[English] 日本語
Yorodumi
- PDB-8hhm: Cryo-EM structure of the Cas12m2-crRNA-target DNA ternary complex... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 8hhm
TitleCryo-EM structure of the Cas12m2-crRNA-target DNA ternary complex intermediate state
Components
  • (DNA (36-MER)) x 2
  • Cas12m2
  • RNA (56-MER)
KeywordsRNA BINDING PROTEIN / CRISPR-Cas / RNA BINDING PROTEIN-DNA COMPLEX
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesMycolicibacterium mucogenicum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsOmura, N.S. / Nakagawa, R. / Wu, Y.W. / Sudfeld, C. / Warren, V.R. / Hirano, H. / Kusakizako, T. / Kise, Y. / Lebbink, H.G.J. / Itoh, Y. ...Omura, N.S. / Nakagawa, R. / Wu, Y.W. / Sudfeld, C. / Warren, V.R. / Hirano, H. / Kusakizako, T. / Kise, Y. / Lebbink, H.G.J. / Itoh, Y. / Oost, V.D.J. / Nureki, O.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED) Japan
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Mechanistic and evolutionary insights into a type V-M CRISPR-Cas effector enzyme.
Authors: Satoshi N Omura / Ryoya Nakagawa / Christian Südfeld / Ricardo Villegas Warren / Wen Y Wu / Hisato Hirano / Charlie Laffeber / Tsukasa Kusakizako / Yoshiaki Kise / Joyce H G Lebbink / ...Authors: Satoshi N Omura / Ryoya Nakagawa / Christian Südfeld / Ricardo Villegas Warren / Wen Y Wu / Hisato Hirano / Charlie Laffeber / Tsukasa Kusakizako / Yoshiaki Kise / Joyce H G Lebbink / Yuzuru Itoh / John van der Oost / Osamu Nureki /
Abstract: RNA-guided type V CRISPR-Cas12 effectors provide adaptive immunity against mobile genetic elements (MGEs) in bacteria and archaea. Among diverse Cas12 enzymes, the recently identified Cas12m2 (CRISPR- ...RNA-guided type V CRISPR-Cas12 effectors provide adaptive immunity against mobile genetic elements (MGEs) in bacteria and archaea. Among diverse Cas12 enzymes, the recently identified Cas12m2 (CRISPR-Cas type V-M) is highly compact and has a unique RuvC active site. Although the non-canonical RuvC triad does not permit dsDNA cleavage, Cas12m2 still protects against invading MGEs through transcriptional silencing by strong DNA binding. However, the molecular mechanism of RNA-guided genome inactivation by Cas12m2 remains unknown. Here we report cryo-electron microscopy structures of two states of Cas12m2-CRISPR RNA (crRNA)-target DNA ternary complexes and the Cas12m2-crRNA binary complex, revealing structural dynamics during crRNA-target DNA heteroduplex formation. The structures indicate that the non-target DNA strand is tightly bound to a unique arginine-rich cluster in the recognition (REC) domains and the non-canonical active site in the RuvC domain, ensuring strong DNA-binding affinity of Cas12m2. Furthermore, a structural comparison of Cas12m2 with TnpB, a putative ancestor of Cas12 enzymes, suggests that the interaction of the characteristic coiled-coil REC2 insertion with the protospacer-adjacent motif-distal region of the heteroduplex is crucial for Cas12m2 to engage in adaptive immunity. Collectively, our findings improve mechanistic understanding of diverse type V CRISPR-Cas effectors and provide insights into the evolution of TnpB to Cas12 enzymes.
History
DepositionNov 16, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 12, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 19, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year
Revision 1.2Aug 9, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.3Aug 30, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 2.0May 22, 2024Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Database references / Derived calculations / Polymer sequence / Source and taxonomy / Structure summary
Category: atom_site / em_entity_assembly ...atom_site / em_entity_assembly / entity / entity_poly / entity_poly_seq / entity_src_gen / ndb_struct_na_base_pair / ndb_struct_na_base_pair_step / pdbx_entity_instance_feature / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_polymer_linkage / struct_asym / struct_conn / struct_ref_seq
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_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.group_PDB / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.label_seq_id / _atom_site.type_symbol / _em_entity_assembly.entity_id_list / _entity_poly.pdbx_seq_one_letter_code / _entity_poly.pdbx_seq_one_letter_code_can / _entity_src_gen.pdbx_end_seq_num / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_unobs_or_zero_occ_residues.auth_comp_id / _pdbx_unobs_or_zero_occ_residues.auth_seq_id / _pdbx_unobs_or_zero_occ_residues.label_comp_id / _pdbx_unobs_or_zero_occ_residues.label_seq_id / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_end / _struct_ref_seq.seq_align_end

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
B: RNA (56-MER)
C: DNA (36-MER)
D: DNA (36-MER)
A: Cas12m2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)107,3266
Polymers107,2364
Non-polymers902
Water00
1


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

-
Components

-
DNA chain , 2 types, 2 molecules CD

#2: DNA chain DNA (36-MER)


Mass: 10976.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mycolicibacterium mucogenicum (bacteria)
#3: DNA chain DNA (36-MER)


Mass: 11180.160 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mycolicibacterium mucogenicum (bacteria)

-
RNA chain / Protein , 2 types, 2 molecules BA

#1: RNA chain RNA (56-MER)


Mass: 18623.137 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium mucogenicum (bacteria)
Production host: Escherichia coli (E. coli)
#4: Protein Cas12m2


Mass: 66457.000 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium mucogenicum (bacteria)
Production host: Escherichia coli (E. coli)

-
Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

-
Details

Has ligand of interestY

-
Experimental details

-
Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

-
Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Cas12m2-crRNA-target DNACOMPLEX#2-#40MULTIPLE SOURCES
2crRNA-target DNACOMPLEX#2-#31MULTIPLE SOURCES
3Cas12m2COMPLEX#41RECOMBINANT
4crRNACOMPLEX#12RECOMBINANT
5target DNACOMPLEX#2-#32SYNTHETIC
Molecular weightExperimental value: NO
Source (natural)Organism: Mycolicibacterium mucogenicum (bacteria)
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
23Escherichia coli (E. coli)562
34Escherichia coli (E. coli)562
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

-
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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

-
Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 180921 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0045821
ELECTRON MICROSCOPYf_angle_d0.518234
ELECTRON MICROSCOPYf_dihedral_angle_d17.7761364
ELECTRON MICROSCOPYf_chiral_restr0.036951
ELECTRON MICROSCOPYf_plane_restr0.005803

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more