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- EMDB-36646: Cryo-EM structure of GeoCas9-sgRNA-dsDNA ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-36646
TitleCryo-EM structure of GeoCas9-sgRNA-dsDNA ternary complex
Map data
Sample
  • Complex: type II CRISPR RNA-guided endonuclease Cas9
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: RNA (139-MER)
    • DNA: DNA (29-MER)
    • DNA: DNA (5'-D(P*GP*GP*GP*CP*GP*CP*GP*AP*A)-3')
KeywordsRNA-binding / DNA-binding / complex / DNA 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, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / HNH endonuclease / CRISPR-associated endonuclease Cas9 / 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 speciesGeobacillus stearothermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsShen PP / Liu BB / Li X / Zhang LL / Chen C-C / Guo R-T
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Acs Catalysis / Year: 2024
Title: Structure of Geobacillus stearothermophilus Cas9: Insights into the Catalytic Process and Thermostability of CRISPR-Cas9
Authors: Shen P / Zhang L / Liu B / Li X / Wang C / Min J / Huang JW / Chen CC / Guo RT
History
DepositionJun 21, 2023-
Header (metadata) releaseJun 5, 2024-
Map releaseJun 5, 2024-
UpdateDec 18, 2024-
Current statusDec 18, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_36646.png

Downloads & links

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Map

FileDownload / File: emd_36646.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 256 pix.
= 217.6 Å		0.85 Å/pix.
x 256 pix.
= 217.6 Å		0.85 Å/pix.
x 256 pix.
= 217.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.095
Minimum - Maximum-0.7086643 - 1.0927348
Average (Standard dev.)0.0010637485 (±0.028471377)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 217.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_36646_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_36646_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 : type II CRISPR RNA-guided endonuclease Cas9

EntireName: type II CRISPR RNA-guided endonuclease Cas9
Components
  • Complex: type II CRISPR RNA-guided endonuclease Cas9
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: RNA (139-MER)
    • DNA: DNA (29-MER)
    • DNA: DNA (5'-D(P*GP*GP*GP*CP*GP*CP*GP*AP*A)-3')

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Supramolecule #1: type II CRISPR RNA-guided endonuclease Cas9

SupramoleculeName: type II CRISPR RNA-guided endonuclease Cas9 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 130 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
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 128.171031 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MRYKIGLDIG ITSVGWAVMN LDIPRIEDLG VRIFDRAENP QTGESLALPR RLARSARRRL RRRKHRLERI RRLVIREGIL TKEELDKLF EEKHEIDVWQ LRVEALDRKL NNDELARVLL HLAKRRGFKS NRKSERSNKE NSTMLKHIEE NRAILSSYRT V GEMIVKDP ...String:
MRYKIGLDIG ITSVGWAVMN LDIPRIEDLG VRIFDRAENP QTGESLALPR RLARSARRRL RRRKHRLERI RRLVIREGIL TKEELDKLF EEKHEIDVWQ LRVEALDRKL NNDELARVLL HLAKRRGFKS NRKSERSNKE NSTMLKHIEE NRAILSSYRT V GEMIVKDP KFALHKRNKG ENYTNTIARD DLEREIRLIF SKQREFGNMS CTEEFENEYI AIWASQRPVA SKDDIEKKVG FC AFEPKEK RAPKATYTFQ SFIAWEHINK LRLISPSGAR GLTDEERRLL YEQAFQKNKI TYHDIRTLLH LPDDTYFKGI VYD RGESRK QNENIRFLEL DAYHQIRKAV DKVYGKEKSS SFLPIDFDTF GYALTLFKDD ADIHSYLRNE YEQNGKRMPN LANK VYDNE LIEELLNLSF TKFGHLSLKA LRSILPYMEQ GEVYSSACER AGYTFTGPKK KQKTMLLPNI PPIANPVVMR ALTQA RKVV NAIIKKYGSP VSIHIELARD LSQTFDERRK TKKEQDENRK KNETAIRQLM EYGLTLNPTG HDIVKFKLWS EQNGRC AYS LQPIEIERLL EPGYVEVDAV IPYSRSLDDS YTNKVLVLTR ENREKGNRIP AEYLGVGTER WQQFETFVLT NKQFSKK KR DRLLRLHYDE NEETEFKNRN LNDTRYISRF FANFIREHLK FAESDDKQKV YTVNGRVTAH LRSRWEFNKN REESDLHH A VDAVIVACTT PSDIAKVTAF YQRREQNKEL AKKTEPHFPQ PWPHFADELR ARLSKHPKES IKALNLGNYD DQKLESLQP VFVSRMPKRS VTGAAHQETL RRYVGIDERS GKIQTVVKTK LSEIKLDASG HFPMYGKESD PRTYEAIRQR LLEHNNDPKK AFQEPLYKP KKNGEPGPVI RTVKIIDTKN QVIPLNDGKT VAYNSNIVRV DVFEKDGKYY CVPVYTMDIM KGILPNKAIE P NKPYSEWK EMTEDYTFRF SLYPNDLIRI ELPREKTVKT AAGEEINVKD VFVYYKTIDS ANGGLELISH DHRFSLRGVG SR TLKRFEK YQVDVLGNIY KVRGEKRVGL ASSAHSKPGK TIRPLQSTRD LEHHHHHH

UniProtKB: CRISPR-associated endonuclease Cas9

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Macromolecule #2: RNA (139-MER)

MacromoleculeName: RNA (139-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 44.697402 KDa
SequenceString:
GGCGCAUAAA GAUGAGACGC GGUCAUAGUU CCCCUGAGAA AUCAGGGUUA CUAUGAUAAG GGCUUUCUGC CUAAGGCAGA CUGACCCGC GGCGUUGGGG AUCGCCUGUC GCCCGCUUUU GGCGGGCAUU CCCCAUCCUU

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Macromolecule #3: DNA (29-MER)

MacromoleculeName: DNA (29-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 8.764607 KDa
SequenceString:
(DT)(DT)(DC)(DG)(DC)(DG)(DC)(DC)(DC)(DG) (DC)(DG)(DT)(DC)(DT)(DC)(DA)(DT)(DC)(DT) (DT)(DT)(DA)(DT)(DG)(DC)(DG)(DC)(DC)

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Macromolecule #4: DNA (5'-D(P*GP*GP*GP*CP*GP*CP*GP*AP*A)-3')

MacromoleculeName: DNA (5'-D(P*GP*GP*GP*CP*GP*CP*GP*AP*A)-3') / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 2.805849 KDa
SequenceString:
(DG)(DG)(DG)(DC)(DG)(DC)(DG)(DA)(DA)

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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
Details: 20 mM Tris-HCl, 0.1 M KCl, 5 mM MgCl2, 1 mM DTT, 0.5% Glycerol
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 389973
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8jtj:
Cryo-EM structure of GeoCas9-sgRNA-dsDNA ternary complex

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