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- EMDB-26083: Cascade complex from type I-A CRISPR-Cas system -

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Basic information

Entry
Database: EMDB / ID: EMD-26083
TitleCascade complex from type I-A CRISPR-Cas system
Map data
Sample
  • Complex: Partial R-loop state of Cas3-Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system
    • Protein or peptide: Cas8a
    • Protein or peptide: Cas11a
    • Protein or peptide: Cas7a
    • Protein or peptide: Cas5a
    • RNA: crRNA (45-MER)
    • Protein or peptide: CRISPR-associated helicase Cas3
    • Protein or peptide: CRISPR-associated endonuclease Cas3-HD
    • DNA: Target strand DNA
    • DNA: Non-Target strand DNA
  • Ligand: NICKEL (II) ION
Function / homology
Function and homology information


defense response to virus
Similarity search - Function
CRISPR-associated protein, MJ0385 / CRISPR-associated protein (Cas_Csa4) / CRISPR-associated protein, Cas5a type / CRISPR-associated protein Cas7/Cst2/DevR, subtype I-a/Apern / CRISPR-associated protein Cas7/Cst2/DevR / CRISPR-associated negative auto-regulator DevR/Csa2 / CRISPR-associated protein Cas5, N-terminal
Similarity search - Domain/homology
Type I-A CRISPR-associated protein Cas5 / Uncharacterized protein / Type I-A CRISPR-associated protein Cas7/Csa2 / Type I-A CRISPR-associated protein Cas8a2/Csa4
Similarity search - Component
Biological speciesGeobacter sulfurreducens (bacteria) / Pyrococcus furiosus DSM 3638 (archaea) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsHu C / Ni D / Nam KH / Majumdar S / McLean J / Stahlberg H / Terns M / Ke A
Funding support United States, Switzerland, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118117 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118160 United States
Swiss National Science FoundationNCCR Switzerland
CitationJournal: Mol Cell / Year: 2022
Title: Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools.
Authors: Chunyi Hu / Dongchun Ni / Ki Hyun Nam / Sonali Majumdar / Justin McLean / Henning Stahlberg / Michael P Terns / Ailong Ke /
Abstract: Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme ...Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme Cas3 is then recruited in trans for processive DNA degradation. Contrary to this model, here, we show that type I-A Cascade and Cas3 function as an integral effector complex. We provide four cryoelectron microscopy (cryo-EM) snapshots of the Pyrococcus furiosus (Pfu) type I-A effector complex in different stages of DNA recognition and degradation. The HD nuclease of Cas3 is autoinhibited inside the effector complex. It is only allosterically activated upon full R-loop formation, when the entire targeted region has been validated by the RNA guide. The mechanistic insights inspired us to convert Pfu Cascade-Cas3 into a high-sensitivity, low-background, and temperature-activated nucleic acid detection tool. Moreover, Pfu CRISPR-Cas3 shows robust bi-directional deletion-editing activity in human cells, which could find usage in allele-specific inactivation of disease-causing mutations.
History
DepositionJan 28, 2022-
Header (metadata) releaseAug 10, 2022-
Map releaseAug 10, 2022-
UpdateAug 24, 2022-
Current statusAug 24, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26083.png

Downloads & links

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Map

FileDownload / File: emd_26083.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.24 Å
Density
Contour LevelBy AUTHOR: 0.155
Minimum - Maximum-0.28079575 - 0.8262551
Average (Standard dev.)-0.0011118895 (±0.035560507)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 372.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Partial R-loop state of Cas3-Cascade complex from Pyrococcus furi...

EntireName: Partial R-loop state of Cas3-Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system
Components
  • Complex: Partial R-loop state of Cas3-Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system
    • Protein or peptide: Cas8a
    • Protein or peptide: Cas11a
    • Protein or peptide: Cas7a
    • Protein or peptide: Cas5a
    • RNA: crRNA (45-MER)
    • Protein or peptide: CRISPR-associated helicase Cas3
    • Protein or peptide: CRISPR-associated endonuclease Cas3-HD
    • DNA: Target strand DNA
    • DNA: Non-Target strand DNA
  • Ligand: NICKEL (II) ION

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Supramolecule #1: Partial R-loop state of Cas3-Cascade complex from Pyrococcus furi...

