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- PDB-7yoj: Structure of CasPi with guide RNA and target DNA -

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Basic information

Entry
Database: PDB / ID: 7yoj
TitleStructure of CasPi with guide RNA and target DNA
Components
  • CasPi
  • DNA (30-MER)
  • DNA (5'-D(P*CP*GP*GP*GP*AP*TP*GP*CP*CP*CP*AP*G)-3')
  • RNA (174-MER)
KeywordsRNA BINDING PROTEIN/DNA/RNA / CasPi complex / RNA BINDING PROTEIN / RNA BINDING PROTEIN-DNA-RNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / Transposase
Function and homology information
Biological speciesArmatimonadota bacterium (bacteria)
Armatimonadetes bacterium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsLi, C.P. / Wang, J. / Liu, J.J.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Cell Res / Year: 2023
Title: The compact Casπ (Cas12l) 'bracelet' provides a unique structural platform for DNA manipulation.
Authors: Ao Sun / Cheng-Ping Li / Zhihang Chen / Shouyue Zhang / Dan-Yuan Li / Yun Yang / Long-Qi Li / Yuqian Zhao / Kaichen Wang / Zhaofu Li / Jinxia Liu / Sitong Liu / Jia Wang / Jun-Jie Gogo Liu /
Abstract: CRISPR-Cas modules serve as the adaptive nucleic acid immune systems for prokaryotes, and provide versatile tools for nucleic acid manipulation in various organisms. Here, we discovered a new ...CRISPR-Cas modules serve as the adaptive nucleic acid immune systems for prokaryotes, and provide versatile tools for nucleic acid manipulation in various organisms. Here, we discovered a new miniature type V system, CRISPR-Casπ (Cas12l) (~860 aa), from the environmental metagenome. Complexed with a large guide RNA (~170 nt) comprising the tracrRNA and crRNA, Casπ (Cas12l) recognizes a unique 5' C-rich PAM for DNA cleavage under a broad range of biochemical conditions, and generates gene editing in mammalian cells. Cryo-EM study reveals a 'bracelet' architecture of Casπ effector encircling the DNA target at 3.4 Å resolution, substantially different from the canonical 'two-lobe' architectures of Cas12 and Cas9 nucleases. The large guide RNA serves as a 'two-arm' scaffold for effector assembly. Our study expands the knowledge of DNA targeting mechanisms by CRISPR effectors, and offers an efficient but compact platform for DNA manipulation.
History
DepositionAug 1, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 15, 2023Provider: repository / Type: Initial release
Revision 2.0Mar 22, 2023Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Data processing / Database references / Derived calculations / Polymer sequence / Refinement description / Source and taxonomy / Structure summary
Category: atom_site / em_3d_reconstruction ...atom_site / em_3d_reconstruction / em_ctf_correction / em_image_processing / em_image_recording / em_single_particle_entity / em_software / entity / entity_name_com / entity_poly / entity_poly_seq / entity_src_gen / pdbx_contact_author / pdbx_poly_seq_scheme / pdbx_unobs_or_zero_occ_atoms / refine / struct_ref_seq_dif / struct_sheet_range
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_comp_id / _atom_site.label_comp_id / _em_image_recording.avg_electron_dose_per_image / _entity.formula_weight / _entity.pdbx_mutation / _entity_poly.pdbx_seq_one_letter_code / _entity_poly.pdbx_seq_one_letter_code_can / _entity_poly_seq.mon_id / _entity_src_gen.pdbx_gene_src_scientific_name / _pdbx_poly_seq_scheme.mon_id / _pdbx_poly_seq_scheme.pdb_mon_id / _refine.B_iso_mean / _refine.ls_d_res_high / _refine.pdbx_stereochemistry_target_values / _struct_sheet_range.end_auth_comp_id / _struct_sheet_range.end_label_comp_id
Description: Sequence discrepancy
Details: In the first version, we uploaded an incorrect primary sequence in which A537 and A643 were mistakenly identified as D537 and E643, respectively. These sequence mistakes have already been corrected.
Provider: author / Type: Coordinate replacement
Revision 2.1Mar 29, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 2.2Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CasPi
C: DNA (5'-D(P*CP*GP*GP*GP*AP*TP*GP*CP*CP*CP*AP*G)-3')
D: DNA (30-MER)
B: RNA (174-MER)


Theoretical massNumber of molelcules
Total (without water)168,0244
Polymers168,0244
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CasPi / Reverse transcriptase domain-containing protein


Mass: 98731.234 Da / Num. of mol.: 1 / Mutation: D537A, E643A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Armatimonadota bacterium (bacteria) / Gene: DCC45_12200, EDM73_08925 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A399WQY8
#2: DNA chain DNA (5'-D(P*CP*GP*GP*GP*AP*TP*GP*CP*CP*CP*AP*G)-3')


Mass: 3688.405 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Armatimonadetes bacterium (bacteria) / Production host: Escherichia coli (E. coli)
#3: DNA chain DNA (30-MER)


Mass: 9192.878 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Armatimonadetes bacterium (bacteria) / Production host: Escherichia coli (E. coli)
#4: RNA chain RNA (174-MER)


Mass: 56411.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Armatimonadetes bacterium (bacteria) / Production host: Escherichia coli (E. coli)

