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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-32389 | |||||||||
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Title | PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state | |||||||||
![]() | PlmCasX-sgRNAv1-dsDNA-StateI | |||||||||
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![]() | CRISPR / CasX / sgRNA / R-loop complex / RNA BINDING PROTEIN-DNA-RNA complex / RNA BINDING PROTEIN / DNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex | |||||||||
Function / homology | Transposase![]() | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.9 Å | |||||||||
![]() | Zhang S / Liu JJG | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 5.7 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 15.2 KB 15.2 KB | Display Display | ![]() |
Images | ![]() | 63.6 KB | ||
Filedesc metadata | ![]() | 6.2 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7wayMC ![]() 7wazC ![]() 7wb0C ![]() 7wb1C M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | PlmCasX-sgRNAv1-dsDNA-StateI | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.94 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state
Entire | Name: PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state |
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Components |
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-Supramolecule #1: PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state
Supramolecule | Name: PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() |
-Supramolecule #2: dsDNA
Supramolecule | Name: dsDNA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: ![]() |
-Supramolecule #3: sgRNAv1
Supramolecule | Name: sgRNAv1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3 |
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Source (natural) | Organism: ![]() |
-Supramolecule #4: PlmCasX
Supramolecule | Name: PlmCasX / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4 |
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-Macromolecule #1: DNA (33-MER)
Macromolecule | Name: DNA (33-MER) / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 12.283895 KDa |
Sequence | String: (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) |
-Macromolecule #2: DNA (27-MER)
Macromolecule | Name: DNA (27-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 12.193829 KDa |
Sequence | String: (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) |
-Macromolecule #3: RNA
Macromolecule | Name: RNA / type: rna / ID: 3 / Number of copies: 1 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 39.272391 KDa |
Sequence | String: GGCGCGUUUA UUCCAUUACU UUGGAGCCAG UCCCAGCGAC UAUGUCGUAU GGACGAAGCG CUUAUUUAUC GGAGAGAAAC CGAUAAGUA AAACGCAUCA AAGUCCUGCA GCAGAAAAUC AAA |
-Macromolecule #4: dPlmCasX
Macromolecule | Name: dPlmCasX / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 112.68257 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 UniProtKB: Transposase |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Startup model | Type of model: NONE |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 520000 |
Initial angle assignment | Type: OTHER |
Final angle assignment | Type: OTHER / Software - Name: cryoSPARC (ver. 2) |
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Overall B value: 57 |
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Output model | ![]() PDB-7way: |