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

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Basic information

Entry
Database: PDB / ID: 8dc2
TitleCryo-EM structure of CasLambda (Cas12l) bound to crRNA and DNA
Components
  • CasLambda
  • DNA NTS
  • DNA TS
  • RNA (51-MER)
KeywordsRNA BINDING PROTEIN/RNA/DNA / CRISPR / RNA Binding Protein / DNA binding protein / phage / viral protein / enzyme / ribonucleoprotein / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesuncultured virus (environmental samples)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsAl-Shayeb, B. / Skopintsev, P. / Soczek, K. / Doudna, J.
Funding support United States, Switzerland, 2items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 14, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CasLambda
B: RNA (51-MER)
C: DNA TS
D: DNA NTS


Theoretical massNumber of molelcules
Total (without water)132,4894
Polymers132,4894
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CasLambda


Mass: 87337.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) uncultured virus (environmental samples)
Production host: Escherichia coli (E. coli)
#2: RNA chain RNA (51-MER)


Mass: 16483.719 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) uncultured virus (environmental samples)
#3: DNA chain DNA TS


Mass: 14296.228 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) uncultured virus (environmental samples)
#4: DNA chain DNA NTS


Mass: 14371.228 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) uncultured virus (environmental samples)

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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: CasLambda-crRNA-dsDNA ternary structure / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: uncultured virus (environmental samples)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1Topazparticle selectionas implemented in cryoSPARC
2SerialEM3.8.7image acquisition
4cryoSPARC3.2.0CTF correctionPatchCTF
9PHENIX1.19.2-4158model refinement
10cryoSPARC3.2.0initial Euler assignment
11cryoSPARC3.2.0final Euler assignment
12cryoSPARC3.2.0classification
13cryoSPARC3.2.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 369389 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037617
ELECTRON MICROSCOPYf_angle_d0.52710657
ELECTRON MICROSCOPYf_dihedral_angle_d18.7531689
ELECTRON MICROSCOPYf_chiral_restr0.0371214
ELECTRON MICROSCOPYf_plane_restr0.0031042

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