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

2026-04-08 REL RCSB INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY DATABASE_REFERENCES citation citation_author em_admin _citation.country _citation.journal_abbrev _citation.journal_id_CSD _citation.journal_id_ISSN _citation.pdbx_database_id_DOI _citation.pdbx_database_id_PubMed _citation.title _citation.year _citation_author.identifier_ORCID _em_admin.last_update EXPERIMENTAL_SUMMARY citation citation_author em_admin _citation.country _citation.journal_abbrev _citation.journal_id_CSD _citation.journal_id_ISSN _citation.pdbx_database_id_DOI _citation.pdbx_database_id_PubMed _citation.title _citation.year _citation_author.identifier_ORCID _em_admin.last_update DATABASE_REFERENCES citation em_admin _citation.journal_volume _em_admin.last_update EXPERIMENTAL_SUMMARY citation em_admin _citation.journal_volume _em_admin.last_update RCSB RCSB 2025-04-02 2026-02-25 2026-02-25 2026-04-08 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 CryoEM structure of M. mazei topoisomerase VI(A-E342Q)-minicircle DNA complex in cleavage state Richman DE Wendorff TJ Rashid F Beck C Yan Q Johnson HR Eckerty RA Fogg JM Baker ML Zechiedrich L Berger JM TOPRIM, GHKL, minicircle DNA, Spo11, ISOMERASE-DNA complex Richman DE Wendorff TJ Rashid F Beck C Yan Q Johnson HR Eckerty RA Fogg JM Baker ML Zechiedrich L Berger JM Supercoiled DNA recognition and cleavage control in topoisomerase VI. Nat Commun UK 17 2026 41698922 doi:10.1038/s41467-026-69491-0 2041-1723 9o0g FULLOVERLAP EMDB EMD-49972 associated EM volume

Topoisomerase VI heterotetramer (A2B2) bound to minicircle DNA and ADPNP Topoisomerase VI heterotetramer (A2B2) bound to minicircle DNA and ADPNP 0 1 2 3 4 5 6

Top6A subunits contain E342Q mutation

0.2 Top6 heterotetramer (A2B2) 1 1 2

Top6A subunits contain E342Q mutation

Methanosarcina mazei Go1 ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88 306 bp minicircle DNA 1 3 4 5 6

Approximately 80 bp of the 306 bp minicircle are visible in EM imaging of the Top6-DNA complex

unidentified Type 2 DNA topoisomerase 6 subunit A Methanosarcina mazei Go1 ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88 0.042178309 2 Escherichia coli BL21(DE3) LEVO MEGEKGSKTRKGDALAREKLLEIAEKIYNQFEEEVVPSVSLPSRTKANLEYSDESDVWVYGDRESERSAKTVKGAFQLLK TTYATDFLINEHLARNRGSTLRELYYISEGWDYAKFKEQGESDRLIEDLEILTSLQREYFHMRPEEDGATMFGPIEITEQ TKRGERNIHCQKDVGEGGYQIPFNVENIEFQKHDASMIIAIETGGMYARLMENGFDEAYNAILVHLKGQPARSTRRIIKR MNEELGIPVAVFTDGDPWSYRIYASVAYGAIKSAHLSEFMATPAAKFLGLQPSDIVEYELSTDKLTEQDVSALRSELSDP RFESDYWKEQIQLQLDIGKKAQQQAFAGKGLDFVTEVYLPNRLKEMGMI Q8PUB7 5.6.2.2 Type 2 DNA topoisomerase 6 subunit B Methanosarcina mazei Go1 ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88 0.068853367 2 Escherichia coli BL21(DE3) LEVO METPIAEELAKKQKSISVAEFFEKNRQILGFDSAPRSLITTVKEAVDNALDACEEAGILPDILVQVERTGPDYVTVIIED NGPGIVREQIPKVFAKLLYGSRFHALKQSRGQQGIGISAAVLYAQMTAGRHTKILSKTSPTAPAHYYELMINTSTNEPDI LVDEVRDWFRPHGTQIELEMRAAYVKGRRQSIYEYLKATAIVNPHARITLIDPDGNEEVFERATDKMPEPAEEILPHPEG IELGTLMKMLHYTERQKLAPFLRYSFCKIGLLTAEEICKAAGLDPEIDPHALGRHEARKLIEAFEKVKIMAPPTDCLSPI GEDLIYRGLEKETTVDFIATSTRKPAVYSGNPFVVEVGMAYGGNLPKEEKISIMRFANRVPLLYQQGGCVTTHAVEDIKW KQYGLNQPGGGIPVGPVILLIHVASINVPFTSESKDAIADIPVIKEEIDLAIKEVARKLKHYLSKQSNLKKRREKEIIIT KVLPKLAAKVAHVLEKDVPDINPVVAKIMGNLLVHRVIKNNGDGTVDVAIKVKNFGTSAYSFRVHEMLPCKVSGAKPEPK VVTMGNDYDYVWDISASAGSSKVLSYKIESASEEELQKLPQLIVEGIEEELVTGAKAFKGV Q8PUB8 5.6.2.2 Minicircle DNA sequence unidentified 0.010781896000000001 1 (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) DNA Minicircle DNA sequence unidentified 0.012011685999999999 1 (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) DNA Minicircle DNA sequence unidentified 0.011360259 1 (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) DNA Minicircle DNA sequence unidentified 0.011402311 1 (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) DNA CALCIUM ION 4.0078e-05 4 CA PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER 0.000506196 2 ANP POTASSIUM ION 3.9098e-05 2 K singleParticle particle 0.1 7.5 20.0 C8H18N2O4S HEPES 50.0 C5H8KNO4 potassium glutamate 10.0 CaCl2 calcium chloride 1.0 C3H8O3 glycerol 1.0 C9H15O6P TCEP 0.25 C29H32O13 etoposide 1.0 C10H17N6O12P3 ADPNP

