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- EMDB-24976: Structure of type I-D Cascade bound to a ssRNA target -

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Basic information

Entry
Database: EMDB / ID: EMD-24976
TitleStructure of type I-D Cascade bound to a ssRNA target
Map dataCRISPR-Cas type I-D complex bound to an ssRNA target
Sample
  • Complex: Type I-D Cascade bound to a ssRNA target
    • Protein or peptide: Cas7d
    • Protein or peptide: Cas5d
    • Protein or peptide: Cas10d
    • Protein or peptide: Cas11d
    • RNA: ssRNA target
    • RNA: crRNA
KeywordsCRISPR / Complex / Ribonucleoprotein complex / type I-D / type ID / type I / cyanobacteria / synechocystis / RNA binding protein / RNA BINDING PROTEIN-RNA complex
Function / homologyCRISPR-associated protein Csc1 / CRISPR associated protein Csc3 / CRISPR-associated protein Csc2 / Csc2 Crispr / Slr7013 protein / Slr7012 protein / CRISPR-associated protein Csc3
Function and homology information
Biological speciesSynechocystis sp. PCC 6803 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSchwartz EA / Taylor DW
Funding support United States, 1 items
OrganizationGrant numberCountry
Welch FoundationF-1938 United States
CitationJournal: Nat Commun / Year: 2022
Title: Structural rearrangements allow nucleic acid discrimination by type I-D Cascade.
Authors: Evan A Schwartz / Tess M McBride / Jack P K Bravo / Daniel Wrapp / Peter C Fineran / Robert D Fagerlund / David W Taylor /
Abstract: CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. ...CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. Interestingly, the type I-D interference proteins contain characteristic features of both type I and type III systems. Here, we present the structures of type I-D Cascade bound to both a double-stranded (ds)DNA and a single-stranded (ss)RNA target at 2.9 and 3.1 Å, respectively. We show that type I-D Cascade is capable of specifically binding ssRNA and reveal how PAM recognition of dsDNA targets initiates long-range structural rearrangements that likely primes Cas10d for Cas3' binding and subsequent non-target strand DNA cleavage. These structures allow us to model how binding of the anti-CRISPR protein AcrID1 likely blocks target dsDNA binding via competitive inhibition of the DNA substrate engagement with the Cas10d active site. This work elucidates the unique mechanisms used by type I-D Cascade for discrimination of single-stranded and double stranded targets. Thus, our data supports a model for the hybrid nature of this complex with features of type III and type I systems.
History
DepositionSep 24, 2021-
Header (metadata) releaseJun 8, 2022-
Map releaseJun 8, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24976.png

Downloads & links

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Map

FileDownload / File: emd_24976.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCRISPR-Cas type I-D complex bound to an ssRNA target
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 440 pix.
= 459.8 Å		1.05 Å/pix.
x 440 pix.
= 459.8 Å		1.05 Å/pix.
x 440 pix.
= 459.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.045 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.0017209093 - 1.6268128
Average (Standard dev.)0.0005333639 (±0.014780736)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 459.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Type I-D Cascade bound to a ssRNA target

EntireName: Type I-D Cascade bound to a ssRNA target
Components
  • Complex: Type I-D Cascade bound to a ssRNA target
    • Protein or peptide: Cas7d
    • Protein or peptide: Cas5d
    • Protein or peptide: Cas10d
    • Protein or peptide: Cas11d
    • RNA: ssRNA target
    • RNA: crRNA

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Supramolecule #1: Type I-D Cascade bound to a ssRNA target

SupramoleculeName: Type I-D Cascade bound to a ssRNA target / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 450 KDa

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Macromolecule #1: Cas7d

MacromoleculeName: Cas7d / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Molecular weightTheoretical: 36.527109 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MLDSLKSQFQ PSFPRLASGH YVHFLMLRHS QSFPVFQTDG VLNTTRTQAG LLEKTDQLSR LVMFKRKQTT PERLAGRELL RNLGLTSAD KSAKNLCEYN GEGSCKQCPD CILYGFAIGD SGSERSKVYS DSAFSLGAYE QSHRSFTFNA PFEGGTMSEA G VMRSAINE ...String:
MLDSLKSQFQ PSFPRLASGH YVHFLMLRHS QSFPVFQTDG VLNTTRTQAG LLEKTDQLSR LVMFKRKQTT PERLAGRELL RNLGLTSAD KSAKNLCEYN GEGSCKQCPD CILYGFAIGD SGSERSKVYS DSAFSLGAYE QSHRSFTFNA PFEGGTMSEA G VMRSAINE LDHILPEVTF PTVESLRDAT YEGFIYVLGN LLRTKRYGAQ ESRTGTMKNH LVGIVFADGE IFSNLHLTQA LY DQMGGEL NKPISELCET AATVAQDLLN KEPVRKSELI FGAHLDTLLQ EVNDIYQNDA ELTKLLGSLY QQTQDYATEF GAL SGGKKK AKS

