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- EMDB-32104: Cryo-EM structure of SaCas9-sgRNA-DNA ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-32104
TitleCryo-EM structure of SaCas9-sgRNA-DNA ternary complex
Map data
Sample
  • Complex: SaCas9-sgRNA-target DNA ternary complex
    • Complex: SaCas9
      • Protein or peptide: SaCas9
    • Complex: sgRNA-target DNA
      • RNA: single-guide RNA (sgRNA)
      • DNA: Target DNA strand(TS)
      • DNA: Non-target DNA strand (NTS)
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
: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease ...: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI 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 speciesStaphylococcus aureus (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsDu WH / Huang Q / Zhu HX / Xue DM / Zheng S
Funding support China, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31971377 China
National Natural Science Foundation of China (NSFC)31671386 China
CitationJournal: Int J Mol Sci / Year: 2023
Title: Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State.
Authors: Wenhao Du / Haixia Zhu / Jiaqiang Qian / Dongmei Xue / Sen Zheng / Qiang Huang /
Abstract: Cas9 (SaCas9) is a widely used genome editing tool. Understanding its molecular mechanisms of DNA cleavage could effectively guide the engineering optimization of this system. Here, we determined ... Cas9 (SaCas9) is a widely used genome editing tool. Understanding its molecular mechanisms of DNA cleavage could effectively guide the engineering optimization of this system. Here, we determined the first cryo-electron microscopy structure of the SaCas9-sgRNA-DNA ternary complex. This structure reveals that the HNH nuclease domain is tightly bound to the cleavage site of the target DNA strand, and is in close contact with the WED and REC domains. Moreover, it captures the complete structure of the sgRNA, including the previously unresolved stem-loop 2. Based on this structure, we build a full-length model for the ternary complex in cleavage state. This model enables identification of the residues for the interactions between the HNH domain and the WED and REC domains. Moreover, we found that the stem-loop 2 of the sgRNA tightly binds to the PI and RuvC domains and may also regulate the position shift of the RuvC domain. Further mutagenesis and molecular dynamics simulations supported the idea that the interactions of the HNH domain with the WED and REC domains play an important role in the DNA cleavage. Thus, this study provides new mechanistic insights into the DNA cleavage of SaCas9 and is also useful for guiding the future engineering of SaCas9-mediated gene editing systems.
History
DepositionOct 25, 2021-
Header (metadata) releaseMar 15, 2023-
Map releaseMar 15, 2023-
UpdateMar 15, 2023-
Current statusMar 15, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32104.png

Downloads & links

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Map

FileDownload / File: emd_32104.map.gz / Format: CCP4 / Size: 48.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.0042
Minimum - Maximum-0.025862101 - 0.049840245
Average (Standard dev.)0.00023336892 (±0.0016744635)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions234234234
Spacing234234234
CellA=B=C: 198.90001 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : SaCas9-sgRNA-target DNA ternary complex

EntireName: SaCas9-sgRNA-target DNA ternary complex
Components
  • Complex: SaCas9-sgRNA-target DNA ternary complex
    • Complex: SaCas9
      • Protein or peptide: SaCas9
    • Complex: sgRNA-target DNA
      • RNA: single-guide RNA (sgRNA)
      • DNA: Target DNA strand(TS)
      • DNA: Non-target DNA strand (NTS)

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Supramolecule #1: SaCas9-sgRNA-target DNA ternary complex

SupramoleculeName: SaCas9-sgRNA-target DNA ternary complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Staphylococcus aureus (bacteria)

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Supramolecule #2: SaCas9

SupramoleculeName: SaCas9 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1

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Supramolecule #3: sgRNA-target DNA

SupramoleculeName: sgRNA-target DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#4

