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- PDB-9ud1: Cryo-EM structure of STITCHR (R2Tg and Cas9 nickase) bound to gui... -

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Basic information

Entry
Database: PDB / ID: 9ud1
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
Components
  • (DNA non-target strand (bound to ...) x 2
  • CRISPR-associated endonuclease Cas9
  • DNA non target strand (priming strand bound to R2)
  • R2Tg 3'UTR RNA
  • Reverse transcriptase-like protein
  • guide RNA
KeywordsDNA BINDING PROTEIN / retrotransposon / LINE / reverse transcriptase / endonuclease / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex
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
THYMIDINE-5'-TRIPHOSPHATE / : / DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesTaeniopygia guttata (zebra finch)
Streptococcus pyogenes (bacteria)
Cloning vector pET-J23100-lacO-GFP-LVA
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKomatsu, D. / Hiraizumi, M. / Fujimatsu, K. / Yamada, S. / Yamashita, K. / Nishimasu, H.
Funding support Japan, 1items
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, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 14, 2026Provider: repository / Type: Initial release
Revision 1.0Oct 14, 2026Data content type: EM metadata / Data content type: EM metadata / 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: Reverse transcriptase-like protein
B: R2Tg 3'UTR RNA
C: DNA non-target strand (bound to R2)
D: DNA non-target strand (bound to R2)
E: DNA non target strand (priming strand bound to R2)
F: CRISPR-associated endonuclease Cas9
G: guide RNA
H: DNA non-target strand (bound to R2)
I: DNA non-target strand (bound to R2)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)424,31516
Polymers423,5239
Non-polymers7927
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 2 molecules AF

#1: Protein Reverse transcriptase-like protein


Mass: 146346.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Taeniopygia guttata (zebra finch) / Production host: Spodoptera frugiperda (fall armyworm)
#6: Protein CRISPR-associated endonuclease Cas9


Mass: 158457.578 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Gene: dcas9, cas9 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A386IRG9, Hydrolases; Acting on ester bonds

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RNA chain , 2 types, 2 molecules BG

#2: RNA chain R2Tg 3'UTR RNA


Mass: 25219.021 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Taeniopygia guttata (zebra finch)
#7: RNA chain guide RNA


Mass: 31607.787 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria)
Production host: in vitro transcription vector pT7-TP(deltai) (others)

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DNA non-target strand (bound to ... , 2 types, 4 molecules CHDI

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


Mass: 18388.766 Da / Num. of mol.: 2 / Source method: obtained synthetically
Source: (synth.) Cloning vector pET-J23100-lacO(SymR+1)-GFP-LVA (others)
References: GenBank: 1820520299
#4: DNA chain DNA non-target strand (bound to R2)


Mass: 6273.067 Da / Num. of mol.: 2 / Source method: obtained synthetically
Source: (synth.) Cloning vector pET-J23100-lacO(SymR+1)-GFP-LVA (others)

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DNA chain , 1 types, 1 molecules E

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


Mass: 12568.069 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Cloning vector pET-J23100-lacO(SymR+1)-GFP-LVA (others)

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Non-polymers , 3 types, 7 molecules

#8: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE


Mass: 482.168 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O14P3 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
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