UniProtKB: Slr7012 protein

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Macromolecule #2: Cas5d

MacromoleculeName: Cas5d / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Molecular weightTheoretical: 28.942748 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTKIYRCKLT LHDNVFFASR EMGILYETEK YFHNWALSYA FFKGTIIPHP YGLVGQNAQT PAYLDRDREQ NLLHLNDSGI YVFPAQPIH WSYQINTFKA AQSAYYGRSV QFGGKGATKN YPINYGRAKE LAVGSEFLTY IVSQKELDLP VWIRLGKWSS K IRVEVEAI ...String:
MTKIYRCKLT LHDNVFFASR EMGILYETEK YFHNWALSYA FFKGTIIPHP YGLVGQNAQT PAYLDRDREQ NLLHLNDSGI YVFPAQPIH WSYQINTFKA AQSAYYGRSV QFGGKGATKN YPINYGRAKE LAVGSEFLTY IVSQKELDLP VWIRLGKWSS K IRVEVEAI APDQIKTASG VYVCNHPLNP LDCPANQQIL LYNRVVMPPS SLFSQSQLQG DYWQIDRNTF LPQGFHYGAT TA IAQDSPQ LSLLDTN

UniProtKB: Slr7013 protein

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Macromolecule #3: Cas10d

MacromoleculeName: Cas10d / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Molecular weightTheoretical: 112.067508 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTTLLQTLLI RTLSEQKDYI LLEYFQTILP ALEEHFGNTS GLGGSFISHQ KHFGTQGYDT EKAKKMAQGF AKKGDQTLAA HILNALLTT WNVMQELEFP LNDIERRLLC LGITLHDYDK HCHAQDMAAP EPDNIQEIIN ICLELGKRLN FDEFWADWRD Y IAEISYLA ...String:
MTTLLQTLLI RTLSEQKDYI LLEYFQTILP ALEEHFGNTS GLGGSFISHQ KHFGTQGYDT EKAKKMAQGF AKKGDQTLAA HILNALLTT WNVMQELEFP LNDIERRLLC LGITLHDYDK HCHAQDMAAP EPDNIQEIIN ICLELGKRLN FDEFWADWRD Y IAEISYLA QNTHGKQHTN LISSNWSNAG YPFTIKERKL DHPLRHLLTF GDVAVHLSSP HDLVSSTMGD RLRDLLNRLG IE KRFVYHH LRDTTGILSN AIHNVILRTV QKLDWKPLLF FAQGVIYFAP QDTEIPERNE IKQIVWQGIS QELGKKMSAG DVG FKRDGK GLKVSPQTSE LLAAADIVRI LPQVISVKVN NAKSPATPKR LEKLELGDAE REKLYEVADL RCDRLAELLG LVQK EIFLL PEPFIEWVLK DLELTSVIMP EETQVQSGGV NYGWYRVAAH YVANHATWDL EEFQEFLQGF GDRLATWAEE EGYFA EHQS PTRQIFEDYL DRYLEIQGWE SDHQAFIQEL ENYVNAKTKK SKQPICSLSS GEFPSEDQMD SVVLFKPQQY SNKNPL GGG QIKRGISKIW SLEMLLRQAF WSVPSGKFED QQPIFIYLYP AYVYAPQVVE AIRELVYGIA SVNLWDVRKH WVNNKMD LT SLKSLPWLNE EVEAGTNAQL KYTKEDLPFL ATVYTTTREK TDTDAWVKPA FLALLLPYLL GVKAIATRSM VPLYRSDQ D FRESIHLDGV AGFWSLLGIP TDLRVEDITP ALNKLLAIYT LHLAARSSPP KARWQDLPKT VQEVMTDVLN VFALAEQGL RREKRDRPYE SEVTEYWQFA ELFSQGNIVM TEKLKLTKRL VEEYRRFYQV ELSKKPSTHA ILLPLSKALE QILSVPDDWD EEELILQGS GQLQAALDRQ EVYTRPIIKD KSVAYETRQL QELEAIQIFM TTCVRDLFGE MCKGDRAILQ EQRNRIKSGA E FAYRLLAL EAQQNQN

UniProtKB: CRISPR-associated protein Csc3

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Macromolecule #4: Cas11d

MacromoleculeName: Cas11d / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Molecular weightTheoretical: 17.024494 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MTEKLKLTKR LVEEYRRFYQ VELSKKPSTH AILLPLSKAL EQILSVPDDW DEEELILQGS GQLQAALDRQ EVYTRPIIKD KSVAYETRQ LQELEAIQIF MTTCVRDLFG EMCKGDRAIL QEQRNRIKSG AEFAYRLLAL EAQQNQN

UniProtKB: CRISPR-associated protein Csc3

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Macromolecule #5: ssRNA target

MacromoleculeName: ssRNA target / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 10.700537 KDa
SequenceString:
AGGCAUUGAA AGCGACCACC AGGGGCACAA CAA

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Macromolecule #6: crRNA

MacromoleculeName: crRNA / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Molecular weightTheoretical: 13.699081 KDa
SequenceString:
ACUGAAACGA UUGUUGUGCC CCUGGCGGUC GCUUUCAAUG CCU

GENBANK: GENBANK: CP073020.1

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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

Concentration0.16 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
100.0 mMNaClsodium chloride
20.0 mMHEPES
1.0 mMDTT
5.0 %Glycerol
GridModel: C-flat / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Blot force 0, blot time 4s, no wait time between sample application and blotting..
DetailsMonodisperse sample of elongated particles.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Average exposure time: 3.0 sec. / Average electron dose: 41.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4100000
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 167000
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsModel was built de novo using coot then flexibly refined using ISOLDE.
Output model

PDB-7sbb:
Structure of type I-D Cascade bound to a ssRNA target

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