Etoposide was not observed in the structure

Au-flat 1.2/1.3 GOLD 300 CARBON CONTINUOUS 4.0 GOLD HOLEY 45.0 GLOW DISCHARGE 15 AIR 0.035

Current set to 15 milliamp

ETHANE 100 283 FEI VITROBOT MARK IV

Vitrification carried out in air.

Summary of order of assembly: minicircle DNA, Top6, then additional buffer components, ending with ADPNP. Sample was soluble and monodisperse.

TFS KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 50.0 2.7 0.8 0.8 1.8 1.8 130000.0 FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN TFS Selectris 10 TFS FALCON 4i (4k x 4k) 4096 4096 1 26728 4.0 30.0 1 2479118 cryoSPARC 4.4.1 PHASE FLIPPING AND AMPLITUDE CORRECTION

Ab initio reconstruction

FOURIER SPACE 2.5 FSC 0.143 CUT-OFF cryoSPARC 4.6.0 EMReady 2.0 329731 MAXIMUM LIKELIHOOD cryoSPARC 4.4.1 MAXIMUM LIKELIHOOD cryoSPARC 4.6.0 emd_49972.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 360 360 360 0 0 0 360 360 360 334.8 334.8 334.8 90.0 90.0 90.0 X Y Z -0.12787469 27.282429 -0.013860122 0.4856933 0.92999995 0.92999995 0.92999995 4.5 AUTHOR ::::EMDATABANK.org::::EMD-49972:::: EMReady2 sharpened map derived from CryoSPARC raw map Other other

ModelAngelo

2Q2E PDB experimental model FLEXIBLE FIT Coot 0.9.8.91 REAL emd_49972_msk_1.map emd_49972_additional_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 360 360 360 0 0 0 360 360 360 334.8 334.8 334.8 90.0 90.0 90.0 X Y Z -1.4625255 2.6302097 0.00039170185 0.04263063 0.92999995 0.92999995 0.92999995 AUTHOR ::::EMDATABANK.org::::EMD-49972:::: CryoSPARC auto-sharpened map emd_49972_half_map_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 360 360 360 0 0 0 360 360 360 334.8 334.8 334.8 90.0 90.0 90.0 X Y Z -0.560132 1.3182684 0.000682538 0.065674536 0.92999995 0.92999995 0.92999995 AUTHOR ::::EMDATABANK.org::::EMD-49972:::: emd_49972_half_map_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 360 360 360 0 0 0 360 360 360 334.8 334.8 334.8 90.0 90.0 90.0 X Y Z -0.5495515 1.1899918 0.0006826625 0.06565803 0.92999995 0.92999995 0.92999995 AUTHOR ::::EMDATABANK.org::::EMD-49972::::