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- EMDB-27144: Structure of Acidothermus cellulolyticus Cas9 ternary complex (Po... -

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Basic information

Entry
Database: EMDB / ID: EMD-27144
TitleStructure of Acidothermus cellulolyticus Cas9 ternary complex (Post-cleavage 2)
Map dataAccas9 ternary complex (Post-cleavage2)
Sample
  • Complex: CryoEM Structure of AceCas9 (Post-cleavage 2)
    • Complex: CRISPR-associated endonuclease, Csn1 family/RNA
      • Protein or peptide: CRISPR-associated endonuclease, Csn1 family
      • RNA: Single guide RNA (106-MER)
      • DNA: DNA target strand (5'-D(*AP*GP*CP*TP*TP*GP*GP*TP*GP*TP*AP*TP*A)-3')
    • Complex: DNA
      • DNA: DNA target strand (5'-D(P*CP*CP*AP*GP*GP*AP*TP*CP*TP*TP*G)-3')
      • DNA: DNA non-target strand (5'-D(P*AP*TP*AP*CP*AP*CP*CP*AP*AP*GP*CP*T)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsCas9 / AcCas9 / Crispr / Post-cleavage 2 / RNA BINDING PROTEIN / RNA BINDING PROTEIN-DNA-RNA complex
Function / homology
Function and homology information


defense response to virus / endonuclease activity / DNA binding / RNA binding / zinc ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, beta-hairpin domain / Cas9 C-terminal domain / Cas9, topo homolgy domain / CRISPR-associated endonuclease Cas9 beta-hairpin domain / Topo homolgy domain in CRISPR-associated endonuclease Cas9 / Cas9 C-terminal domain / Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / HNH endonuclease / HNH endonuclease ...CRISPR-associated endonuclease Cas9, beta-hairpin domain / Cas9 C-terminal domain / Cas9, topo homolgy domain / CRISPR-associated endonuclease Cas9 beta-hairpin domain / Topo homolgy domain in CRISPR-associated endonuclease Cas9 / Cas9 C-terminal domain / Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / HNH endonuclease / HNH endonuclease / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / HNH nucleases / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
CRISPR-associated endonuclease, Csn1 family
Similarity search - Component
Biological speciesAcidothermus cellulolyticus 11B (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsRai J / Das A / Li H
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM101343 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM099604 United States
CitationJournal: Nat Catal / Year: 2023
Title: Coupled catalytic states and the role of metal coordination in Cas9
Authors: Das A / Rai J / Roth MO / Shu Y / Medina ML / Barakat MR / Li H
History
DepositionMay 30, 2022-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateDec 20, 2023-
Current statusDec 20, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27144.png

Downloads & links

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Map

FileDownload / File: emd_27144.map.gz / Format: CCP4 / Size: 107.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAccas9 ternary complex (Post-cleavage2)
Voxel sizeX=Y=Z: 0.825 Å
Density
Contour LevelBy AUTHOR: 0.016
Minimum - Maximum-0.105928265 - 0.20932604
Average (Standard dev.)0.00009882948 (±0.0047566313)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions304304304
Spacing304304304
CellA=B=C: 250.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map2

Fileemd_27144_half_map_1.map
AnnotationHalf map2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map1

Fileemd_27144_half_map_2.map
AnnotationHalf map1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CryoEM Structure of AceCas9 (Post-cleavage 2)

EntireName: CryoEM Structure of AceCas9 (Post-cleavage 2)
Components
  • Complex: CryoEM Structure of AceCas9 (Post-cleavage 2)
    • Complex: CRISPR-associated endonuclease, Csn1 family/RNA
      • Protein or peptide: CRISPR-associated endonuclease, Csn1 family
      • RNA: Single guide RNA (106-MER)
      • DNA: DNA target strand (5'-D(*AP*GP*CP*TP*TP*GP*GP*TP*GP*TP*AP*TP*A)-3')
    • Complex: DNA
      • DNA: DNA target strand (5'-D(P*CP*CP*AP*GP*GP*AP*TP*CP*TP*TP*G)-3')
      • DNA: DNA non-target strand (5'-D(P*AP*TP*AP*CP*AP*CP*CP*AP*AP*GP*CP*T)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: CryoEM Structure of AceCas9 (Post-cleavage 2)

SupramoleculeName: CryoEM Structure of AceCas9 (Post-cleavage 2) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5

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Supramolecule #2: CRISPR-associated endonuclease, Csn1 family/RNA

SupramoleculeName: CRISPR-associated endonuclease, Csn1 family/RNA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: Acidothermus cellulolyticus 11B (bacteria)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #4-#5

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Macromolecule #1: CRISPR-associated endonuclease, Csn1 family