ComponentName: Cas9-R2Tg-guide RNA-R2 RNA-DNA complex / Type: COMPLEX / Entity ID: #1-#3, #5-#7 / Source: RECOMBINANT
Source (natural)Organism: Taeniopygia guttata (zebra finch)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
4cryoSPARCCTF correction
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11RELIONclassification
12cryoSPARC3D reconstruction
13Servalcatmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35803 / Symmetry type: POINT
RefinementResolution: 3.3→3.3 Å / Num. reflection obs: 2132770 / Average fsc work: 0.6748
Displacement parametersBiso mean: 109.68 Å2
Refine LS restraints
Refine-IDTypeDev idealNumberWeight
ELECTRON MICROSCOPYs_bond_nonh_d0.0049258550.0116
ELECTRON MICROSCOPYs_angle_nonh_deg1.3919362191.8423
ELECTRON MICROSCOPYs_dihedral_angle_1_deg5.894327315.1589
ELECTRON MICROSCOPYs_dihedral_angle_2_deg3.60378355
ELECTRON MICROSCOPYs_dihedral_angle_3_deg13.0699520510
ELECTRON MICROSCOPYs_dihedral_angle_6_deg15.2948101410
ELECTRON MICROSCOPYs_chiral_restr0.141343560.1447
ELECTRON MICROSCOPYs_planes0.0044310420.02
ELECTRON MICROSCOPYs_nbd0.1958318650.2
ELECTRON MICROSCOPYs_nbtor0.2119384840.2
ELECTRON MICROSCOPYs_hbond_nbd0.14915670.2
LS refinement shell
Resolution (Å)Refine-IDNum. reflection obsFsc work
3.3-3.337ELECTRON MICROSCOPY693250.3884
3.337-3.37ELECTRON MICROSCOPY610450.4166
3.371-3.405ELECTRON MICROSCOPY611490.4422
3.405-3.44ELECTRON MICROSCOPY593830.4664
3.44-3.476ELECTRON MICROSCOPY579450.4815
3.476-3.513ELECTRON MICROSCOPY561610.4935
3.513-3.55ELECTRON MICROSCOPY553630.5155
3.551-3.589ELECTRON MICROSCOPY542730.5463
3.589-3.628ELECTRON MICROSCOPY531730.5774
3.628-3.668ELECTRON MICROSCOPY518850.5922
3.669-3.709ELECTRON MICROSCOPY512950.6076
3.71-3.751ELECTRON MICROSCOPY498450.6211
3.751-3.794ELECTRON MICROSCOPY486370.6358
3.794-3.838ELECTRON MICROSCOPY472390.6435
3.838-3.883ELECTRON MICROSCOPY465690.6495
3.883-3.929ELECTRON MICROSCOPY457690.6615
3.929-3.976ELECTRON MICROSCOPY440670.6815
3.976-4.024ELECTRON MICROSCOPY432730.6979
4.024-4.073ELECTRON MICROSCOPY423330.7117
4.074-4.124ELECTRON MICROSCOPY415930.7232
4.124-4.176ELECTRON MICROSCOPY400150.7288
4.176-4.229ELECTRON MICROSCOPY391890.7338
4.229-4.283ELECTRON MICROSCOPY378610.7413
4.284-4.34ELECTRON MICROSCOPY374550.7461
4.34-4.397ELECTRON MICROSCOPY363730.756
4.397-4.456ELECTRON MICROSCOPY350130.7646
4.456-4.517ELECTRON MICROSCOPY348490.7591
4.517-4.579ELECTRON MICROSCOPY333430.7616
4.579-4.643ELECTRON MICROSCOPY328650.7715
4.643-4.709ELECTRON MICROSCOPY312850.7783
4.709-4.776ELECTRON MICROSCOPY306030.7835
4.777-4.846ELECTRON MICROSCOPY303130.7819
4.847-4.918ELECTRON MICROSCOPY287410.7859
4.919-4.991ELECTRON MICROSCOPY284550.7897
4.993-5.068ELECTRON MICROSCOPY271570.79
5.069-5.147ELECTRON MICROSCOPY270450.7865
5.147-5.228ELECTRON MICROSCOPY257770.7849
5.23-5.311ELECTRON MICROSCOPY244350.783
5.312-5.397ELECTRON MICROSCOPY242770.7765
5.399-5.487ELECTRON MICROSCOPY233170.7713
5.488-5.579ELECTRON MICROSCOPY228510.7677
5.58-5.674ELECTRON MICROSCOPY217450.7646
5.675-5.773ELECTRON MICROSCOPY210330.761
5.774-5.875ELECTRON MICROSCOPY207130.7602
5.876-5.981ELECTRON MICROSCOPY196470.7672
5.982-6.091ELECTRON MICROSCOPY188410.7675
6.092-6.204ELECTRON MICROSCOPY182170.7622
6.206-6.323ELECTRON MICROSCOPY177270.7593
6.324-6.446ELECTRON MICROSCOPY171250.7551
6.448-6.572ELECTRON MICROSCOPY161250.7496
6.575-6.706ELECTRON MICROSCOPY158710.7522
6.707-6.844ELECTRON MICROSCOPY152250.7648
6.846-6.988ELECTRON MICROSCOPY145330.7719
6.99-7.139ELECTRON MICROSCOPY137530.7801
7.14-7.294ELECTRON MICROSCOPY130590.7843
7.297-7.459ELECTRON MICROSCOPY129730.7977
7.463-7.631ELECTRON MICROSCOPY120210.8114
7.633-7.808ELECTRON MICROSCOPY117310.8156
7.812-7.998ELECTRON MICROSCOPY109810.8129
8.001-8.196ELECTRON MICROSCOPY107250.8139
8.198-8.403ELECTRON MICROSCOPY101170.8153
8.406-8.621ELECTRON MICROSCOPY93750.8198
8.624-8.848ELECTRON MICROSCOPY91330.8239
8.854-9.093ELECTRON MICROSCOPY85770.8257
9.097-9.349ELECTRON MICROSCOPY82270.8333
9.353-9.616ELECTRON MICROSCOPY75970.841
9.624-9.907ELECTRON MICROSCOPY73770.8417
9.912-10.212ELECTRON MICROSCOPY69010.8432
10.217-10.536ELECTRON MICROSCOPY63030.8549
10.541-10.881ELECTRON MICROSCOPY60730.8687
10.887-11.243ELECTRON MICROSCOPY56130.8726
11.256-11.644ELECTRON MICROSCOPY52750.8788
11.651-12.067ELECTRON MICROSCOPY49810.8897
12.075-12.513ELECTRON MICROSCOPY45330.8984
12.531-13.012ELECTRON MICROSCOPY43030.9027
13.022-13.543ELECTRON MICROSCOPY40170.8997
13.554-14.118ELECTRON MICROSCOPY35650.902
14.131-14.745ELECTRON MICROSCOPY33010.9088
14.759-15.413ELECTRON MICROSCOPY29910.9173
15.446-16.181ELECTRON MICROSCOPY28570.9163
16.2-17.009ELECTRON MICROSCOPY25170.9091
17.054-17.9ELECTRON MICROSCOPY21790.9087
17.952-18.948ELECTRON MICROSCOPY20850.9238
18.979-20.094ELECTRON MICROSCOPY18610.9274
20.13-21.386ELECTRON MICROSCOPY16690.927
21.52-22.856ELECTRON MICROSCOPY13110.9251
22.91-24.475ELECTRON MICROSCOPY12490.9337
24.609-26.496ELECTRON MICROSCOPY10890.9351
26.667-28.788ELECTRON MICROSCOPY9070.9232
28.897-31.232ELECTRON MICROSCOPY7290.9175
31.655-34.803ELECTRON MICROSCOPY6250.9239
34.996-38.858ELECTRON MICROSCOPY5710.9076
39.127-43.974ELECTRON MICROSCOPY3810.9069
44.365-50.63ELECTRON MICROSCOPY3010.934
51.229-58.69ELECTRON MICROSCOPY2250.9216
60.615-72.448ELECTRON MICROSCOPY1750.8989
74.237-92.08ELECTRON MICROSCOPY1050.9371
95.84-117.38ELECTRON MICROSCOPY490.9272
135.538-332ELECTRON MICROSCOPY400.8478

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