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- EMDB-29879: Exploiting Activation and Inactivation Mechanisms in Type I-C CRI... -

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Basic information

Entry
Database: EMDB / ID: EMD-29879
TitleExploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications
Map data
Sample
  • Complex: Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-loop formation
    • Protein or peptide: x 5 types
    • RNA: x 1 types
    • DNA: x 3 types
  • Ligand: x 2 types
KeywordsCRISPR / type I-C / Cascade / Anti-CRISPR / DNA BINDING PROTEIN
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding
Similarity search - Function
CRISPR-associated protein Csd2 / CRISPR-associated protein Cas7, subtype I-B/I-C / CRISPR-associated protein Cas7 / CRISPR-associated protein, Csd1-type / CRISPR-associated protein (Cas_Csd1) / CRISPR pre-crRNA endoribonuclease Cas5d / CRISPR-associated protein, Cas5 / CRISPR-associated protein (Cas_Cas5) / CRISPR-associated protein Cas5, N-terminal
Similarity search - Domain/homology
pre-crRNA processing endonuclease / CRISPR-associated protein, Csd1 family / CRISPR-associated protein, Csd2 family
Similarity search - Component
Biological speciesNeisseria lactamica (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsHu C / Nam KH / Ke A
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Research Resources (NIH/NCRR)GM118174 United States
CitationJournal: Mol Cell / Year: 2024
Title: Exploiting activation and inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications.
Authors: Chunyi Hu / Mason T Myers / Xufei Zhou / Zhonggang Hou / Macy L Lozen / Ki Hyun Nam / Yan Zhang / Ailong Ke /
Abstract: Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size ...Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here, we use four cryoelectron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9 that strongly inhibit Neisseria lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through allosteric inhibition, whereas AcrIC9 achieves so through direct competition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for type I CRISPR eukaryotic genome engineering.
History
DepositionFeb 22, 2023-
Header (metadata) releaseMar 6, 2024-
Map releaseMar 6, 2024-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29879.png

Downloads & links

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Map

FileDownload / File: emd_29879.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.27 Å/pix.
x 300 pix.
= 381.3 Å		1.27 Å/pix.
x 300 pix.
= 381.3 Å		1.27 Å/pix.
x 300 pix.
= 381.3 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.271 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.111
Minimum - Maximum-0.27639133 - 0.9470426
Average (Standard dev.)-0.0001906625 (±0.026237516)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 381.30002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #2

Fileemd_29879_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_29879_additional_2.map
Projections & Slices
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Histogram (log scale)

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Half map: #2

Fileemd_29879_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_29879_half_map_2.map
Projections & Slices
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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-lo...

EntireName: Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-loop formation
Components
  • Complex: Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-loop formation
    • Protein or peptide: Cas3
    • Protein or peptide: CRISPR-associated protein, Csd2 family
    • Protein or peptide: Phage associated protein
    • Protein or peptide: CRISPR-associated protein, Csd1 family
    • RNA: crRNA (43-MER)
    • DNA: Traget strand DNA (53-MER)
    • Protein or peptide: pre-crRNA processing endonuclease
    • DNA: proximal nontarget strand DNA (5'-D(P*AP*TP*GP*AP*AP*CP*TP*TP*CP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
    • DNA: Distal nontarget DNA (5'-D(P*TP*TP*AP*TP*AP*TP*TP*AP*AP*TP*AP*TP*TP*AP*TP*AP*TP*TP*TP*A)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHATE ION

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Supramolecule #1: Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-lo...

SupramoleculeName: Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-loop formation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 400 KDa

