(DA)(DC)(DC) (DG)(DC)(DC)(DG)(DC)(DC)(DG)(DG)(DG)(DA) (DT)(DC)(DA)(DC)(DT)(DC)(DT)(DC)(DG) (DG)(DC)(DA)(DT)(DG)(DG)(DA)(DC)

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Macromolecule #6: Minicircle DNA sequence

MacromoleculeName: Minicircle DNA sequence / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 11.402311 KDa
SequenceString:
(DG)(DA)(DG)(DC)(DT)(DG)(DT)(DA)(DC)(DA) (DA)(DG)(DG)(DG)(DG)(DC)(DC)(DC)(DG)(DC) (DC)(DC)(DC)(DA)(DA)(DC)(DT)(DG)(DG) (DG)(DG)(DT)(DA)(DA)(DC)(DC)(DT)

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Macromolecule #7: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 7 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #8: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 8 / Number of copies: 2 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #9: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 9 / Number of copies: 2 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
50.0 mMC5H8KNO4potassium glutamate
10.0 mMCaCl2calcium chloride
1.0 percent (v/v)C3H8O3glycerol
1.0 mMC9H15O6PTCEP
0.25 mMC29H32O13etoposide
1.0 mMC10H17N6O12P3ADPNP

Details: Etoposide was not observed in the structure
GridModel: Au-flat 1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: CONTINUOUS / Support film - #0 - Film thickness: 4 / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: GOLD / Support film - #1 - topology: HOLEY / Support film - #1 - Film thickness: 45 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.035 kPa / Details: Current set to 15 milliamp
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV / Details: Vitrification carried out in air.
DetailsSummary of order of assembly: minicircle DNA, Top6, then additional buffer components, ending with ADPNP. Sample was soluble and monodisperse.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 26728 / Average exposure time: 4.0 sec. / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 1.8 µm / Calibrated defocus min: 0.8 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2479118
CTF correctionSoftware - Name: cryoSPARC (ver. 4.4.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: Ab initio reconstruction
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF
Software: (Name: cryoSPARC (ver. 4.6.0), EMReady (ver. 2.0))
Number images used: 329731
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
ChainDetailsPDB ID
source_name: Other, initial_model_type: otherModelAngelo
source_name: PDB, initial_model_type: experimental model

2q2e

SoftwareName: Coot (ver. 0.9.8.91)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9o0g:
CryoEM structure of M. mazei topoisomerase VI(A-E342Q)-minicircle DNA complex in cleavage state

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