SupramoleculeName: Partial R-loop state of Cas3-Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#9 / Details: Cascade alone
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Molecular weightExperimental: 450 KDa

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Macromolecule #1: Cas8a

MacromoleculeName: Cas8a / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1
Molecular weightTheoretical: 38.800719 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: FNEFKTPQID PIFDLYVAYG YVVSLIRGGA KEATLIPHGA SYLIQTDVSN EEFRHGLVDA LSSMLSLHIA LARHSPREGG KLVSDADFS AGANINNVYW DSVPRNLEKL MKDLEKKRSV KGTATIPITL MPSAGKYMLK HFGVQGGNPI KVDLLNYALA W VGFHYYTP ...String:
FNEFKTPQID PIFDLYVAYG YVVSLIRGGA KEATLIPHGA SYLIQTDVSN EEFRHGLVDA LSSMLSLHIA LARHSPREGG KLVSDADFS AGANINNVYW DSVPRNLEKL MKDLEKKRSV KGTATIPITL MPSAGKYMLK HFGVQGGNPI KVDLLNYALA W VGFHYYTP YIKYAKGDTT WIHIYQIAPV EEVDMISILS LKDLKMHLPH YYESNLDFLI NRRLALLYHL LHSEALELFT EK EFVIHSY TLERSGNNQA IRSFEEEEIG KLMDFLWKLK RRDFYHAIKF IDDLLKKATE GALALIDAIM NERLEGFYTA LKL GKKAGV VSSREIVAAL EDIIC

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Macromolecule #2: Cas11a

MacromoleculeName: Cas11a / type: protein_or_peptide / ID: 2 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1
Molecular weightTheoretical: 12.208933 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GGWIRNIGRY LSYLVDDTFE EYAYDVVDGI AKARTQEELL EGVYKALRLA PKLKKKAESK GCPPPRIPSP EDIEALEEKV EQLSNPKDL RKLAVSLALW AFASWNNCP

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Macromolecule #3: Cas7a

MacromoleculeName: Cas7a / type: protein_or_peptide / ID: 3 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1
Molecular weightTheoretical: 36.989148 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MYVRISGRIR LNAHSLNAQG GGGTNYIEIT KTKVTVRTEN GWTVVEVPAI TGNMLKHWHF VGFVDYFKTT PYGVNLTERA LRYNGTRFG QGETTATKAN GATVQLNDEA TIIKELADAD VHGFLAPKTG RRRVSLVKAS FILPTEDFIK EVEGERLITA I KHNRVDVD ...String:
MYVRISGRIR LNAHSLNAQG GGGTNYIEIT KTKVTVRTEN GWTVVEVPAI TGNMLKHWHF VGFVDYFKTT PYGVNLTERA LRYNGTRFG QGETTATKAN GATVQLNDEA TIIKELADAD VHGFLAPKTG RRRVSLVKAS FILPTEDFIK EVEGERLITA I KHNRVDVD EKGAIGSSKE GTAQMLFSRE YATGLYGFSI VLDLGLVGIP QGLPVKFEEN QPRPNIVIDP NERKARIESA LK ALIPMLS GYIGANLARS FPVFKVEELV AIASEGPIPA LVHGFYEDYI EANRSIIKNA RALGFNIEVF TYNVDLGEDI EAT KVSSVE ELVANLVKMV

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Macromolecule #4: Cas5a

MacromoleculeName: Cas5a / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1
Molecular weightTheoretical: 29.147219 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDILLVCLRF PFFSVAKRSY QVRTSFLLPP PSALKGALAK GLILLKPEKY ASSSLDEAAL KAIKEIESKL VDIKAVSVAP LSPLIRNAF LLKRLRNLES GSNAEKSDAM RREYTFTREL LVAYIFKNLT QEEKNLYLKA AMLIDVIGDT ESLATPVWAS F VKPEDKKA ...String:
MDILLVCLRF PFFSVAKRSY QVRTSFLLPP PSALKGALAK GLILLKPEKY ASSSLDEAAL KAIKEIESKL VDIKAVSVAP LSPLIRNAF LLKRLRNLES GSNAEKSDAM RREYTFTREL LVAYIFKNLT QEEKNLYLKA AMLIDVIGDT ESLATPVWAS F VKPEDKKA PLAFSAPYTE IYSLLSSKIQ AKGKIRMYIE KMRVSPEYSK TKGPQEEIFY LPIEERRYKR IVYYARIYPP EV EKALTVD GEVLGIWIP

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Macromolecule #6: CRISPR-associated helicase Cas3

