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- EMDB-35035: Cryo-EM structure of the EvCas9-sgRNA-target DNA ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-35035
TitleCryo-EM structure of the EvCas9-sgRNA-target DNA ternary complex
Map data
Sample
  • Complex: Ternary complex of EvCas9-sgRNA-dsDNA
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: sgRNA
    • DNA: Target DNA strand
    • DNA: Non-target DNA strand
KeywordsRNA / DNA / RNA BINDING PROTEIN/RNA/DNA / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. ...Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesEubacterium ventriosum ATCC 27560 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.43 Å
AuthorsTang N / Wu Z / Gao Y / Chen W / Su M / Wang Z / Ji Q
Funding support China, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)21922705 China
National Natural Science Foundation of China (NSFC)22277078 China
CitationJournal: ACS Synth Biol / Year: 2024
Title: Molecular Basis and Genome Editing Applications of a Compact CRISPR-Cas9 System.
Authors: Na Tang / Zhaowei Wu / Yan Gao / Weizhong Chen / Zixiao Wang / Mengjiao Su / Wenxin Ji / Quanjiang Ji /
Abstract: CRISPR-Cas9 systems have been widely harnessed for diverse genome editing applications because of their ease of use and high efficiency. However, the large molecular sizes and strict PAM requirements ...CRISPR-Cas9 systems have been widely harnessed for diverse genome editing applications because of their ease of use and high efficiency. However, the large molecular sizes and strict PAM requirements of commonly used CRISPR-Cas9 systems restrict their broad applications in therapeutics. Here, we report the molecular basis and genome editing applications of a novel compact type II-A CRISPR-Cas9 system (EvCas9) with 1107 residues and distinct 5'-NNGDGN-3' (where D represents A, T, or G) PAM specificity. We determine the cryo-EM structure of EvCas9 in a complex with an sgRNA and a target DNA, revealing the detailed PAM recognition and sgRNA and target DNA association mechanisms. Additionally, we demonstrate the robust genome editing capacity of EvCas9 in bacteria and human cells with superior fidelity compared to SaCas9 and SpCas9, and we engineer it to be efficient base editors by fusing a cytidine or adenosine deaminase. Collectively, our results facilitate further understanding of CRISPR-Cas9 working mechanisms and expand the compact CRISPR-Cas9 toolbox.
History
DepositionDec 23, 2022-
Header (metadata) releaseDec 27, 2023-
Map releaseDec 27, 2023-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_35035.png

Downloads & links

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Map

FileDownload / File: emd_35035.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 384 pix.
= 314.88 Å		0.82 Å/pix.
x 384 pix.
= 314.88 Å		0.82 Å/pix.
x 384 pix.
= 314.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-1.8336205 - 3.260326
Average (Standard dev.)-0.000088828965 (±0.04034778)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 314.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_35035_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35035_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 : Ternary complex of EvCas9-sgRNA-dsDNA

EntireName: Ternary complex of EvCas9-sgRNA-dsDNA
Components
  • Complex: Ternary complex of EvCas9-sgRNA-dsDNA
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: sgRNA
    • DNA: Target DNA strand
    • DNA: Non-target DNA strand

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Supramolecule #1: Ternary complex of EvCas9-sgRNA-dsDNA

SupramoleculeName: Ternary complex of EvCas9-sgRNA-dsDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Eubacterium ventriosum ATCC 27560 (bacteria)
Molecular weightTheoretical: 156.5 KDa

