2025-12-24 REL PDBe 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.journal_volume _citation.page_first _citation.page_last _citation_author.identifier_ORCID _em_admin.last_update EXPERIMENTAL_SUMMARY citation citation_author em_admin _citation.journal_volume _citation.page_first _citation.page_last _citation_author.identifier_ORCID _em_admin.last_update PDBe PDBe 2025-03-04 2025-12-17 2025-12-17 2025-12-24 Not funded Manakova EN Zaremba M Jurgelaitis E short prokaryotic Argonaute, bacterial defence system, DREN domain, DREN-APAZ, RNA BINDING PROTEIN Jurgelaitis E Zagorskaite E Kopustas A Asmontas S Manakova E Dalgediene I Tylenyte U Silanskas A Toliusis P Grybauskas A Tutkus M Venclovas C Zaremba M Cell Res. CN 35 1056 1078 2025 41298897 doi:10.1038/s41422-025-01198-1 1001-0602 9qcc FULLOVERLAP PDB 9QBQ unspecified

apo XauSPARDA

PDB 9QBL unspecified

apo XauSPARDA deltaDREN mutant

PDB 9QBP unspecified

apo EaeSPARDA

EMDB EMD-53008 associated EM volume

Activated XauSPARDA filament assembly with bound dsDNA substrate

Filament assembly of activated XauSPARDA binary monomers in the presence of dsDNA substrate Filament assembly of activated XauSPARDA binary monomers in the presence of dsDNA substrate 0 1 2 3 4 5 6

SPARDA binary monomers are activated upon binding of guide 5'p-RNA and complementary DNA target (component 1). Activated monomers oligomerise into filament-like structure, that supports the tetrameric assembly of DREN-domains (component 2) which unspecifically binds dsDNA substrate

Xanthobacter autotrophicus Py2 Activated XauSPARDA binary monomer containing bound 5'p-RNA guide and complementary DNA target 1 1 2 3 4

XauSPARDA binary monomer binds 5'p-RNA guide and complementary DNA target strand in the presence of calcium ion

Xanthobacter autotrophicus Py2 DREN-domains tetramer with bound dsDNA substrate 1 1 5 6

DREN-domains tetramer assembled upon oligomerization of activated binary monomers in the presence of dsDNA and calcium

Xanthobacter autotrophicus Py2 DUF4365 domain-containing protein Xanthobacter autotrophicus Py2 0.052420254

N-terminal purification His-tag

8 Escherichia coli LEVO MGGSHHHHHHGMASMSKRITDSQVLGELGETAIKKIVLETGFLYEQRGRLEAGTDGIIELRDPKSGAPLGKLLGVQVKST ESGQYVRENDNSFEYLLKPDDLKYWRTSNIPVIIVLWRKSDETAYWKDVSDCVRGEERRLKFDKGTDVFDPRSADRIGAL TIDRRTPGVFLPPLNKGEDAIINLLRIRLPDEIFISTSPFGSGRDAVPELVKHGNVRFDWVIRKRRFVSFFDPREYGTRA IVDLDQVEAVDTKLIAFNDEQDDLNDTMDLLRRTVERQTATQLSFLRKDRLFHFKAVGVGKSRSYRYMSNVNETSAKVVS AYSSKKKDGWGYVRHHAARLRFERLADEWFLVIDPDFHFTTDGFQPHRYPEALLAGKKRLERNAAVRGQVTMWQHLLVES GKHEVGLFDADKPAPLLQFERLPVIQLSQAVPESSWNRTDPRAKEMEAQDLFEEGGVG A7ICE8 Protein argonaute Xanthobacter autotrophicus Py2 0.053589663999999995 8 Escherichia coli BL21(DE3) LEVO MTFKAHVFDEPMLEFGDGGQHCDPRQGLREHGPLQPRSGDVIRVGVIGTDDTVAGFTEFLAETGRGIESGNKQLINLNPD FPGLGNQNPFRCKFEVPDGATVTISRRQVNDITGIGRHDEAVRHAVELISSQLSALVEGSAKPDVIVLALPIPLIEKLVN AKSEGEDSDDDVDGGDMLNFRDLLKAKTLHLPVPTQIVWPDTWDDAAKIPRKIKRDSNRQTQVKATRAWNLLNALFYKAG KVPWRLLPDQAEYRTSFLGIGFYRDLDGQQLWTSTAQMFDERGRGLILRGARAQTETRGRHPYLTAKDAEDLVVQSIAAY KAHHRHVPARLVVLKTSRFRSEEAEGIDAALGKSGIEMSDLVWVQESSPIAIFRDGNYPVLRGTFVDLDGKGLLYTRGSV PFYGTFPGLRVPRPLLLVPHENSDSTILTLAKDVLALTKVNWNTTQFDQKLPAPIKAAREVGRILKHVEFGTAVSSDFRR YT A7ICE9 DNA (5'-D(*AP*TP*TP*CP*GP*GP*CP*CP*GP*TP*GP*TP*AP*CP*AP*AP*T)-3') Escherichia coli 0.005202384

