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- EMDB-29639: Cas9 with double-stranded guide RNA and target DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-29639
TitleCas9 with double-stranded guide RNA and target DNA
Map dataDeepEMhancer HiRes map
Sample
  • Complex: CRISPR-associated endonuclease Cas9 complex with double stranded guide RNA and target DNA
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • RNA: CrE2
    • RNA: tracrRNA
    • DNA: E2_TS
    • DNA: E2-NTS
KeywordsCas9 / RNA / DNA / HYDROLASE
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. ...CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / 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/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.03 Å
AuthorsKorolev S / Gagnon K
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: To Be Published
Title: SpCas9 with dual-guide RNA and target DNA
Authors: Korolev S / Gagnon K
History
DepositionJan 30, 2023-
Header (metadata) releaseMay 31, 2023-
Map releaseMay 31, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29639.png

Downloads & links

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Map

FileDownload / File: emd_29639.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeepEMhancer HiRes map
Voxel sizeX=Y=Z: 0.7 Å
Density
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.03135217 - 1.9954342
Average (Standard dev.)0.0013514948 (±0.024339993)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions380380380
Spacing380380380
CellA=B=C: 266.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29639_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_29639_half_map_1.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_29639_half_map_2.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CRISPR-associated endonuclease Cas9 complex with double stranded ...

EntireName: CRISPR-associated endonuclease Cas9 complex with double stranded guide RNA and target DNA
Components
  • Complex: CRISPR-associated endonuclease Cas9 complex with double stranded guide RNA and target DNA
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • RNA: CrE2
    • RNA: tracrRNA
    • DNA: E2_TS
    • DNA: E2-NTS

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Supramolecule #1: CRISPR-associated endonuclease Cas9 complex with double stranded ...

SupramoleculeName: CRISPR-associated endonuclease Cas9 complex with double stranded guide RNA and target DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Streptococcus pyogenes (bacteria)

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Macromolecule #1: CRISPR-associated endonuclease Cas9/Csn1

MacromoleculeName: CRISPR-associated endonuclease Cas9/Csn1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 160.774281 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNE MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA DLRLIYLALA H MIKFRGHF ...String:
MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNE MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA DLRLIYLALA H MIKFRGHF LIEGDLNPDN SDVDKLFIQL VQTYNQLFEE NPINASGVDA KAILSARLSK SRRLENLIAQ LPGEKKNGLF GN LIALSLG LTPNFKSNFD LAEDAKLQLS KDTYDDDLDN LLAQIGDQYA DLFLAAKNLS DAILLSDILR VNTEITKAPL SAS MIKRYD EHHQDLTLLK ALVRQQLPEK YKEIFFDQSK NGYAGYIDGG ASQEEFYKFI KPILEKMDGT EELLVKLNRE DLLR KQRTF DNGSIPHQIH LGELHAILRR QEDFYPFLKD NREKIEKILT FRIPYYVGPL ARGNSRFAWM TRKSEETITP WNFEE VVDK GASAQSFIER MTNFDKNLPN EKVLPKHSLL YEYFTVYNEL TKVKYVTEGM RKPAFLSGEQ KKAIVDLLFK TNRKVT VKQ LKEDYFKKIE CFDSVEISGV EDRFNASLGT YHDLLKIIKD KDFLDNEENE DILEDIVLTL TLFEDREMIE ERLKTYA HL FDDKVMKQLK RRRYTGWGRL SRKLINGIRD KQSGKTILDF LKSDGFANRN FMQLIHDDSL TFKEDIQKAQ VSGQGDSL H EHIANLAGSP AIKKGILQTV KVVDELVKVM GRHKPENIVI EMARENQTTQ KGQKNSRERM KRIEEGIKEL GSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNA KLITQRKFDN LTKAERGGLS ELDKAGFIKR QLVETRQITK HVAQILDSRM NTKYDENDKL IREVKVITLK S KLVSDFRK DFQFYKVREI NNYHHAHDAY LNAVVGTALI KKYPKLESEF VYGDYKVYDV RKMIAKSEQE IGKATAKYFF YS NIMNFFK TEITLANGEI RKRPLIETNG ETGEIVWDKG RDFATVRKVL SMPQVNIVKK TEVQTGGFSK ESILPKRNSD KLI ARKKDW DPKKYGGFDS PTVAYSVLVV AKVEKGKSKK LKSVKELLGI TIMERSSFEK NPIDFLEAKG YKEVKKDLII KLPK YSLFE LENGRKRMLA SAGELQKGNE LALPSKYVNF LYLASHYEKL KGSPEDNEQK QLFVEQHKHY LDEIIEQISE FSKRV ILAD ANLDKVLSAY NKHRDKPIRE QAENIIHLFT LTNLGAPAAF KYFDTTIDRK RYTSTKEVLD ATLIHQSITG LYETRI DLS QLGGDPKKKR KVMDKHHHHH H

UniProtKB: CRISPR-associated endonuclease Cas9/Csn1

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Macromolecule #2: CrE2

MacromoleculeName: CrE2 / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 13.370906 KDa
SequenceString:
UACCAGCAAA ACACUCCGAU GUUUUAGAGC UAUGCUGUUU UG

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Macromolecule #3: tracrRNA

MacromoleculeName: tracrRNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 22.574494 KDa
SequenceString:
AAACAGCAUA GCAAGUUAAA AUAAGGCUAG UCCGUUAUCA ACUUGAAAAA GUGGCACCGA GUCGGUGCUU

GENBANK: GENBANK: LS483330.1

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Macromolecule #4: E2_TS

MacromoleculeName: E2_TS / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.334909 KDa
SequenceString:
(DC)(DT)(DA)(DA)(DT)(DC)(DG)(DC)(DC)(DA) (DA)(DT)(DC)(DG)(DG)(DA)(DG)(DT)(DG)(DT) (DT)(DT)(DT)(DG)(DC)(DT)(DG)(DG)(DT) (DA)(DT)(DA)(DC)(DG)(DC)(DA)(DC)(DT)(DG) (DG)

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Macromolecule #5: E2-NTS

MacromoleculeName: E2-NTS / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.290917 KDa
SequenceString:
(DC)(DC)(DA)(DG)(DT)(DG)(DC)(DG)(DT)(DA) (DT)(DA)(DC)(DC)(DA)(DG)(DC)(DA)(DA)(DA) (DA)(DC)(DA)(DC)(DT)(DC)(DC)(DG)(DA) (DT)(DT)(DG)(DG)(DC)(DG)(DA)(DT)(DT)(DA) (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

Concentration1.4 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsChromatic aberration corrector: CEOS manufactured Cc corrector
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 11890 / Average exposure time: 4.28 sec. / Average electron dose: 51.37 e/Å2 / Details: Movies were collected in counted-mode
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4un3

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 108043
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8fzt:
SpCas9 with dual-guide RNA and target DNA

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