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Macromolecule #1: Cas3

MacromoleculeName: Cas3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 84.57275 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: DYIAHARQDS SNAWHSHPLQ KHLQKVAQLA KRFAGRYGSL FAEYAGLLHD LGKFQESFQK YIRNASGFEK ENAHLEDVES TKLRKIPHS TAGAKYAVER LNPFFGHLLA YLIAGHHAGL ADWYDKGSLK RRLQQADDEL AASLSGFVES SLPEDFFPLS D DDLMRDFF ...String:
DYIAHARQDS SNAWHSHPLQ KHLQKVAQLA KRFAGRYGSL FAEYAGLLHD LGKFQESFQK YIRNASGFEK ENAHLEDVES TKLRKIPHS TAGAKYAVER LNPFFGHLLA YLIAGHHAGL ADWYDKGSLK RRLQQADDEL AASLSGFVES SLPEDFFPLS D DDLMRDFF AFWEDGAKLE ELHIWMRFLF SCLVDADFLD TEAFMNGYAD ADAAAAAAAA AAAAAAAARR YAEQYAQLSA AE DADKNSS LNQERHAILQ QCFSAAETDR TLFSLTVPTG GGKTLASLGF ALKHALKFGK KRIIYAIPFT SIIEQNANVF RNA LGDDVV LEHHSNLEVK EDKETAKTRL ATENWDAPLI VTTNVQLFES LFAAKTSRCR KIHNIADSVV ILDEAQQLPR DFQK PITDM MRVLARDYGV TFVLCTATQP ELGKNIDAFG RTILEGLPDV REIVADKIAL SEKLRRVRIK MPPPNGETQS WQKIA DEIA ARPCVLAVVN TRKHAQKLFA ALPSNGIKLH LSANMCATHC SEVIALVRRY LALYRAGSLH KPLWLVSTQL IEAGVD LDF PCVYRAMAGL DSIAQAAGRC NREGKLPQLG EVVVFRAEEG APSGSLKQGQ DITEEMLKAG LLDDPLSPLA FAEYFRR FN GKGDVDKHDI TRLLTAEASN ENPLAIKFRT AAERFHLIDN QGVALIVPFI PLAHWEKDGS PQIVEANELD DFFRRHLA A AAAAAAWAAA AAAAAAAFPQ PPDNPDNPFG QTDQPLLAAA AAAAAAAAAA

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Macromolecule #2: CRISPR-associated protein, Csd2 family

MacromoleculeName: CRISPR-associated protein, Csd2 family / type: protein_or_peptide / ID: 2 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 32.208111 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: TIEKRYDFVF LFDVQDGNPN GDPDAGNLPR IDPQTGEGLV TDVCLKRKVR NFIQMTQNDE HHDIFIREKG ILNNLIDEAH EQENVKGKE KGEKTEAARQ YMCSRYYDIR TFGAVMTTGK NAGQVRGPVQ LTFSRSIDPI MTLEHSITRM AVTNEKDASE T GDNRTMGR ...String:
TIEKRYDFVF LFDVQDGNPN GDPDAGNLPR IDPQTGEGLV TDVCLKRKVR NFIQMTQNDE HHDIFIREKG ILNNLIDEAH EQENVKGKE KGEKTEAARQ YMCSRYYDIR TFGAVMTTGK NAGQVRGPVQ LTFSRSIDPI MTLEHSITRM AVTNEKDASE T GDNRTMGR KFTVPYGLYR CHGFISTHFA KQTGFSENDL ELFWQALVNM FDHDHSAARG QMNARGLYVF EHSNNLGDAP AD SLFKRIQ VVKKDGVEVV RSFDDYLVSV DDKNLEETKL LRKLGG

UniProtKB: CRISPR-associated protein, Csd2 family

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Macromolecule #3: Phage associated protein

MacromoleculeName: Phage associated protein / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 14.245184 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GLDRNRQDIG YVLGRLFAVL EKIQAEANPG LNATIADRYF GSASSTPIAV FGTLMRLLPH HLNKLEFEGR AVQLQWEIRQ ILEHCQRFP NHLNLEQQGL FAIGYYHETQ FLFTKDALKN LFNEA

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Macromolecule #4: CRISPR-associated protein, Csd1 family

MacromoleculeName: CRISPR-associated protein, Csd1 family / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 64.718613 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MILHALTQYY QRKAESDGGI AQEGFENKEI PFIIVIDKQG NFIQLEDTRE LKVKKKVGRT FLVPKGLGRS GSKSYEVSNL LWDHYGYVL AYAGEKGQEQ ADKQHASFTA KVNELKQALP DDAGVTAVAA FLSSAEEKSK VMQAANWAEC AKVKGCNLSF R LVDEAVDL ...String:
MILHALTQYY QRKAESDGGI AQEGFENKEI PFIIVIDKQG NFIQLEDTRE LKVKKKVGRT FLVPKGLGRS GSKSYEVSNL LWDHYGYVL AYAGEKGQEQ ADKQHASFTA KVNELKQALP DDAGVTAVAA FLSSAEEKSK VMQAANWAEC AKVKGCNLSF R LVDEAVDL VCQSKAVREY VSQANQTQSD NAQKGICLVT GKAAPIARLH NAVKGVNAKP APFASVNLSA FESYGKEQGF AF PIGEQAM FEYTTALNTL LAGENRFRIG DVTTVCWGAK RTPLEESLAS MINGGGKDNP DAHIDAVKAL YKSLYNGQYC KPD GEDKFY LLGLSPNSAR IVVRFWHETT VAALSESIAA WYDDLQMVRG ENSPYPEYMP LPRLLGNLVL DGKMENLPSD LIAQ ITDAA LNNRVLPVSL LQAALRRNKA EQKITYGRAS LLKAYINRAI RAGRLKNMKE LTMGLDRNRQ DIGYVLGRLF AVLEK IQAE ANPGLNATIA DRYFGSASST PIAVFGTLMR LLPHHLNKLE FEGRAVQLQW EIRQILEHCQ RFPNHLNLEQ QGLFAI GYY HETQFLFTKD ALKNLFNEA

