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- EMDB-64058: Cryo-EM structure of STITCHR (R2Tg and Cas9 nickase) bound to gui... -

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Basic information

Entry
Database: EMDB / ID: EMD-64058
TitleCryo-EM structure of STITCHR (R2Tg and Cas9 nickase) bound to guide RNA, template RNA, and target DNA in the target-primed reverse transcription initiation state
Map data
Sample
  • Complex: Cas9-R2Tg-guide RNA-R2 RNA-DNA complex
    • Protein or peptide: Reverse transcriptase-like protein
    • RNA: R2Tg 3'UTR RNA
    • DNA: DNA non-target strand (bound to R2)
    • DNA: DNA non target strand (priming strand bound to R2)
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: guide RNA
  • DNA: DNA non-target strand (bound to R2)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
Keywordsretrotransposon / LINE / reverse transcriptase / endonuclease / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex / DNA BINDING PROTEIN
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / metal ion binding / DNA binding / RNA binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesTaeniopygia guttata (zebra finch) / Streptococcus pyogenes (bacteria) / Cloning vector pET-J23100-lacO(SymR+1)-GFP-LVA (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKomatsu D / Hiraizumi M / Fujimatsu K / Yamada S / Yamashita K / Nishimasu H
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Science and Technology Japan
CitationJournal: To Be Published
Title: Structure mechanism of the avian R2 retrotransposon for programmable gene integration
Authors: Komatsu D / Nishimasu H
History
DepositionApr 6, 2025-
Header (metadata) releaseOct 14, 2026-
Map releaseOct 14, 2026-
UpdateOct 14, 2026-
Current statusOct 14, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_64058.png

Downloads & links

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Map

FileDownload / File: emd_64058.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.38 Å/pix.
x 240 pix.
= 332. Å		1.38 Å/pix.
x 240 pix.
= 332. Å		1.38 Å/pix.
x 240 pix.
= 332. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.38333 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.47007078 - 1.0871264
Average (Standard dev.)-0.0002480194 (±0.029769102)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 331.9999 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_64058_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_64058_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_64058_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 : Cas9-R2Tg-guide RNA-R2 RNA-DNA complex

EntireName: Cas9-R2Tg-guide RNA-R2 RNA-DNA complex
Components
  • Complex: Cas9-R2Tg-guide RNA-R2 RNA-DNA complex
    • Protein or peptide: Reverse transcriptase-like protein
    • RNA: R2Tg 3'UTR RNA
    • DNA: DNA non-target strand (bound to R2)
    • DNA: DNA non target strand (priming strand bound to R2)
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: guide RNA
  • DNA: DNA non-target strand (bound to R2)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE

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Supramolecule #1: Cas9-R2Tg-guide RNA-R2 RNA-DNA complex

SupramoleculeName: Cas9-R2Tg-guide RNA-R2 RNA-DNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3, #5-#7
Source (natural)Organism: Taeniopygia guttata (zebra finch)

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Macromolecule #1: Reverse transcriptase-like protein