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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: CasPi / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.17728 MDa / Experimental value: NO
Source (natural)Organism: Armatimonadetes bacterium (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 1000 nm / Cs: 0.01 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
Image processing
IDImage recording-ID
11
21
CTF correction
IDEM image processing-IDType
11PHASE FLIPPING ONLY
22PHASE FLIPPING ONLY
Symmetry
IDImage processing-IDEntry-IDPoint symmetry
117YOJC1 (asymmetric)
217YOJC1 (asymmetric)
317YOJC1 (asymmetric)
417YOJC1 (asymmetric)
517YOJC1 (asymmetric)
617YOJC1 (asymmetric)
717YOJC1 (asymmetric)
817YOJC1 (asymmetric)
917YOJC1 (asymmetric)
1017YOJC1 (asymmetric)
1117YOJC1 (asymmetric)
1217YOJC1 (asymmetric)
1317YOJC1 (asymmetric)
1417YOJC1 (asymmetric)
1517YOJC1 (asymmetric)
1617YOJC1 (asymmetric)
1717YOJC1 (asymmetric)
1817YOJC1 (asymmetric)
1917YOJC1 (asymmetric)
2017YOJC1 (asymmetric)
2117YOJC1 (asymmetric)
2217YOJC1 (asymmetric)
2317YOJC1 (asymmetric)
2417YOJC1 (asymmetric)
2517YOJC1 (asymmetric)
2617YOJC1 (asymmetric)
2717YOJC1 (asymmetric)
2817YOJC1 (asymmetric)
2917YOJC1 (asymmetric)
3017YOJC1 (asymmetric)
3117YOJC1 (asymmetric)
3217YOJC1 (asymmetric)
3317YOJC1 (asymmetric)
3417YOJC1 (asymmetric)
3517YOJC1 (asymmetric)
3617YOJC1 (asymmetric)
3717YOJC1 (asymmetric)
3817YOJC1 (asymmetric)
3917YOJC1 (asymmetric)
4017YOJC1 (asymmetric)
4117YOJC1 (asymmetric)
4217YOJC1 (asymmetric)
4317YOJC1 (asymmetric)
4417YOJC1 (asymmetric)
4517YOJC1 (asymmetric)
4617YOJC1 (asymmetric)
4717YOJC1 (asymmetric)
4817YOJC1 (asymmetric)
4917YOJC1 (asymmetric)
5017YOJC1 (asymmetric)
5117YOJC1 (asymmetric)
5217YOJC1 (asymmetric)
5317YOJC1 (asymmetric)
5417YOJC1 (asymmetric)
5517YOJC1 (asymmetric)
5617YOJC1 (asymmetric)
5717YOJC1 (asymmetric)
5817YOJC1 (asymmetric)
5917YOJC1 (asymmetric)
6017YOJC1 (asymmetric)
6117YOJC1 (asymmetric)
6217YOJC1 (asymmetric)
6317YOJC1 (asymmetric)
6417YOJC1 (asymmetric)
6527YOJC1 (asymmetric)
6627YOJC1 (asymmetric)
6727YOJC1 (asymmetric)
6827YOJC1 (asymmetric)
6927YOJC1 (asymmetric)
7027YOJC1 (asymmetric)
7127YOJC1 (asymmetric)
7227YOJC1 (asymmetric)
7327YOJC1 (asymmetric)
7427YOJC1 (asymmetric)
7527YOJC1 (asymmetric)
7627YOJC1 (asymmetric)
7727YOJC1 (asymmetric)
7827YOJC1 (asymmetric)
7927YOJC1 (asymmetric)
8027YOJC1 (asymmetric)
8127YOJC1 (asymmetric)
8227YOJC1 (asymmetric)
8327YOJC1 (asymmetric)
8427YOJC1 (asymmetric)
8527YOJC1 (asymmetric)
8627YOJC1 (asymmetric)
8727YOJC1 (asymmetric)
8827YOJC1 (asymmetric)
8927YOJC1 (asymmetric)
9027YOJC1 (asymmetric)
9127YOJC1 (asymmetric)
9227YOJC1 (asymmetric)
9327YOJC1 (asymmetric)
9427YOJC1 (asymmetric)
9527YOJC1 (asymmetric)
9627YOJC1 (asymmetric)
9727YOJC1 (asymmetric)
9827YOJC1 (asymmetric)
9927YOJC1 (asymmetric)
10027YOJC1 (asymmetric)
10127YOJC1 (asymmetric)
10227YOJC1 (asymmetric)
10327YOJC1 (asymmetric)
10427YOJC1 (asymmetric)
10527YOJC1 (asymmetric)
10627YOJC1 (asymmetric)
10727YOJC1 (asymmetric)
10827YOJC1 (asymmetric)
10927YOJC1 (asymmetric)
11027YOJC1 (asymmetric)
11127YOJC1 (asymmetric)
11227YOJC1 (asymmetric)
11327YOJC1 (asymmetric)
11427YOJC1 (asymmetric)
11527YOJC1 (asymmetric)
11627YOJC1 (asymmetric)
11727YOJC1 (asymmetric)
11827YOJC1 (asymmetric)
11927YOJC1 (asymmetric)
12027YOJC1 (asymmetric)
12127YOJC1 (asymmetric)
12227YOJC1 (asymmetric)
12327YOJC1 (asymmetric)
12427YOJC1 (asymmetric)
12527YOJC1 (asymmetric)
12627YOJC1 (asymmetric)
12727YOJC1 (asymmetric)
12827YOJC1 (asymmetric)
3D reconstruction

Algorithm: BACK PROJECTION / Entry-ID: 7YOJ / Num. of particles: 39500 / Resolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Symmetry type: POINT

IDImage processing-ID
11
22
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL
RefinementHighest resolution: 3.36 Å / Cross valid method: NONE
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00912258
ELECTRON MICROSCOPYf_angle_d1.18717603
ELECTRON MICROSCOPYf_dihedral_angle_d19.1843477
ELECTRON MICROSCOPYf_chiral_restr0.0462051
ELECTRON MICROSCOPYf_plane_restr0.0081476

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