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- EMDB-26081: Cascade complex from type I-A CRISPR-Cas system -

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Basic information

Entry
Database: EMDB / ID: EMD-26081
TitleCascade complex from type I-A CRISPR-Cas system
Map data
Sample
  • Complex: 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)
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: 3.4 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 17, 2022-
Current statusAug 17, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26081.png

Downloads & links

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Map

FileDownload / File: emd_26081.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.99 Å/pix.
x 384 pix.
= 381.3 Å		0.99 Å/pix.
x 384 pix.
= 381.3 Å		0.99 Å/pix.
x 384 pix.
= 381.3 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.99297 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.4645081 - 1.1186278
Average (Standard dev.)-0.00048190198 (±0.033241946)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 381.3 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_26081_additional_1.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 : Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system

EntireName: Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system
Components
  • Complex: 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)

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Supramolecule #1: Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system

SupramoleculeName: Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Cascade alone
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Molecular weightExperimental: 350 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 #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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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
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Pretreatment - Type: PLASMA CLEANING
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
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 800 / 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 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

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Image processing

CTF correctionSoftware - Name: EMAN
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 79651
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final 3D classificationSoftware - Name: cryoSPARC

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Atomic model buiding 1

RefinementProtocol: OTHER
Output model

PDB-7tr6:
Cascade complex from type I-A CRISPR-Cas system

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