MacromoleculeName: CRISPR-associated endonuclease, Csn1 family / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Acidothermus cellulolyticus 11B (bacteria) / Strain: ATCC 43068 / DSM 8971 / 11B
Molecular weightTheoretical: 127.498094 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGGSEVGTVP VTWRLGVDVG ERSIGLAAVS YEEDKPKEIL AAVSWIHDGG VGDERSGASR LALRGMARRA RRLRRFRRAR LRDLDMLLS ELGWTPLPDK NVSPVDAWLA RKRLAEEYVV DETERRRLLG YAVSHMARHR GWRNPWTTIK DLKNLPQPSD S WERTRESL ...String:
MGGSEVGTVP VTWRLGVDVG ERSIGLAAVS YEEDKPKEIL AAVSWIHDGG VGDERSGASR LALRGMARRA RRLRRFRRAR LRDLDMLLS ELGWTPLPDK NVSPVDAWLA RKRLAEEYVV DETERRRLLG YAVSHMARHR GWRNPWTTIK DLKNLPQPSD S WERTRESL EARYSVSLEP GTVGQWAGYL LQRAPGIRLN PTQQSAGRRA ELSNATAFET RLRQEDVLWE LRCIADVQGL PE DVVSNVI DAVFCQKRPS VPAERIGRDP LDPSQLRASR ACLEFQEYRI VAAVANLRIR DGSGSRPLSL EERNAVIEAL LAQ TERSLT WSDIALEILK LPNESDLTSV PEEDGPSSLA YSQFAPFDET SARIAEFIAK NRRKIPTFAQ WWQEQDRTSR SDLV AALAD NSIAGEEEQE LLVHLPDAEL EALEGLALPS GRVAYSRLTL SGLTRVMRDD GVDVHNARKT CFGVDDNWRP PLPAL HEAT GHPVVDRNLA ILRKFLSSAT MRWGPPQSIV VELARGASES RERQAEEEAA RRAHRKANDR IRAELRASGL SDPSPA DLV RARLLELYDC HCMYCGAPIS WENSELDHIV PRTDGGSNRH ENLAITCGAC NKEKGRRPFA SWAETSNRVQ LRDVIDR VQ KLKYSGNMYW TRDEFSRYKK SVVARLKRRT SDPEVIQSIE STGYAAVALR DRLLSYGEKN GVAQVAVFRG GVTAEARR W LDISIERLFS RVAIFAQSTS TKRLDRRHHA VDAVVLTTLT PGVAKTLADA RSRRVSAEFW RRPSDVNRHS TEEPQSPAY RQWKESCSGL GDLLISTAAR DSIAVAAPLR LRPTGALHEE TLRAFSEHTV GAAWKGAELR RIVEPEVYAA FLALTDPGGR FLKVSPSED VLPADENRHI VLSDRVLGPR DRVKLFPDDR GSIRVRGGAA YIASFHHARV FRWGSSHSPS FALLRVSLAD L AVAGLLRD GVDVFTAELP PWTPAWRYAS IALVKAVESG DAKQVGWLVP GDELDFGPEG VTTAAGDLSM FLKYFPERHW VV TGFEDDK RINLKPAFLS AEQAEVLRTE RSDRPDTLTE AGEILAQFFP RCWRATVAKV LCHPGLTVIR RTALGQPRWR RGH LPYSWR PWSADPWSGG TP

UniProtKB: CRISPR-associated endonuclease, Csn1 family

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Macromolecule #2: Single guide RNA (106-MER)

MacromoleculeName: Single guide RNA (106-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Acidothermus cellulolyticus 11B (bacteria) / Strain: ATCC 43068 / DSM 8971 / 11B
Molecular weightTheoretical: 34.190301 KDa
SequenceString:
GGUAGGAUGG CAAGAUCCUG GUAUGCUGGG GAGCCUGAAA AGGCUACCUA GCAAGACCCC UUCGUGGGGU CGCAUUCUUC ACCCCCUCG CAGCAGCGAG GGGUUCC

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Macromolecule #3: DNA target strand (5'-D(*AP*GP*CP*TP*TP*GP*GP*TP*GP*TP*AP*TP*A)-3')

MacromoleculeName: DNA target strand (5'-D(*AP*GP*CP*TP*TP*GP*GP*TP*GP*TP*AP*TP*A)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.021632 KDa
SequenceString:
(DA)(DG)(DC)(DT)(DT)(DG)(DG)(DT)(DG)(DT) (DA)(DT)(DA)

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Macromolecule #4: DNA target strand (5'-D(P*CP*CP*AP*GP*GP*AP*TP*CP*TP*TP*G)-3')

MacromoleculeName: DNA target strand (5'-D(P*CP*CP*AP*GP*GP*AP*TP*CP*TP*TP*G)-3')
type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.349197 KDa
SequenceString:
(DC)(DC)(DA)(DG)(DG)(DA)(DT)(DC)(DT)(DT) (DG)

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Macromolecule #5: DNA non-target strand (5'-D(P*AP*TP*AP*CP*AP*CP*CP*AP*AP*GP*CP*T)-3')

MacromoleculeName: DNA non-target strand (5'-D(P*AP*TP*AP*CP*AP*CP*CP*AP*AP*GP*CP*T)-3')
type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.615395 KDa
SequenceString:
(DA)(DT)(DA)(DC)(DA)(DC)(DC)(DA)(DA)(DG) (DC)(DT)

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 3 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / 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: 2.2 µm / Nominal defocus min: 1.0 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: OTHER
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 169296

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