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- EMDB-27320: Cryo-EM structure of CasLambda (Cas12l) bound to crRNA and DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-27320
TitleCryo-EM structure of CasLambda (Cas12l) bound to crRNA and DNA
Map dataLocSpiral map from cryoSPARC half maps
Sample
  • Complex: CasLambda-crRNA-dsDNA ternary structure
    • Protein or peptide: CasLambda
    • RNA: RNA (51-MER)
    • DNA: DNA TS
    • DNA: DNA NTS
KeywordsCRISPR / RNA Binding Protein / DNA binding protein / phage / viral protein / enzyme / ribonucleoprotein / RNA BINDING PROTEIN-RNA-DNA complex
Biological speciesuncultured virus (environmental samples)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsAl-Shayeb B / Skopintsev P / Soczek K / Doudna J
Funding support United States, Switzerland, 2 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States) United States
Swiss National Science FoundationP2EZP3_195621 Switzerland
CitationJournal: Cell / Year: 2022
Title: Diverse virus-encoded CRISPR-Cas systems include streamlined genome editors.
Authors: Basem Al-Shayeb / Petr Skopintsev / Katarzyna M Soczek / Elizabeth C Stahl / Zheng Li / Evan Groover / Dylan Smock / Amy R Eggers / Patrick Pausch / Brady F Cress / Carolyn J Huang / Brian ...Authors: Basem Al-Shayeb / Petr Skopintsev / Katarzyna M Soczek / Elizabeth C Stahl / Zheng Li / Evan Groover / Dylan Smock / Amy R Eggers / Patrick Pausch / Brady F Cress / Carolyn J Huang / Brian Staskawicz / David F Savage / Steven E Jacobsen / Jillian F Banfield / Jennifer A Doudna /
Abstract: CRISPR-Cas systems are host-encoded pathways that protect microbes from viral infection using an adaptive RNA-guided mechanism. Using genome-resolved metagenomics, we find that CRISPR systems are ...CRISPR-Cas systems are host-encoded pathways that protect microbes from viral infection using an adaptive RNA-guided mechanism. Using genome-resolved metagenomics, we find that CRISPR systems are also encoded in diverse bacteriophages, where they occur as divergent and hypercompact anti-viral systems. Bacteriophage-encoded CRISPR systems belong to all six known CRISPR-Cas types, though some lack crucial components, suggesting alternate functional roles or host complementation. We describe multiple new Cas9-like proteins and 44 families related to type V CRISPR-Cas systems, including the Casλ RNA-guided nuclease family. Among the most divergent of the new enzymes identified, Casλ recognizes double-stranded DNA using a uniquely structured CRISPR RNA (crRNA). The Casλ-RNA-DNA structure determined by cryoelectron microscopy reveals a compact bilobed architecture capable of inducing genome editing in mammalian, Arabidopsis, and hexaploid wheat cells. These findings reveal a new source of CRISPR-Cas enzymes in phages and highlight their value as genome editors in plant and human cells.
History
DepositionJun 15, 2022-
Header (metadata) releaseDec 14, 2022-
Map releaseDec 14, 2022-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27320.png

Downloads & links

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Map

FileDownload / File: emd_27320.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocSpiral map from cryoSPARC half maps
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.12 Å/pix.
x 200 pix.
= 223. Å		1.12 Å/pix.
x 200 pix.
= 223. Å		1.12 Å/pix.
x 200 pix.
= 223. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.115 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 10.0
Minimum - Maximum0.0 - 30.251477999999999
Average (Standard dev.)0.17878546 (±1.0602404)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 223.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27320_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: cryoSPARC sharp map

Fileemd_27320_additional_1.map
AnnotationcryoSPARC sharp map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: cryoSPARC map

Fileemd_27320_additional_2.map
AnnotationcryoSPARC map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27320_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_27320_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CasLambda-crRNA-dsDNA ternary structure

EntireName: CasLambda-crRNA-dsDNA ternary structure
Components
  • Complex: CasLambda-crRNA-dsDNA ternary structure
    • Protein or peptide: CasLambda
    • RNA: RNA (51-MER)
    • DNA: DNA TS
    • DNA: DNA NTS

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Supramolecule #1: CasLambda-crRNA-dsDNA ternary structure

