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- EMDB-39082: Cas12h1-crRNA binary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-39082
TitleCas12h1-crRNA binary complex
Map data
Sample
  • Complex: Binary complex of Cas12h1 with crRNA
    • Protein or peptide: Cas12h1
    • RNA: crRNA
  • Ligand: MAGNESIUM ION
KeywordsCas effector / IMMUNE SYSTEM / IMMUNE SYSTEM-RNA complex
Biological speciesunidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsZheng WW / Liu MX
Funding support China, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)82225028 China
National Natural Science Foundation of China (NSFC)82172287 China
CitationJournal: Signal Transduct Target Ther / Year: 2025
Title: Molecular insights and rational engineering of a compact CRISPR-Cas effector Cas12h1 with a broad-spectrum PAM.
Authors: Weiwei Zheng / Hongyu Li / Mengxi Liu / Yuhang Wei / Bo Liu / Zekai Li / Chenyang Xiong / Shiqing Huang / Chunyi Hu / Songying Ouyang /
Abstract: Cas12h1 is a compact CRISPR-associated nuclease from functionally diverse type V CRISPR-Cas effectors and recognizes a purine-rich protospacer adjacent motif (PAM) distinct from that of other type V ...Cas12h1 is a compact CRISPR-associated nuclease from functionally diverse type V CRISPR-Cas effectors and recognizes a purine-rich protospacer adjacent motif (PAM) distinct from that of other type V Cas effectors. Here, we report the nickase preference of Cas12h1, which predominantly cleaves the nontarget strand (NTS) of a double-stranded DNA (dsDNA) substrate. In addition, Cas12h1 acts as a nickase in human cells. We further determined the cryo-EM structures of Cas12h1 in the surveillance, R-loop formation, and interference states, revealing the molecular mechanisms involved in the crRNA maturation, target recognition, R-loop formation, nuclease activation and target degradation. Cas12h1 notably recognizes a broad 5'-DHR-3' PAM (D is A, G, or T; H is A, C, or T; R is A or G) both in vitro and in human cells. In addition, Cas12h1 utilizes a distinct activation mechanism that the lid motif undergoes a "flexible to stable" transition to expose the catalytic site to the substrate. A high-fidelity nucleic acid detector, Cas12h1, was developed through rational engineering, which distinguishes single-base mismatches and retains comparable on-target activities. Our results shed light on the molecular mechanisms underlying Cas12h1 nickase, improve the understanding of type V Cas effectors, and expand the CRISPR toolbox for genome editing and molecular diagnosis.
History
DepositionFeb 7, 2024-
Header (metadata) releaseFeb 12, 2025-
Map releaseFeb 12, 2025-
UpdateMar 19, 2025-
Current statusMar 19, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_39082.png

Downloads & links

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Map

FileDownload / File: emd_39082.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 256 pix.
= 218.88 Å		0.86 Å/pix.
x 256 pix.
= 218.88 Å		0.86 Å/pix.
x 256 pix.
= 218.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.855 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.257
Minimum - Maximum-2.3431616 - 3.5536456
Average (Standard dev.)0.00045726993 (±0.077260405)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 218.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_39082_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_39082_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Binary complex of Cas12h1 with crRNA

EntireName: Binary complex of Cas12h1 with crRNA
Components
  • Complex: Binary complex of Cas12h1 with crRNA
    • Protein or peptide: Cas12h1
    • RNA: crRNA
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Binary complex of Cas12h1 with crRNA

SupramoleculeName: Binary complex of Cas12h1 with crRNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 100 KDa

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Macromolecule #1: Cas12h1

MacromoleculeName: Cas12h1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 100.025758 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKVHEIPRSQ LLKIKQYEGS FVEWYRDLQE DRKKFASLLF RWAAFGYAAR EDDGATYISP SQALLERRLL LGDAEDVAIK FLDVLFKGG APSSSCYSLF YEDFALRDKA KYSGAKREFI EGLATMPLDK IIERIRQDEQ LSKIPAEEWL ILGAEYSPEE I WEQVAPRI ...String:
MKVHEIPRSQ LLKIKQYEGS FVEWYRDLQE DRKKFASLLF RWAAFGYAAR EDDGATYISP SQALLERRLL LGDAEDVAIK FLDVLFKGG APSSSCYSLF YEDFALRDKA KYSGAKREFI EGLATMPLDK IIERIRQDEQ LSKIPAEEWL ILGAEYSPEE I WEQVAPRI VNVDRSLGKQ LRERLGIKCR RPHDAGYCKI LMEVVARQLR SHNETYHEYL NQTHEMKTKV ANNLTNEFDL VC EFAEVLE EKNYGLGWYV LWQGVKQALK EQKKPTKIQI AVDQLRQPKF AGLLTAKWRA LKGAYDTWKL KKRLEKRKAF PYM PNWDND YQIPVGLTGL GVFTLEVKRT EVVVDLKEHG KLFCSHSHYF GDLTAEKHPS RYHLKFRHKL KLRKRDSRVE PTIG PWIEA ALREITIQKK PNGVFYLGLP YALSHGIDNF QIAKRFFSAA KPDKEVINGL PSEMVVGAAD LNLSNIVAPV KARIG KGLE GPLHALDYGY GELIDGPKIL TPDGPRCGEL ISLKRDIVEI KSAIKEFKAC QREGLTMSEE TTTWLSEVES PSDSPR CMI QSRIADTSRR LNSFKYQMNK EGYQDLAEAL RLLDAMDSYN SLLESYQRMH LSPGEQSPKE AKFDTKRASF RDLLRRR VA HTIVEYFDDC DIVFFEDLDG PSDSDSRNNA LVKLLSPRTL LLYIRQALEK RGIGMVEVAK DGTSQNNPIS GHVGWRNK Q NKSEIYFYED KELLVMDADE VGAMNILCRG LNHSVCPYSF VTKAPEKKND EKKEGDYGKR VKRFLKDRYG SSNVRFLVA SMGFVTVTTK RPKDALVGKR LYYHGGELVT HDLHNRMKDE IKYLVEKEVL ARRVSLSDST IKSYKSFAHV

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Macromolecule #2: crRNA

MacromoleculeName: crRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 20.041965 KDa
SequenceString:
GUGCUGGCCG CUCUCGCUAG AGGGAGGUCA GAGCACAUAA UAUCAAUGGA AUAUAGCAAG CU

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (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: 1.8 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 86736
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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