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- EMDB-32391: PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state with f... -

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Basic information

Entry
Database: EMDB / ID: EMD-32391
TitlePlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state with flexible H2 domain
Map dataPlmcasX-sgRNAv1-dsDNA at stateIII
Sample
  • Complex: PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state
    • Complex: DNA
      • DNA: TS-DNA
      • DNA: NTS-DNA
    • Complex: RNA
      • RNA: RNA (115-MER)
    • Complex: PlmCasX
      • Protein or peptide: dPlmCasX
Function / homologyTransposase
Function and homology information
Biological speciesPlanctomycetes bacterium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsZhang S / Liu JJG
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Mol Cell / Year: 2022
Title: Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity.
Authors: Connor A Tsuchida / Shouyue Zhang / Mohammad Saffari Doost / Yuqian Zhao / Jia Wang / Elizabeth O'Brien / Huan Fang / Cheng-Ping Li / Danyuan Li / Zhuo-Yan Hai / Jonathan Chuck / Julian ...Authors: Connor A Tsuchida / Shouyue Zhang / Mohammad Saffari Doost / Yuqian Zhao / Jia Wang / Elizabeth O'Brien / Huan Fang / Cheng-Ping Li / Danyuan Li / Zhuo-Yan Hai / Jonathan Chuck / Julian Brötzmann / Araz Vartoumian / David Burstein / Xiao-Wei Chen / Eva Nogales / Jennifer A Doudna / Jun-Jie Gogo Liu /
Abstract: A compact protein with a size of <1,000 amino acids, the CRISPR-associated protein CasX is a fundamentally distinct RNA-guided nuclease when compared to Cas9 and Cas12a. Although it can induce RNA-guided genome editing in mammalian cells, the activity of CasX is less robust than that of the widely used S. pyogenes Cas9. Here, we show that structural features of two CasX homologs and their guide RNAs affect the R-loop complex assembly and DNA cleavage activity. Cryo-EM-based structural engineering of either the CasX protein or the guide RNA produced two new CasX genome editors (DpbCasX-R3-v2 and PlmCasX-R1-v2) with significantly improved DNA manipulation efficacy. These results advance both the mechanistic understanding of CasX and its application as a genome-editing tool.
History
DepositionDec 15, 2021-
Header (metadata) releaseMar 16, 2022-
Map releaseMar 16, 2022-
UpdateMar 30, 2022-
Current statusMar 30, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.325
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.325
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7wb0
  • Surface level: 0.325
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_32391.png

Downloads & links

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Map

FileDownload / File: emd_32391.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPlmcasX-sgRNAv1-dsDNA at stateIII
Voxel sizeX=Y=Z: 0.94 Å
Density
Contour LevelBy AUTHOR: 0.325 / Movie #1: 0.325
Minimum - Maximum-1.0964792 - 1.9233286
Average (Standard dev.)-0.00041752317 (±0.054100566)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 240.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.940.940.94
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z240.640240.640240.640
α/β/γ90.00090.00090.000
start NX/NY/NZ139118109
NX/NY/NZ123164187
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-1.0961.923-0.000

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Supplemental data

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Sample components

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Entire : PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state

EntireName: PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state
Components
  • Complex: PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state
    • Complex: DNA
      • DNA: TS-DNA
      • DNA: NTS-DNA
    • Complex: RNA
      • RNA: RNA (115-MER)
    • Complex: PlmCasX
      • Protein or peptide: dPlmCasX

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Supramolecule #1: PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state

SupramoleculeName: PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Planctomycetes bacterium (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #2: DNA

SupramoleculeName: DNA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Planctomycetes bacterium (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: RNA

SupramoleculeName: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Planctomycetes bacterium (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #4: PlmCasX

SupramoleculeName: PlmCasX / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4

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Macromolecule #1: TS-DNA

MacromoleculeName: TS-DNA / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Planctomycetes bacterium (bacteria)
Molecular weightTheoretical: 12.283895 KDa
SequenceString:
(DA)(DT)(DC)(DG)(DT)(DT)(DA)(DT)(DA)(DC) (DT)(DT)(DT)(DG)(DA)(DT)(DT)(DT)(DT)(DC) (DT)(DG)(DC)(DT)(DG)(DC)(DA)(DG)(DG) (DA)(DT)(DG)(DA)(DA)(DA)(DT)(DC)(DC)(DC) (DG)