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Macromolecule #1: SaCas9

MacromoleculeName: SaCas9 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MKRNYILGLA IGITSVGYGI IDYETRDVID AGVRLFKEAN VENNEGRRSK RGARRLKRRR RHRIQRVKKL LFDYNLLTDH SELSGINPYE ARVKGLSQKL SEEEFSAALL HLAKRRGVHN VNEVEEDTGN ELSTKEQISR NSKALEEKYV ...String:
MGSSHHHHHH SSGLVPRGSH MKRNYILGLA IGITSVGYGI IDYETRDVID AGVRLFKEAN VENNEGRRSK RGARRLKRRR RHRIQRVKKL LFDYNLLTDH SELSGINPYE ARVKGLSQKL SEEEFSAALL HLAKRRGVHN VNEVEEDTGN ELSTKEQISR NSKALEEKYV AELQLERLKK DGEVRGSINR FKTSDYVKEA KQLLKVQKAY HQLDQSFIDT YIDLLETRRT YYEGPGEGSP FGWKDIKEWY EMLMGHCTYF PEELRSVKYA YNADLYNALN DLNNLVITRD ENEKLEYYEK FQIIENVFKQ KKKPTLKQIA KEILVNEEDI KGYRVTSTGK PEFTNLKVYH DIKDITARKE IIENAELLDQ IAKILTIYQS SEDIQEELTN LNSELTQEEI EQISNLKGYT GTHNLSLKAI NLILDELWHT NDNQIAIFNR LKLVPKKVDL SQQKEIPTTL VDDFILSPVV KRSFIQSIKV INAIIKKYGL PNDIIIELAR EKNSKDAQKM INEMQKRNRQ TNERIEEIIR TTGKENAKYL IEKIKLHDMQ EGKCLYSLEA IPLEDLLNNP FNYEVDHIIP RSVSFDNSFN NKVLVKQEEA SKKGNRTPFQ YLSSSDSKIS YETFKKHILN LAKGKGRISK TKKEYLLEER DINRFSVQKD FINRNLVDTR YATRGLMNLL RSYFRVNNLD VKVKSINGGF TSFLRRKWKF KKERNKGYKH HAEDALIIAN ADFIFKEWKK LDKAKKVMEN QMFEEKQAES MPEIETEQEY KEIFITPHQI KHIKDFKDYK YSHRVDKKPN RELINDTLYS TRKDDKGNTL IVNNLNGLYD KDNDKLKKLI NKSPEKLLMY HHDPQTYQKL KLIMEQYGDE KNPLYKYYEE TGNYLTKYSK KDNGPVIKKI KYYGNKLNAH LDITDDYPNS RNKVVKLSLK PYRFDVYLDN GVYKFVTVKN LDVIKKENYY EVNSKCYEEA KKLKKISNQA EFIASFYNND LIKINGELYR VIGVNNDLLN RIEVNMIDIT YREYLENMND KRPPRIIKTI ASKTQSIKKY STDILGNLYE VKSKKHPQII KKG

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Macromolecule #2: single-guide RNA (sgRNA)

MacromoleculeName: single-guide RNA (sgRNA) / type: rna / ID: 2
Source (natural)Organism: synthetic construct (others)
SequenceString:
GGUACCGCUC CAGUCGUUCA UGGUUUUAGU ACUCUGGAAA CAGAAUCUAC UAAAACAAGG CAAAAUGCCG UGUUUAUCUC GUCAACUUGU UGGCGAGAUU UUUUU

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Macromolecule #3: Target DNA strand(TS)

MacromoleculeName: Target DNA strand(TS) / type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
TCCAGAGTAC TAAAACATTC AACATGAACG ACTGGAGCGG TACTATAGTG AGTCGTATT

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Macromolecule #4: Non-target DNA strand (NTS)

MacromoleculeName: Non-target DNA strand (NTS) / type: dna / ID: 4 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
AATACGACTC ACTATAGTAC CGCTCCAGTC GTTCATGTTG AATGTTTTAG TACTCTGGA

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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

Concentration0.22 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMTristrihydroxymethyl aminomethane
100.0 mMKClpotassium chloride
5.0 mMMgCl2magnesium chloride
1.0 mMDTTdithiothreitol

Details: 20mM Tris-Cl (pH 7.5), 100mM KCl, 5mM MgCl2, 1mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK IV
Detailsblot for 4 seconds before pluging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number real images: 10389 / Average electron dose: 38.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3872337
Details: We used the program PARSED developed in our previous study to pick the complex particles from the micrographs(Yao R, et al. Bioinformatics. 2020, 36:1252-1259).
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5czz

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 217173
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.0.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final 3D classificationSoftware - Name: RELION (ver. 3.0.8)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

5czz

,

5y36

)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: correlation coefficient
Output model

PDB-7vw3:
Cryo-EM structure of SaCas9-sgRNA-DNA ternary complex

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