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- PDB-8i54: Lb2Cas12a RNA DNA complex -

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Basic information

Entry
Database: PDB / ID: 8i54
TitleLb2Cas12a RNA DNA complex
Components
  • DNA (25-MER)
  • DNA (5'-D(*AP*GP*TP*GP*CP*TP*TP*TP*A)-3')
  • Lb2Cas12a
  • RNA (33-MER)
KeywordsANTIMICROBIAL PROTEIN / complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesLachnospiraceae bacterium MA2020 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.95 Å
AuthorsLi, J. / Sivaraman, J. / Satoru, M.
Funding support Singapore, 1items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore)R154-000-A39-112 Singapore
CitationJournal: PLoS Biol / Year: 2023
Title: Structures of apo Cas12a and its complex with crRNA and DNA reveal the dynamics of ternary complex formation and target DNA cleavage.
Authors: Li Jianwei / Chacko Jobichen / Satoru Machida / Sun Meng / Randy J Read / Chen Hongying / Shi Jian / Yuren Adam Yuan / J Sivaraman /
Abstract: Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR-Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium ...Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR-Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium MA2020 (Lb2) and the Lb2Cas12a+crRNA complex, as well as the cryo-EM structure and functional studies of the Lb2Cas12a+crRNA+DNA complex. We demonstrate that apo Lb2Cas12a assumes a unique, elongated conformation, whereas the Lb2Cas12a+crRNA binary complex exhibits a compact conformation that subsequently rearranges to a semi-open conformation in the Lb2Cas12a+crRNA+DNA ternary complex. Notably, in solution, apo Lb2Cas12a is dynamic and can exist in both elongated and compact forms. Residues from Met493 to Leu523 of the WED domain undergo major conformational changes to facilitate the required structural rearrangements. The REC lobe of Lb2Cas12a rotates 103° concomitant with rearrangement of the hinge region close to the WED and RuvC II domains to position the RNA-DNA duplex near the catalytic site. Our findings provide insight into crRNA recognition and the mechanism of target DNA cleavage.
History
DepositionJan 24, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 22, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 5, 2023Group: Structure summary / Category: audit_author / Item: _audit_author.name
Revision 1.2May 3, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Lb2Cas12a
B: RNA (33-MER)
C: DNA (25-MER)
D: DNA (5'-D(*AP*GP*TP*GP*CP*TP*TP*TP*A)-3')


Theoretical massNumber of molelcules
Total (without water)162,1794
Polymers162,1794
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Lb2Cas12a


Mass: 141200.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lachnospiraceae bacterium MA2020 (bacteria)
Production host: Escherichia coli (E. coli)
#2: RNA chain RNA (33-MER)


Mass: 10452.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lachnospiraceae bacterium MA2020 (bacteria)
Production host: synthetic construct (others)
#3: DNA chain DNA (25-MER)


Mass: 7780.007 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#4: DNA chain DNA (5'-D(*AP*GP*TP*GP*CP*TP*TP*TP*A)-3')


Mass: 2745.821 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)

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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: LbCas12a-crRNA-DNA ternary complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.14 MDa / Experimental value: NO
Source (natural)Organism: Lachnospiraceae bacterium MA2020 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 K

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 150 K / Temperature (min): 140 K
Image recordingAverage exposure time: 3.49 sec. / Electron dose: 45 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2560

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 173902 / Symmetry type: POINT
RefinementHighest resolution: 3.95 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00510938
ELECTRON MICROSCOPYf_angle_d0.8315033
ELECTRON MICROSCOPYf_dihedral_angle_d20.0611953
ELECTRON MICROSCOPYf_chiral_restr0.0431666
ELECTRON MICROSCOPYf_plane_restr0.0051703

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