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- PDB-8kgf: Structure of AmCas12a with crRNA -

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Basic information

Entry
Database: PDB / ID: 8kgf
TitleStructure of AmCas12a with crRNA
Components
  • CRISPR-associated endonuclease Cas12a
  • RNA (44-MER)
KeywordsDNA BINDING PROTEIN/RNA / CRISPR/Cas12a / DNA binding protein / DNA BINDING PROTEIN-RNA complex
Function / homologyRNA / RNA (> 10)
Function and homology information
Biological speciesAnaeroglobus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsFeng, Y. / Zhang, X. / Shi, J. / Ma, P. / Tang, J. / Huang, X.
Funding support China, 2items
OrganizationGrant numberCountry
Other government2021YFA0804702
National Natural Science Foundation of China (NSFC)22177073 China
CitationJournal: Nat Commun / Year: 2025
Title: Discovery of CRISPR-Cas12a clades using a large language model.
Authors: Yuanyuan Feng / Junchao Shi / Zhanwei Li / Yongqian Li / Jiaxi Yang / Shisheng Huang / Jinfang Zheng / Wei Han / Yunbo Qiao / Jun Zhang / Qi Liu / Yao Yang / Chunyi Hu / Lina Wu / Xiaokang ...Authors: Yuanyuan Feng / Junchao Shi / Zhanwei Li / Yongqian Li / Jiaxi Yang / Shisheng Huang / Jinfang Zheng / Wei Han / Yunbo Qiao / Jun Zhang / Qi Liu / Yao Yang / Chunyi Hu / Lina Wu / Xiaokang Zhang / Jin Tang / Xingxu Huang / Peixiang Ma /
Abstract: CRISPR-Cas systems revolutionize life science. Metagenomes contain millions of unknown Cas proteins. Traditional mining relies on protein sequence alignments. In this work, we employ an evolutionary ...CRISPR-Cas systems revolutionize life science. Metagenomes contain millions of unknown Cas proteins. Traditional mining relies on protein sequence alignments. In this work, we employ an evolutionary scale language model (ESM) to learn the information beyond sequences. Trained with CRISPR-Cas data, ESM accurately identifies Cas proteins without alignment. Limited experimental data restricts feature prediction, but integrating with machine learning enables trans-cleavage activity prediction of uncharacterized Cas12a. We discover 7 undocumented Cas12a subtypes with unique CRISPR loci. Structural analyses reveal 8 subtypes of Cas1, Cas2, and Cas4. Cas12a subtypes display distinct 3D-folds. CryoEM analyses unveil unique RNA interactions with the uncharacterized Cas12a. These proteins show distinct double-strand and single-strand DNA cleavage preferences and broad PAM recognition. Finally, we establish a specific detection strategy for the oncogene SNP without traditional Cas12a PAM. This study highlights the potential of language models in exploring undocumented Cas protein function via gene cluster classification.
History
DepositionAug 18, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
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Revision 1.0Sep 4, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.1Jul 2, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
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Revision 1.1Jul 2, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name
Revision 1.2Sep 24, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas12a
G: RNA (44-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)171,4864
Polymers171,4382
Non-polymers492
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CRISPR-associated endonuclease Cas12a


Mass: 157312.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Anaeroglobus (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: RNA chain RNA (44-MER)


Mass: 14125.415 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Anaeroglobus (bacteria)
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
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: Structure of AmCas12a with crRNA / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Anaeroglobus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: NICKEL/TITANIUM / Grid mesh size: 300 divisions/in.
VitrificationCryogen name: ETHANE / Humidity: 100 %

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 46.63 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.1particle selection
9PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 196390 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038828
ELECTRON MICROSCOPYf_angle_d0.58711866
ELECTRON MICROSCOPYf_dihedral_angle_d5.2841145
ELECTRON MICROSCOPYf_chiral_restr0.0411276
ELECTRON MICROSCOPYf_plane_restr0.0051509

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