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Macromolecule #2: NTS-DNA

MacromoleculeName: NTS-DNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Planctomycetes bacterium (bacteria)
Molecular weightTheoretical: 12.193829 KDa
SequenceString:
(DC)(DG)(DG)(DG)(DA)(DT)(DT)(DT)(DC)(DA) (DT)(DC)(DC)(DT)(DG)(DC)(DA)(DG)(DC)(DA) (DT)(DC)(DC)(DC)(DC)(DG)(DA)(DC)(DC) (DC)(DG)(DT)(DA)(DT)(DA)(DA)(DC)(DG)(DA) (DT)

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Macromolecule #3: RNA (115-MER)

MacromoleculeName: RNA (115-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Planctomycetes bacterium (bacteria)
Molecular weightTheoretical: 39.272391 KDa
SequenceString:
GGCGCGUUUA UUCCAUUACU UUGGAGCCAG UCCCAGCGAC UAUGUCGUAU GGACGAAGCG CUUAUUUAUC GGAGAGAAAC CGAUAAGUA AAACGCAUCA AAGUCCUGCA GCAGAAAAUC AAA

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Macromolecule #4: dPlmCasX

MacromoleculeName: dPlmCasX / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Planctomycetes bacterium (bacteria)
Molecular weightTheoretical: 112.68257 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQEIKRINKI RRRLVKDSNT KKAGKTGPMK TLLVRVMTPD LRERLENLRK KPENIPQPIS NTSRANLNKL LTDYTEMKKA ILHVYWEEF QKDPVGLMSR VAQPAPKNID QRKLIPVKDG NERLTSSGFA CSQCCQPLYV YKLEQVNDKG KPHTNYFGRC N VSEHERLI ...String:
MQEIKRINKI RRRLVKDSNT KKAGKTGPMK TLLVRVMTPD LRERLENLRK KPENIPQPIS NTSRANLNKL LTDYTEMKKA ILHVYWEEF QKDPVGLMSR VAQPAPKNID QRKLIPVKDG NERLTSSGFA CSQCCQPLYV YKLEQVNDKG KPHTNYFGRC N VSEHERLI LLSPHKPEAN DELVTYSLGK FGQRALDFYS IHVTRESNHP VKPLEQIGGN SCASGPVGKA LSDACMGAVA SF LTKYQDI ILEHQKVIKK NEKRLANLKD IASANGLAFP KITLPPQPHT KEGIEAYNNV VAQIVIWVNL NLWQKLKIGR DEA KPLQRL KGFPSFPLVE RQANEVDWWD MVCNVKKLIN EKKEDGKVFW QNLAGYKRQE ALLPYLSSEE DRKKGKKFAR YQFG DLLLH LEKKHGEDWG KVYDEAWERI DKKVEGLSKH IKLEEERRSE DAQSKAALTD WLRAKASFVI EGLKEADKDE FCRCE LKLQ KWYGDLRGKP FAIEAENSIL DISGFSKQYN CAFIWQKDGV KKLNLYLIIN YFKGGKLRFK KIKPEAFEAN RFYTVI NKK SGEIVPMEVN FNFDDPNLII LPLAFGKRQG REFIWNDLLS LETGSLKLAN GRVIEKTLYN RRTRQDEPAL FVALTFE RR EVLDSSNIKP MNLIGIARGE NIPAVIALTD PEGCPLSRFK DSLGNPTHIL RIGESYKEKQ RTIQAAKEVE QRRAGGYS R KYASKAKNLA DDMVRNTARD LLYYAVTQDA MLIFANLSRG FGRQGKRTFM AERQYTRMED WLTAKLAYEG LPSKTYLSK TLAQYTSKTC SNCGFTITSA DYDRVLEKLK KTATGWMTTI NGKELKVEGQ ITYYNRYKRQ NVVKDLSVEL DRLSEESVNN DISSWTKGR SGEALSLLKK RFSHRPVQEK FVCLNCGFET HAAEQAALNI ARSWLFLRSQ EYKKYQTNKT TGNTDKRAFV E TWQSFYRK KLKEVWKPAV

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 2)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 710824

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 57
Output model

PDB-7wb0:
PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state with flexible H2 domain

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