MacromoleculeName: Reverse transcriptase-like protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Taeniopygia guttata (zebra finch)
Molecular weightTheoretical: 146.346734 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSYYHHHHHH DGASWSHPQF EKGGGSGGGS GGSAWSHPQF EKGGSTTLEV LFQGPELFVP EEGSSEKESE DAPKTSPPTP GKHGLEQTG EEKVMVTVPD KNPPCPCCGT RVNSVLNLIE HLKVSHGKRG VCFRCAKCGK ENSNYHSVVC HFPKCRGPET E KAPAGEWI ...String:
MSYYHHHHHH DGASWSHPQF EKGGGSGGGS GGSAWSHPQF EKGGSTTLEV LFQGPELFVP EEGSSEKESE DAPKTSPPTP GKHGLEQTG EEKVMVTVPD KNPPCPCCGT RVNSVLNLIE HLKVSHGKRG VCFRCAKCGK ENSNYHSVVC HFPKCRGPET E KAPAGEWI CEVCNRDFTT KIGLGQHKRL AHPAVRNQER IVASQPKETS NRGAHKRCWT KEEEELLIRL EAQFEGNKNI NK LIAEHIT TKTAKQISDK RRLLSRKPAE EPREEPGTCH HTRRAAASLR TEPEMSHHAQ AEDRDNGPGR RPLPGRAAAG GRT MDEIRR HPDKGNGQQR PTKQKSEEQL QAYYKKTLEE RLSAGALNTF PRAFKQVMEG RDIKLVINQT AQDCFGCLES ISQI RTATR DKKDTVTREK HPKKPFQKWM KDRAIKKGNY LRFQRLFYLD RGKLAKIILD DIECLSCDIP LSEIYSVFKT RWETT GSFK SLGDFKTYGK ADNTAFRELI TAKEIEKNVQ EMSKGSAPGP DGITLGDVVK MDPEFSRTME IFNLWLTTGK IPDMVR GCR TVLIPKSSKP DRLKDINNWR PITIGSILLR LFSRIVTARL SKACPLNPRQ RGFIRAAGCS ENLKLLQTII WSAKREH RP LGVVFVDIAK AFDTVSHQHI IHALQQREVD PHIVGLVSNM YENISTYITT KRNTHTDKIQ IRVGVKQGDP MSPLLFNL A MDPLLCKLEE SGKGYHRGQS SITAMAFADD LVLLSDSWEN MNTNISILET FCNLTGLKTQ GQKCHGFYIK PTKDSYTIN DCAAWTINGT PLNMIDPGES EKYLGLQFDP WIGIARSGLS TKLDFWLQRI DQAPLKPLQK TDILKTYTIP RLIYIADHSE VKTALLETL DQKIRTAVKE WLHLPPCTCD AILYSSTRDG GLGITKLAGL IPSVQARRLH RIAQSSDDTM KCFMEKEKME Q LHKKLWIQ AGGDRENIPS IWEAPPSSEP PNNVSTNSEW EAPTQKDKFP KPCNWRKNEF KKWTKLASQG RGIVNFERDK IS NHWIQYY RRIPHRKLLT ALQLRANVYP TREFLARGRQ DQYIKACRHC DADIESCAHI IGNCPVTQDA RIKRHNYICE LLL EEAKKK DWVVFKEPHI RDSNKELYKP DLIFVKDARA LVVDVTVRYE AAKSSLEEAA AEKVRKYKHL ETEVRHLTNA KDVT FVGFP LGARGKWHQD NFKLLTELGL SKSRQVKMAE TFSTVALFSS VDIVHMFASR ARKSMVMGGS DYKDHDGDYK DHDID YKDD DDK

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Macromolecule #6: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 158.457578 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: DKKYSIGLAI GTNSVGWAVI TDEYKVPSKK FKVLGNTDRH SIKKNLIGAL LFDSGETAEA TRLKRTARRR YTRRKNRICY LQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH M IKFRGHFL ...String:
DKKYSIGLAI GTNSVGWAVI TDEYKVPSKK FKVLGNTDRH SIKKNLIGAL LFDSGETAEA TRLKRTARRR YTRRKNRICY LQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH M IKFRGHFL IEGDLNPDNS DVDKLFIQLV QTYNQLFEEN PINASGVDAK AILSARLSKS RRLENLIAQL PGEKKNGLFG NL IALSLGL TPNFKSNFDL AEDAKLQLSK DTYDDDLDNL LAQIGDQYAD LFLAAKNLSD AILLSDILRV NTEITKAPLS ASM IKRYDE HHQDLTLLKA LVRQQLPEKY KEIFFDQSKN GYAGYIDGGA SQEEFYKFIK PILEKMDGTE ELLVKLNRED LLRK QRTFD NGSIPHQIHL GELHAILRRQ EDFYPFLKDN REKIEKILTF RIPYYVGPLA RGNSRFAWMT RKSEETITPW NFEEV VDKG ASAQSFIERM TNFDKNLPNE KVLPKHSLLY EYFTVYNELT KVKYVTEGMR KPAFLSGEQK KAIVDLLFKT NRKVTV KQL KEDYFKKIEC FDSVEISGVE DRFNASLGTY HDLLKIIKDK DFLDNEENED ILEDIVLTLT LFEDREMIEE RLKTYAH LF DDKVMKQLKR RRYTGWGRLS RKLINGIRDK QSGKTILDFL KSDGFANRNF MQLIHDDSLT FKEDIQKAQV SGQGDSLH E HIANLAGSPA IKKGILQTVK VVDELVKVMG RHKPENIVIE MARENQTTQK GQKNSRERMK RIEEGIKELG SQILKEHPV ENTQLQNEKL YLYYLQNGRD MYVDQELDIN RLSDYDVDAI VPQSFLKDDS IDNKVLTRSD KNRGKSDNVP SEEVVKKMKN YWRQLLNAK LITQRKFDNL TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS K LVSDFRKD FQFYKVREIN NYHHAHDAYL NAVVGTALIK KYPKLESEFV YGDYKVYDVR KMIAKSEQEI GKATAKYFFY SN IMNFFKT EITLANGEIR KRPLIETNGE TGEIVWDKGR DFATVRKVLS MPQVNIVKKT EVQTGGFSKE SILPKRNSDK LIA RKKDWD PKKYGGFDSP TVAYSVLVVA KVEKGKSKKL KSVKELLGIT IMERSSFEKN PIDFLEAKGY KEVKKDLIIK LPKY SLFEL ENGRKRMLAS AGELQKGNEL ALPSKYVNFL YLASHYEKLK GSPEDNEQKQ LFVEQHKHYL DEIIEQISEF SKRVI LADA NLDKVLSAYN KHRDKPIREQ AENIIHLFTL TNLGAPAAFK YFDTTIDRKR YTSTKEVLDA TLIHQSITGL YETRID LSQ LGGD

