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- EMDB-38856: Ternary structure of dLesCas12e-sgRNA-dsDNA -

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Basic information

Entry
Database: EMDB / ID: EMD-38856
TitleTernary structure of dLesCas12e-sgRNA-dsDNA
Map data
Sample
  • Complex: Ternary complex of dVemCas12e-sgRNA-dsDNA
    • DNA: DNA (35-MER)
    • Protein or peptide: dLesCas12e
    • RNA: sgRNA
    • DNA: DNA (35-MER)
KeywordsCas12e complex / DNA BINDING PROTEIN / DNA BINDING PROTEIN-RNA-DNA COMPLEX / DNA BINDING PROTEIN-DNA-RNA complex
Biological speciesVerrucomicrobiota (bacteria) / Lentisphaerota (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.92 Å
AuthorsZhang S / Lin S / Liu JJG
Funding support China, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32150018 China
National Natural Science Foundation of China (NSFC)32101195 China
CitationJournal: Nat Commun / Year: 2024
Title: Cas12e orthologs evolve variable structural elements to facilitate dsDNA cleavage.
Authors: Danyuan Li / Shouyue Zhang / Shuo Lin / Wenjing Xing / Yun Yang / Fengxia Zhu / Dingding Su / Chunlai Chen / Jun-Jie Gogo Liu /
Abstract: Exceptionally diverse type V CRISPR-Cas systems provide numerous RNA-guided nucleases as powerful tools for DNA manipulation. Two known Cas12e nucleases, DpbCas12e and PlmCas12e, are both effective ...Exceptionally diverse type V CRISPR-Cas systems provide numerous RNA-guided nucleases as powerful tools for DNA manipulation. Two known Cas12e nucleases, DpbCas12e and PlmCas12e, are both effective in genome editing. However, many differences exist in their in vitro dsDNA cleavage activities, reflecting the diversity in Cas12e's enzymatic properties. To comprehensively understand the Cas12e family, we identify and characterize six unreported Cas12e members that vary in their CRISPR-locus architectures, PAM preferences, and cleavage efficacies. Interestingly, among all variants, PlmCas12e exhibits the most robust trans-cleavage activity and the lowest salt sensitivity in cis-cleavage. Further structural comparisons reveal that the unique NTSB domain in PlmCas12e is beneficial to DNA unwinding at high salt concentrations, while some NTSB-lacking Cas12e proteins rely on positively charged loops for dsDNA unwinding. These findings demonstrate how divergent evolution of structural elements shapes the nuclease diversity within the Cas12e family, potentially contributing to their adaptations to varying environmental conditions.
History
DepositionJan 26, 2024-
Header (metadata) releaseOct 23, 2024-
Map releaseOct 23, 2024-
UpdateJan 15, 2025-
Current statusJan 15, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_38856.png

Downloads & links

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Map

FileDownload / File: emd_38856.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-5.2169905 - 8.2609215
Average (Standard dev.)-0.0018987753 (±0.09746386)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_38856_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_38856_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of dVemCas12e-sgRNA-dsDNA

EntireName: Ternary complex of dVemCas12e-sgRNA-dsDNA
Components
  • Complex: Ternary complex of dVemCas12e-sgRNA-dsDNA
    • DNA: DNA (35-MER)
    • Protein or peptide: dLesCas12e
    • RNA: sgRNA
    • DNA: DNA (35-MER)

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Supramolecule #1: Ternary complex of dVemCas12e-sgRNA-dsDNA

SupramoleculeName: Ternary complex of dVemCas12e-sgRNA-dsDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1, #3-#4, #2
Source (natural)Organism: Verrucomicrobiota (bacteria)

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Macromolecule #1: DNA (35-MER)

MacromoleculeName: DNA (35-MER) / type: dna / ID: 1 / Details: Target strand / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Lentisphaerota (bacteria)
Molecular weightTheoretical: 10.709873 KDa
SequenceString:
(DG)(DG)(DA)(DT)(DC)(DG)(DT)(DT)(DC)(DA) (DC)(DC)(DA)(DG)(DG)(DG)(DT)(DG)(DT)(DC) (DG)(DC)(DC)(DC)(DT)(DC)(DA)(DA)(DA) (DA)(DT)(DC)(DC)(DC)(DG)