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Macromolecule #1: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Eubacterium ventriosum ATCC 27560 (bacteria)
Molecular weightTheoretical: 131.110531 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MGYTVGLDIG VASVGVAVLD ENDNIVEAVS NIFDEADTSN NKVRRTLREG RRTKRRQKTR IEDFKQLWET SGYIIPHKLH LNIIELRNK GLTELLSLDE LYCVLLSMLK HRGISYLEDA DDGEKGNAYK KGLAFNEKQL KEKMPCEIQL ERMKKYGKYH G EFIIEIND ...String:
MGYTVGLDIG VASVGVAVLD ENDNIVEAVS NIFDEADTSN NKVRRTLREG RRTKRRQKTR IEDFKQLWET SGYIIPHKLH LNIIELRNK GLTELLSLDE LYCVLLSMLK HRGISYLEDA DDGEKGNAYK KGLAFNEKQL KEKMPCEIQL ERMKKYGKYH G EFIIEIND EKEYQSNVFT TKAYKKELEK IFETQRCNGN KINTKFIKKY MEIYERKREY YIGPGNEKSR TDYGIYTTRT DE EGNFIDE KNIFGKLIGK CSVYPEEYRA SSASYTAQEF NLLNDLNNLK INNEKLTEFQ KKEIVEIIKD ASSVNMRKII KKV IDEDIE QYSGARIDKK GKEIYHTFEI YRKLKKELKT INVDIDSFTR EELDKTMDIL TLNTERESIV KAFDEQKFVY EENL IKKLI EFRKNNQRLF SGWHSFSYKA MLQLIPVMYK EPKEQMQLLT EMNVFKSKKE KYVNYKYIPE NEVVKEIYNP VVVKS IRTT VKILNALIKK YGYPESVVIE MPRDKNSDDE KEKIDMNQKK NQEEYEKILN KIYDEKGIEI TNKDYKKQKK LVLKLK LWN EQEGLCLYSG KKIAIEDLLN HPEFFEIDHI IPKSISLDDS RSNKVLVYKT ENSIKENDTP YHYLTRINGK WGFDEYK AN VLELRRRGKI DDKKVNNLLC MEDITKIDVV KGFINRNLND TRYASRVVLN EMQSFFESRK YCNTKVKVIR GSLTYQMR Q DLHLKKNREE SYSHHAVDAM LIAFSQKGYE AYRKIQKDCY DFETGEILDK EKWNKYIDDD EFDDILYKER MNEIRKKII EAEEKVKYNY KIDKKCNRGL CNQTIYGTRE KDGKIHKISS YNIYDDKECN SLKKMINSGK GSDLLMYNND PKTYRDMLKI LETYSSEKN PFVAYNKETG DYFRKYSKNH NGPKVEKVKY YSGQINSCID ISHKYGHAKN SKKVVLVSLN PYRTDVYYDN D TGKYYLVG VKYNHIKCVG NKYVIDSETY NELLRKEGVL NSDENLEDLN SKNITYKFSL YKNDIIQYEK GGEYYTERFL SR IKEQKNL IETKPINKPN FQRKNKKGEW ENTRNQIALA KTKYVGKLVT DVLGNCYIVN MEKFSLVVDK

UniProtKB: CRISPR-associated endonuclease Cas9

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.97624 KDa
SequenceString:
GGUAAUCGCU CUCCUCCGGC GAUUUUAGUA CCUGAGAAAU CAGAUCUACU AAAACAAGGC UUUAUGCCGA AAUCA

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Macromolecule #3: Target DNA strand

MacromoleculeName: Target DNA strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.641558 KDa
SequenceString:
(DC)(DG)(DC)(DC)(DA)(DC)(DG)(DT)(DC)(DG) (DC)(DC)(DG)(DG)(DA)(DG)(DG)(DA)(DG)(DA) (DG)(DC)(DG)(DA)(DT)(DT)(DA)(DC)

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Macromolecule #4: Non-target DNA strand

MacromoleculeName: Non-target DNA strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.467629 KDa
SequenceString:
(DA)(DC)(DG)(DT)(DG)(DG)(DC)(DG)

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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

Concentration0.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
300.0 mMNaClsodium chloride
5.0 mMMgCl2magnesium chloride
20.0 mMTris-HClTRIS hydrochloride
1.0 mMDTTDithiothreitol
GridModel: Homemade / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 16.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 300599
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6m0x

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.43 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 33599
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT
FSC plot (resolution estimation)

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