DNA target strand

8 (DA)(DT)(DT)(DC)(DG)(DG)(DC)(DC)(DG)(DT)(DG)(DT)(DA)(DC)(DA)(DA)(DT) DNA RNA (5'-R(P*AP*UP*UP*GP*UP*AP*CP*AP*CP*GP*GP*CP*CP*GP*AP*AP*U)-3') Escherichia coli 0.005427286

5'-P RNA guide strand

8 AUUGUACACGGCCGAAU dsDNA substrate, first strand Escherichia coli 0.012994325

first strand of dsDNA substrate

1 (DA)(DT)(DA)(DA)(DT)(DG)(DG)(DT)(DT)(DT)(DC)(DT)(DT)(DA)(DG)(DA)(DC)(DG)(DT)(DC) (DG)(DT)(DT)(DT)(DT)(DA)(DG)(DA)(DG)(DC)(DT)(DG)(DT)(DG)(DT)(DT)(DG)(DT)(DT)(DT) (DC)(DG) DNA dsDNA substrate, second strand Escherichia coli 0.012862349

complementary strand of dsDNA substrate

1 (DC)(DG)(DA)(DA)(DA)(DC)(DA)(DA)(DC)(DA)(DC)(DA)(DG)(DC)(DT)(DC)(DT)(DA)(DA)(DA) (DA)(DC)(DG)(DA)(DC)(DG)(DT)(DC)(DT)(DA)(DA)(DG)(DA)(DA)(DA)(DC)(DC)(DA)(DT)(DT) (DA)(DT) DNA CALCIUM ION 4.0078e-05 12 CA singleParticle particle 1.96 8.0 20.0 tris HCl 100.0 NaCl 2.0 CACl2 Quantifoil R1.2/1.3 COPPER 300 CARBON HOLEY ARRAY GLOW DISCHARGE 45 AIR ETHANE 95 277 FEI VITROBOT MARK IV

Solution containing activated XauSPARDA monomers was mixed with solution containing dsDNA substrate and centrifugated before applying on the grid.

TFS GLACIOS SPOT SCAN BRIGHT FIELD FIELD EMISSION GUN 200 100.0 2.7 1.0 2.0 92000.0 OTHER NITROGEN FEI FALCON III (4k x 4k) COUNTING 1 1946 46.33 30.0 1 6077663

Template picking

cryoSPARC v.4.2.1 PHASE FLIPPING AND AMPLITUDE CORRECTION

Ab-initio reconstruction after blob picking

1 C1 FOURIER SPACE 3.0 FSC 0.143 CUT-OFF cryoSPARC v.4.2.1

Local refinement within mask

204199 MAXIMUM LIKELIHOOD cryoSPARC v.4.2.1 MAXIMUM LIKELIHOOD cryoSPARC v.4.2.1 3 648.0 cryoSPARC v.4.2.1

Heterogenous refinement

9QBQ C PDB experimental model

apo XauSPARDA DREN-APAZ subunit

9QBQ D PDB experimental model

apo XauSPARDA Ago subunit

RIGID BODY FIT UCSF Chimera 1.17 Fitting of monomrs into map REAL 131.0 emd_53008_msk_1.map emd_53008_half_map_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 512 512 512 0 0 0 512 512 512 563.2 563.2 563.2 90.0 90.0 90.0 X Y Z -0.8580628 2.4155216 0.0009662456 0.1052807 1.1 1.1 1.1 AUTHOR ::::EMDATABANK.org::::EMD-53008:::: emd_53008_half_map_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 512 512 512 0 0 0 512 512 512 563.2 563.2 563.2 90.0 90.0 90.0 X Y Z -0.86720103 2.5983515 0.0009611784 0.10529034 1.1 1.1 1.1 AUTHOR ::::EMDATABANK.org::::EMD-53008::::