MacromoleculeName: CRISPR-associated helicase Cas3 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1
Molecular weightTheoretical: 61.412555 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: ELTGFEPYDY QLRAWEKIRE IMNNGGKVII EVPTAGGKTE TAVMPFFAGI YNNNWPVARL VYVLPTRSLV EKQAERLRNL VYKLLQLKG KSKEEAEKLA RELVVVEYGL EKTHAFLGWV VVTTWDAFLY GLAAHRTVGN RFTFPAGAIA QSLVIFDEVQ M YQDESMYM ...String:
ELTGFEPYDY QLRAWEKIRE IMNNGGKVII EVPTAGGKTE TAVMPFFAGI YNNNWPVARL VYVLPTRSLV EKQAERLRNL VYKLLQLKG KSKEEAEKLA RELVVVEYGL EKTHAFLGWV VVTTWDAFLY GLAAHRTVGN RFTFPAGAIA QSLVIFDEVQ M YQDESMYM PRLLSLVVGI LEEANVPLVI MSATIPSKLR EMIAGDTEVI TVDKNDKNKP SKGNVKVRLV EGDITDVLND IK KILKNGK KVLVVRNTVR KAVETYQVLK KKLNDTLANP SDALLIHSRF TIGDRREKER ALDSARLIVA TQVVEAGLDL PNV GLVVTD IAPLDALIQR IGRCARRPGE EGEGIILIPA AAAAAAAAAA AAAAAAAAAA AAAAAAAVVT STNEYDRVVE IHYG EGKKN FVYVGDIDTA RRVLEKKRSK KLPKDLYIIP YSVSPYPDPL VLLTTYDELS KIGEYLADTT KARKALDRVY KFHYE NNIV PKEFASYIYF KELKLFSAPP EYEKAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAI DAKYYNSELA AAAAAA AAA AAAAAAAAA

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Macromolecule #7: CRISPR-associated endonuclease Cas3-HD

MacromoleculeName: CRISPR-associated endonuclease Cas3-HD / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1
Molecular weightTheoretical: 26.057617 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SCKAFQGQTL REHIEAMLAA WEIVKNKYIP SIIRVMKTVG VKFTEEDADK FMKTLIILHD VGKCSEVYQK HLSNNEPLRG FRHELVSAY YAYNILKDMF KDETIAFIGA LVVMMHHEPI LMGQIRSLDK EELTPEVVLD KLRTFNGVME GTESFIKSMI K EKLGVIPK ...String:
SCKAFQGQTL REHIEAMLAA WEIVKNKYIP SIIRVMKTVG VKFTEEDADK FMKTLIILHD VGKCSEVYQK HLSNNEPLRG FRHELVSAY YAYNILKDMF KDETIAFIGA LVVMMHHEPI LMGQIRSLDK EELTPEVVLD KLRTFNGVME GTESFIKSMI K EKLGVIPK VPSPTQEDVL REVIRLSVLA RHRPDSGKLR MVVGALLIPL VCDYKGAAAA AAAAAAAAAA AAAAAAAAA

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Macromolecule #5: crRNA (45-MER)

MacromoleculeName: crRNA (45-MER) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 14.373537 KDa
SequenceString:
AUUGAAAGAG UGCUUCCCCA AACCCUUAAC UGGUUGUAAC AGUUG

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Macromolecule #8: Target strand DNA

MacromoleculeName: Target strand DNA / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 6.929449 KDa
SequenceString:
(DG)(DG)(DG)(DT)(DT)(DG)(DG)(DG)(DG)(DG) (DA)(DA)(DG)(DC)(DA)(DC)(DT)(DG)(DG)(DG) (DT)(DC)

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Macromolecule #9: Non-Target strand DNA

MacromoleculeName: Non-Target strand DNA / type: dna / ID: 9 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 2.411606 KDa
SequenceString:
(DG)(DA)(DC)(DC)(DC)(DA)(DG)(DT)

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Macromolecule #10: NICKEL (II) ION

MacromoleculeName: NICKEL (II) ION / type: ligand / ID: 10 / Number of copies: 2 / Formula: NI
Molecular weightTheoretical: 58.693 Da
Chemical component information

ChemComp-NI:
NICKEL (II) ION / Nickel

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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: NaClSodium chloride / Component - Name: sodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: 6 seconds.

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Electron microscopy

MicroscopeTFS TALOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 1209 / Average exposure time: 2.5 sec. / Average electron dose: 50.0 e/Å2

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Image processing

CTF correctionSoftware - Name: EMAN
Initial angle assignmentType: OTHER
Final 3D classificationSoftware - Name: cryoSPARC
Final angle assignmentType: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 13781

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Atomic model buiding 1

RefinementProtocol: OTHER
Output model

PDB-7tr9:
Cascade complex from type I-A CRISPR-Cas system

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