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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: sgRNASubgenomic mRNA
    • 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 / endonuclease activity / defense response to virus / 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 / Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease ...Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe 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: To Be Published
Title: Cryo-EM structure of the EvCas9-sgRNA-target DNA ternary complex
Authors: Tang N / Wu Z / Gao Y / Chen W / Wang Z / Su M / Ji Q
History
DepositionDec 23, 2022-
Header (metadata) releaseDec 27, 2023-
Map releaseDec 27, 2023-
UpdateDec 27, 2023-
Current statusDec 27, 2023Processing 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)
Voxel sizeX=Y=Z: 0.82 Å
Density
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: sgRNASubgenomic mRNA
    • 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 chloridesodium chloride
5.0 mMMgCl2magnesium chloride
20.0 mMTris-HClTrisTRIS 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 16.8 e/Å2
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

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

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