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Macromolecule #2: DNA (35-MER)

MacromoleculeName: DNA (35-MER) / type: dna / ID: 2 / Details: Non-target strand / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Lentisphaerota (bacteria)
Molecular weightTheoretical: 10.828958 KDa
SequenceString:
(DC)(DG)(DG)(DG)(DA)(DT)(DT)(DT)(DT)(DG) (DA)(DG)(DG)(DG)(DC)(DG)(DA)(DC)(DA)(DC) (DA)(DA)(DG)(DT)(DT)(DG)(DT)(DC)(DC) (DA)(DG)(DA)(DT)(DC)(DC)

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Macromolecule #3: dLesCas12e

MacromoleculeName: dLesCas12e / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lentisphaerota (bacteria)
Molecular weightTheoretical: 100.370016 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPTKNRKTDS TSIHASLRHL LQLGLKRSEA AIPQTITRTA KFKINTAIKP GLIPLLNAQF DAVEGFRRKV LGELEAWWNE DPEAFQKMV KCSMKMKFQG KSSCYAWLYT HFLKGATLAQ GLSRDAANSL LDNMGGGLKS FLTRRAHVAE EIRKRYDQNL G DWDDGLKD ...String:
MPTKNRKTDS TSIHASLRHL LQLGLKRSEA AIPQTITRTA KFKINTAIKP GLIPLLNAQF DAVEGFRRKV LGELEAWWNE DPEAFQKMV KCSMKMKFQG KSSCYAWLYT HFLKGATLAQ GLSRDAANSL LDNMGGGLKS FLTRRAHVAE EIRKRYDQNL G DWDDGLKD LAAEHGLELP PPPPRVNFEK LTAQEIEKYN DWVGRTRAWG NLLLIQKKKV ERRDACLPRY LKGYPGFPGS QR YATASAM AAALAELEQA AREQYGKARA RFAKVSAESW AQTVERFAPA PVRAEHGRPE PRTAHQTVSA RLAALIAAQP GWQ PAQLAE EILAGVLRGA EKLKTHLSKC GSHDRQAVIK LANLYNVAVA FALEPVRVAG DYLSFYAEET PKRKAFGNVR GALH QPSDD TAAIQITGFS INDEGSPNYN GLLVCKQSGD RLHDEWAFLF CHQPGQVFQL AAEDAKLRGK ILTEWLGFGS QGGSR KKAE ASAKKMIRRP VWMNEKTPPT ILPLAFGVRQ GREYLWHFDR NLRTKEGWVL GNGRLLRVMP PGRPHAADFY LTLTLE REA PPLAEVAAEK YIGIARGEAV PAAYAIIDRE GRLLAGGKIA EAFRDQQRKT NDEKRELQRT AGGYTKAFRS KERNRAR AL GGEVTRAIFA LSAAHRAPVI LANLNSSLAT RGGKGTMMSQ MQYERMLVAL EQKFAEAGLY ALPSAPKYRK GDNGFIKL V GPAYTSATCS ACGHVHSSDF YEKLADTLEG KCGSSWCVTL PNGEQQQLPD AYTFWLKGKG EQTKSTHERL EELLKGKSV AKLAKTNRRK LVGLLKSRWL PYRATQADFS CVLCGHTMNA AEQGALNIAR KFLFRTERGK QAGELTEAER RKMRADWQNW YKEKLRTVW RAPERGDGNG

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Macromolecule #4: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Lentisphaerota (bacteria)
Molecular weightTheoretical: 43.299637 KDa
SequenceString:
GCCCGUUAGU AUUUUCUUUG AUUCUGUACG CCUGCCGGCC CCACCGGAUG UGACGUACUU CGCGAACAUC AACGGUUCUU CGGAACCGC UGACGCUCGC GAAGAUGGAU UCAAAGAGGG CGACACCCUG GUGAAC

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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 (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: 1.5 µm / Nominal defocus min: 1.3 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1346569
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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