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- EMDB-33983: Structure of CasPi with guide RNA and target DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-33983
TitleStructure of CasPi with guide RNA and target DNA
Map data
Sample
  • Complex: CasPi
    • Protein or peptide: CasPi
    • DNA: DNA (5'-D(P*CP*GP*GP*GP*AP*TP*GP*CP*CP*CP*AP*G)-3')
    • DNA: DNA (30-MER)
    • RNA: RNA (174-MER)
Function / homologyReverse transcriptase domain-containing protein
Function and homology information
Biological speciesArmatimonadetes bacterium (bacteria) / Armatimonadota bacterium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsLi CP / Wang J / Liu JJ
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Cell Res / Year: 2023
Title: The compact Casπ (Cas12l) 'bracelet' provides a unique structural platform for DNA manipulation.
Authors: Ao Sun / Cheng-Ping Li / Zhihang Chen / Shouyue Zhang / Dan-Yuan Li / Yun Yang / Long-Qi Li / Yuqian Zhao / Kaichen Wang / Zhaofu Li / Jinxia Liu / Sitong Liu / Jia Wang / Jun-Jie Gogo Liu /
Abstract: CRISPR-Cas modules serve as the adaptive nucleic acid immune systems for prokaryotes, and provide versatile tools for nucleic acid manipulation in various organisms. Here, we discovered a new ...CRISPR-Cas modules serve as the adaptive nucleic acid immune systems for prokaryotes, and provide versatile tools for nucleic acid manipulation in various organisms. Here, we discovered a new miniature type V system, CRISPR-Casπ (Cas12l) (~860 aa), from the environmental metagenome. Complexed with a large guide RNA (~170 nt) comprising the tracrRNA and crRNA, Casπ (Cas12l) recognizes a unique 5' C-rich PAM for DNA cleavage under a broad range of biochemical conditions, and generates gene editing in mammalian cells. Cryo-EM study reveals a 'bracelet' architecture of Casπ effector encircling the DNA target at 3.4 Å resolution, substantially different from the canonical 'two-lobe' architectures of Cas12 and Cas9 nucleases. The large guide RNA serves as a 'two-arm' scaffold for effector assembly. Our study expands the knowledge of DNA targeting mechanisms by CRISPR effectors, and offers an efficient but compact platform for DNA manipulation.
History
DepositionAug 1, 2022-
Header (metadata) releaseFeb 15, 2023-
Map releaseFeb 15, 2023-
UpdateMar 29, 2023-
Current statusMar 29, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33983.png

Downloads & links

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Map

FileDownload / File: emd_33983.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.856 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.94826883 - 1.7381262
Average (Standard dev.)0.0045154565 (±0.05623912)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 205.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_33983_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_33983_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 : CasPi

EntireName: CasPi
Components
  • Complex: CasPi
    • Protein or peptide: CasPi
    • DNA: DNA (5'-D(P*CP*GP*GP*GP*AP*TP*GP*CP*CP*CP*AP*G)-3')
    • DNA: DNA (30-MER)
    • RNA: RNA (174-MER)

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Supramolecule #1: CasPi

SupramoleculeName: CasPi / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Armatimonadetes bacterium (bacteria)
Molecular weightTheoretical: 177.28 KDa