UniProtKB: CRISPR-associated protein, Csd1 family

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Macromolecule #7: pre-crRNA processing endonuclease

MacromoleculeName: pre-crRNA processing endonuclease / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 23.870451 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: RFILEISGDL ACFTRSELKV ERVSYPVITP SAARNILMAI LWKPAIRWKV LKIEILKPIQ WTNIRRNEVG TKMSERSGSL YIEDNRQQR ASMLLKDVAY RIHADFDMTS EAGESDNYVK FAEMFKRRAK KGQYFHQPYL GCREFPCDFR LLEKAEDGLP L EDITQDFG ...String:
RFILEISGDL ACFTRSELKV ERVSYPVITP SAARNILMAI LWKPAIRWKV LKIEILKPIQ WTNIRRNEVG TKMSERSGSL YIEDNRQQR ASMLLKDVAY RIHADFDMTS EAGESDNYVK FAEMFKRRAK KGQYFHQPYL GCREFPCDFR LLEKAEDGLP L EDITQDFG FMLYDMDFSK SDPRDSNNAE PMFYQCKAVN GVITVPP

UniProtKB: pre-crRNA processing endonuclease

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Macromolecule #5: crRNA (43-MER)

MacromoleculeName: crRNA (43-MER) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 13.816202 KDa
SequenceString:
GUUGAAACAG GGUCAGCUUG CCGUAGGUGG CAUCGCCCUC GUC

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Macromolecule #6: Traget strand DNA (53-MER)

MacromoleculeName: Traget strand DNA (53-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 16.410494 KDa
SequenceString: (DA)(DG)(DG)(DA)(DG)(DG)(DG)(DC)(DG)(DA) (DG)(DG)(DG)(DC)(DG)(DA)(DT)(DG)(DC)(DC) (DA)(DC)(DC)(DT)(DA)(DC)(DG)(DG)(DC) (DA)(DA)(DG)(DC)(DT)(DG)(DA)(DC)(DC)(DC) (DT) (DG)(DA)(DA)(DG)(DT)(DT) ...String:
(DA)(DG)(DG)(DA)(DG)(DG)(DG)(DC)(DG)(DA) (DG)(DG)(DG)(DC)(DG)(DA)(DT)(DG)(DC)(DC) (DA)(DC)(DC)(DT)(DA)(DC)(DG)(DG)(DC) (DA)(DA)(DG)(DC)(DT)(DG)(DA)(DC)(DC)(DC) (DT) (DG)(DA)(DA)(DG)(DT)(DT)(DC)(DA) (DT)(DC)(DT)(DG)(DC)

GENBANK: GENBANK: MH290751.1

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Macromolecule #8: proximal nontarget strand DNA (5'-D(P*AP*TP*GP*AP*AP*CP*TP*TP*CP*...

MacromoleculeName: proximal nontarget strand DNA (5'-D(P*AP*TP*GP*AP*AP*CP*TP*TP*CP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 6.160087 KDa
SequenceString:
(DA)(DT)(DG)(DA)(DA)(DC)(DT)(DT)(DC)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)

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Macromolecule #9: Distal nontarget DNA (5'-D(P*TP*TP*AP*TP*AP*TP*TP*AP*AP*TP*AP*TP*...

MacromoleculeName: Distal nontarget DNA (5'-D(P*TP*TP*AP*TP*AP*TP*TP*AP*AP*TP*AP*TP*TP*AP*TP*AP*TP*TP*TP*A)-3')
type: dna / ID: 9 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 6.111011 KDa
SequenceString:
(DT)(DT)(DA)(DT)(DA)(DT)(DT)(DA)(DA)(DT) (DA)(DT)(DT)(DA)(DT)(DA)(DT)(DT)(DT)(DA)

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Macromolecule #10: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 10 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #11: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 11 / Number of copies: 4 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION

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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 mg/mL
BufferpH: 7.5 / Component - Concentration: 150.0 mM / Component - Formula: NaCl / Component - Name: sodium chloride / Details: 25mM Tris pH 7.5, 150mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 70.0 K / Max: 100.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1798 / Number real images: 1798 / Average exposure time: 2.5 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 3.0 µm / Calibrated defocus min: 1.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 67000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionNumber classes used: 6 / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 307088
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-8g9u:
Exploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications

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