UniProtKB: CRISPR-associated endonuclease Cas9

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Macromolecule #2: R2Tg 3'UTR RNA

MacromoleculeName: R2Tg 3'UTR RNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Taeniopygia guttata (zebra finch)
Molecular weightTheoretical: 25.219021 KDa
SequenceString:
GGAGGUGGAC GGGCCACCUU UACUUAACCC GGAAAAGGAA CAUAUAUAAU UUAUGUGUGU UCGAUAAAGG GAAACCUG

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Macromolecule #7: guide RNA

MacromoleculeName: guide RNA / type: rna / ID: 7 / Number of copies: 1
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 31.607787 KDa
SequenceString:
GGCACGACAG GUUUCCCGAC GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAG UCGGUGCUU

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Macromolecule #3: DNA non-target strand (bound to R2)

MacromoleculeName: DNA non-target strand (bound to R2) / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Cloning vector pET-J23100-lacO(SymR+1)-GFP-LVA (others)
Molecular weightTheoretical: 18.388766 KDa
SequenceString: (DT)(DC)(DA)(DC)(DT)(DG)(DC)(DC)(DC)(DG) (DC)(DT)(DT)(DT)(DC)(DC)(DA)(DG)(DT)(DC) (DG)(DG)(DG)(DA)(DA)(DA)(DC)(DC)(DT) (DG)(DT)(DC)(DG)(DT)(DG)(DC)(DC)(DA)(DG) (DC) (DT)(DG)(DC)(DA)(DT)(DT) ...String:
(DT)(DC)(DA)(DC)(DT)(DG)(DC)(DC)(DC)(DG) (DC)(DT)(DT)(DT)(DC)(DC)(DA)(DG)(DT)(DC) (DG)(DG)(DG)(DA)(DA)(DA)(DC)(DC)(DT) (DG)(DT)(DC)(DG)(DT)(DG)(DC)(DC)(DA)(DG) (DC) (DT)(DG)(DC)(DA)(DT)(DT)(DA)(DA) (DT)(DG)(DA)(DA)(DT)(DC)(DG)(DG)(DC)(DC) (DA)(DA)

GENBANK: GENBANK: MN865131.1

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Macromolecule #4: DNA non-target strand (bound to R2)

MacromoleculeName: DNA non-target strand (bound to R2) / type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Cloning vector pET-J23100-lacO(SymR+1)-GFP-LVA (others)
Molecular weightTheoretical: 6.273067 KDa
SequenceString:
(DG)(DA)(DC)(DT)(DG)(DG)(DA)(DA)(DA)(DG) (DC)(DG)(DG)(DG)(DC)(DA)(DG)(DT)(DG)(DA)

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Macromolecule #5: DNA non target strand (priming strand bound to R2)

MacromoleculeName: DNA non target strand (priming strand bound to R2) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Cloning vector pET-J23100-lacO(SymR+1)-GFP-LVA (others)
Molecular weightTheoretical: 12.568069 KDa
SequenceString:
(DT)(DT)(DG)(DG)(DC)(DC)(DG)(DA)(DT)(DT) (DC)(DA)(DT)(DT)(DA)(DA)(DT)(DG)(DC)(DA) (DG)(DC)(DT)(DG)(DG)(DC)(DA)(DC)(DG) (DA)(DC)(DA)(DG)(DG)(DT)(DT)(DT)(DC)(DC) (DC) (2DT)

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #10: THYMIDINE-5'-TRIPHOSPHATE

MacromoleculeName: THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 10 / Number of copies: 1 / Formula: TTP
Molecular weightTheoretical: 482.168 Da
Chemical component information

ChemComp-TTP:
THYMIDINE-5'-TRIPHOSPHATE

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 35803
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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