SupramoleculeName: CasLambda-crRNA-dsDNA ternary structure / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: uncultured virus (environmental samples)

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Macromolecule #1: CasLambda

MacromoleculeName: CasLambda / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: uncultured virus (environmental samples)
Molecular weightTheoretical: 87.337969 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASHKKTESN QIIKTFSFKI KNANGLSLDV LNDAITEYQN YYNICSDWIK DHLTMKISEL YKYIPNEKKN SGYALTLISD EWKDKPMYM MFKKGYPANN RDNAIYETLN TCNTEHYTGN ILNFSDTYYR RFGYVASAIS NYVTKISKMS TGSRSKNISN D SDVDTIME ...String:
MASHKKTESN QIIKTFSFKI KNANGLSLDV LNDAITEYQN YYNICSDWIK DHLTMKISEL YKYIPNEKKN SGYALTLISD EWKDKPMYM MFKKGYPANN RDNAIYETLN TCNTEHYTGN ILNFSDTYYR RFGYVASAIS NYVTKISKMS TGSRSKNISN D SDVDTIME QVIYEMEHNG WTSVKDWENQ MEYLESKTDS NPNFVYRMTT LYEFYKSHID EVNSKMETMS IDSLIKFGGC RR KDSKKSM YIMGGSNTPF DITQIGGNSL NIKFSKNLNV DVFGRYDVIK DNTLLVDIIN GHGASFVLKI INDEIYIDIN VSV PFDKKI ATTNKVVGID VNIKHMLLAT NILDDGNVKG YVNIYKEVIN DSDFKKVCNS TVMQYFTDFS KFVTFCPLEF DFLF SRVCN QKGIYNDNSA MEKSFSDVLN KLKWNFIETG DNTKRIYIEN VMKLRSQMKA YAIVKNAYYK QQSEYDFGKS EEFIQ EHPF SNTDKGIEIL NKLDNISKKI LGCRNNIIQY SYNLFEINGY DMVSLEKLTS SQFKKKPFPT VNSLLKYHKI LGCTQE EME KKDIYSVIKK GYYDIIFDND VVTDAKLSAK GELSKFKDDF FNLMIKSIHF ADIKDYFITL SNNGTAGVSL VPSYFTS QM DSIDHKIYFV QDNKSGKLKL ANKHKVRSSQ EKHINGLNAD YNAARNIAYI MENTDCRNMF MKQSRTDKSL YNKPSYET F IKTQGSAVAK LKKEGFVKIL DEASVGSSGH HHHHH

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Macromolecule #2: RNA (51-MER)

MacromoleculeName: RNA (51-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: uncultured virus (environmental samples)
Molecular weightTheoretical: 16.483719 KDa
SequenceString:
AUUGUUGUAA CUCUUAUUUU GUAUGGAGUA AACAACUAGC AUCACCUUCA CC

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Macromolecule #3: DNA TS

MacromoleculeName: DNA TS / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: uncultured virus (environmental samples)
Molecular weightTheoretical: 14.296228 KDa
SequenceString:
(DC)(DA)(DT)(DT)(DA)(DA)(DC)(DA)(DT)(DT) (DA)(DC)(DT)(DA)(DA)(DG)(DA)(DG)(DG)(DG) (DT)(DG)(DA)(DA)(DG)(DG)(DT)(DG)(DA) (DT)(DG)(DC)(DT)(DA)(DC)(DA)(DA)(DA)(DC) (DG) (DG)(DT)(DC)(DA)(DA)(DG)

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Macromolecule #4: DNA NTS

MacromoleculeName: DNA NTS / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: uncultured virus (environmental samples)
Molecular weightTheoretical: 14.371228 KDa
SequenceString:
(DC)(DT)(DT)(DG)(DA)(DC)(DC)(DG)(DT)(DT) (DT)(DG)(DA)(DT)(DC)(DG)(DT)(DA)(DG)(DT) (DG)(DG)(DA)(DA)(DG)(DT)(DG)(DG)(DG) (DA)(DG)(DA)(DT)(DA)(DG)(DT)(DA)(DA)(DT) (DG) (DT)(DT)(DA)(DA)(DT)(DG)

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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
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab-initio map calculated in cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 369389
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.2.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8dc2:
Cryo-EM structure of CasLambda (Cas12l) bound to crRNA and DNA

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