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Macromolecule #1: CasPi

MacromoleculeName: CasPi / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Armatimonadota bacterium (bacteria)
Molecular weightTheoretical: 98.731234 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAKATKEVKS KRVEALRQVA YQRLERLERK AQKIGAHLRK PGKAADLQSL HYLLHKVEVE YHDIARNLEK DPTWTPKPKM RREKRAIVP ESGPAAPLPT TAKGEPGRPA NRHIPPPVPL DSARIPEDQQ SMGQGSGGRS WCSAPFVEVK LPPTQWSNVR E KLLKFRIE ...String:
MAKATKEVKS KRVEALRQVA YQRLERLERK AQKIGAHLRK PGKAADLQSL HYLLHKVEVE YHDIARNLEK DPTWTPKPKM RREKRAIVP ESGPAAPLPT TAKGEPGRPA NRHIPPPVPL DSARIPEDQQ SMGQGSGGRS WCSAPFVEVK LPPTQWSNVR E KLLKFRIE DDADIVRRWA EAKFGSIETA RDGLRASAEI GTSPDVWRSF ISRAISNGKK DFEPLLSLDD DELTADATAE RV VRRWHQI DWVGRMLDSI LETVPSGVSK DTFRSRVESR LKTFHSSVNS FELKKRKDGT VERKRKHTNP QFPYLSPSAV SID PDVVTM EAVELLQMQP EERFAKDPND ANGRMRLRVL QAELGKARRE ALGRRGEKAP PWSGRKVFRG TTTRKREACL VWDK EAQAD GLYFALVMSG GPKIDDKRFV YMDGQPLQSD WQLHNGVAGK AKSCRAMPLI LKHDFLRWYH RHIKNHDVNA PLEKR CVHT TTQFVFVEPD EKKGLQPRLF IRPVFKFYDP VYEVPDSHSI DKKPDCRYLI GIARGVNYPY RAAVYDCETN SIIADK FVD GRKADWERIR NELAYHQRRR DLLRNSRASS AAIQREIRAI ARIRKRERGL NKVETVESIA RLVDWAEENL GKCNYCF VL ADLSSNLNLG RNNRVKHIAA IKEALINQMR KRGYRFKKSG KVDGVREESA WYTSAVAPSG WWAKKEEVDG AWKADKTR P LARKIGSYYC CEEIDGLHLR GVLKGLGRAK RLVLQSDDPS APTRRRGFGS ELFWDPYCTE LCGHAFPQGV VLDADFIGA FNIALRPLVR EELGKKAKAV DLADRHQTLN PTVALRCGVT AYEFVEVGGD PRGGLRKILL NPAEAVI

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Macromolecule #2: DNA (5'-D(P*CP*GP*GP*GP*AP*TP*GP*CP*CP*CP*AP*G)-3')

MacromoleculeName: DNA (5'-D(P*CP*GP*GP*GP*AP*TP*GP*CP*CP*CP*AP*G)-3') / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Armatimonadetes bacterium (bacteria)
Molecular weightTheoretical: 3.688405 KDa
SequenceString:
(DC)(DG)(DG)(DG)(DA)(DT)(DG)(DC)(DC)(DC) (DA)(DG)

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Macromolecule #3: DNA (30-MER)

MacromoleculeName: DNA (30-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Armatimonadetes bacterium (bacteria)
Molecular weightTheoretical: 9.192878 KDa
SequenceString:
(DG)(DT)(DT)(DC)(DA)(DC)(DC)(DA)(DG)(DG) (DG)(DT)(DG)(DT)(DC)(DG)(DC)(DC)(DC)(DT) (DG)(DG)(DG)(DC)(DA)(DT)(DC)(DC)(DC) (DG)

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Macromolecule #4: RNA (174-MER)

MacromoleculeName: RNA (174-MER) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Armatimonadetes bacterium (bacteria)
Molecular weightTheoretical: 56.411703 KDa
SequenceString:
GUCUGCCGAA GACGCCGCAC GGAGCCUGGG CCGGAAUCGU AGAUCGAACG CGGCAUCGAA GCCCUGCAGC CCUUCGGGGC CAAGGCGGC GCAGCAAGCC UCUUUCAGGC GGCAGAGUCC UUUAGAGUGU GAGAGACACU CUAAAGGAAU GAAAGAGGGC G ACACCCUG 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: 8
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing #1

Startup modelType of model: NONE / Details: ab-initial in cryosparc
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 3
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 39500
Image processing ID1

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Image processing #2

Startup modelType of model: NONE / Details: ab-initial in cryosparc
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 3
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 39500
Image processing ID2

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: BACKBONE TRACE
Output model

PDB-7yoj:
Structure of CasPi with guide RNA and target DNA

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