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- EMDB-29671: SpCas9 with sgRNA and target DNA -

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Open data


ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-29671
TitleSpCas9 with sgRNA and target DNA
Map datadeepemhancer HiRes map
Sample
  • Complex: CRISPR-associated endonuclease Cas9 complex with sgRNA and target DNA
    • RNA: sgRNASubgenomic mRNA
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • DNA: DNA E2_TS
    • DNA: DNA E2_NTS
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) / Renilla reniformis (sea pansy)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.12 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 sgRNA and target DNA
Authors: Korolev S / Gagnon K
History
DepositionFeb 2, 2023-
Header (metadata) releaseMay 17, 2023-
Map releaseMay 17, 2023-
UpdateMay 17, 2023-
Current statusMay 17, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29671.png

Downloads & links

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Map

FileDownload / File: emd_29671.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: 0.07
Minimum - Maximum-0.024584439 - 2.0661013
Average (Standard dev.)0.0012308527 (±0.022518996)
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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Half map: #2

Fileemd_29671_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_29671_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 : CRISPR-associated endonuclease Cas9 complex with sgRNA and target DNA

EntireName: CRISPR-associated endonuclease Cas9 complex with sgRNA and target DNA
Components
  • Complex: CRISPR-associated endonuclease Cas9 complex with sgRNA and target DNA
    • RNA: sgRNASubgenomic mRNA
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • DNA: DNA E2_TS
    • DNA: DNA E2_NTS

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Supramolecule #1: CRISPR-associated endonuclease Cas9 complex with sgRNA and target DNA

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

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Macromolecule #1: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 31.543791 KDa
SequenceString:
UACCAGCAAA ACACUCCGAU GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAG UCGGUGCUU

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Macromolecule #2: CRISPR-associated endonuclease Cas9/Csn1

MacromoleculeName: CRISPR-associated endonuclease Cas9/Csn1 / type: protein_or_peptide / ID: 2 / 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

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Macromolecule #3: DNA E2_TS

MacromoleculeName: DNA E2_TS / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Renilla reniformis (sea pansy)
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 #4: DNA E2_NTS

MacromoleculeName: DNA E2_NTS / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Renilla reniformis (sea pansy)
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

Concentration1.2 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 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 opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
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: 5008 / Average exposure time: 4.28 sec. / Average electron dose: 51.37 e/Å2 / Details: Movies were collected in counted-mode
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

Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.0.3)
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 64788

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8g1i:
SpCas9 with sgRNA and target DNA

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