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- EMDB-39859: Cryo-EM structure of NTR-bound type VII CRISPR-Cas complex at sub... -

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Basic information

Entry
Database: EMDB / ID: EMD-39859
TitleCryo-EM structure of NTR-bound type VII CRISPR-Cas complex at substrate-engaged state I
Map data
Sample
  • Complex: protein structure
    • Protein or peptide: Protein structure
    • Protein or peptide: Protein structure
    • Protein or peptide: Protein structure
    • RNA: RNA (48-MER)
    • RNA: RNA (41-MER)
  • Ligand: ZINC ION
Keywordsprotein structure / ANTIVIRAL PROTEIN
Biological speciesmetagenome (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsZhang H / Deng Z / Li X
Funding support China, 1 items
OrganizationGrant numberCountry
Other government China
CitationJournal: Nature / Year: 2024
Title: Structural basis for the activity of the type VII CRISPR-Cas system.
Authors: Jie Yang / Xuzichao Li / Qiuqiu He / Xiaoshen Wang / Jingjing Tang / Tongyao Wang / Yi Zhang / Feiyang Yu / Shuqin Zhang / Zhikun Liu / Lingling Zhang / Fumeng Liao / Hang Yin / Haiyan Zhao ...Authors: Jie Yang / Xuzichao Li / Qiuqiu He / Xiaoshen Wang / Jingjing Tang / Tongyao Wang / Yi Zhang / Feiyang Yu / Shuqin Zhang / Zhikun Liu / Lingling Zhang / Fumeng Liao / Hang Yin / Haiyan Zhao / Zengqin Deng / Heng Zhang /
Abstract: The newly identified type VII CRISPR-Cas candidate system uses a CRISPR RNA-guided ribonucleoprotein complex formed by Cas5 and Cas7 proteins to target RNA. However, the RNA cleavage is executed by a ...The newly identified type VII CRISPR-Cas candidate system uses a CRISPR RNA-guided ribonucleoprotein complex formed by Cas5 and Cas7 proteins to target RNA. However, the RNA cleavage is executed by a dedicated Cas14 nuclease, which is distinct from the effector nucleases of the other CRISPR-Cas systems. Here we report seven cryo-electron microscopy structures of the Cas14-bound interference complex at different functional states. Cas14, a tetrameric protein in solution, is recruited to the Cas5-Cas7 complex in a target RNA-dependent manner. The N-terminal catalytic domain of Cas14 binds a stretch of the substrate RNA for cleavage, whereas the C-terminal domain is primarily responsible for tethering Cas14 to the Cas5-Cas7 complex. The biochemical cleavage assays corroborate the captured functional conformations, revealing that Cas14 binds to different sites on the Cas5-Cas7 complex to execute individual cleavage events. Notably, a plugged-in arginine of Cas7 sandwiched by a C-shaped clamp of C-terminal domain precisely modulates Cas14 binding. More interestingly, target RNA cleavage is altered by a complementary protospacer flanking sequence at the 5' end, but not at the 3' end. Altogether, our study elucidates critical molecular details underlying the assembly of the interference complex and substrate cleavage in the type VII CRISPR-Cas system, which may help rational engineering of the type VII CRISPR-Cas system for biotechnological applications.
History
DepositionApr 23, 2024-
Header (metadata) releaseAug 21, 2024-
Map releaseAug 21, 2024-
UpdateNov 27, 2024-
Current statusNov 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_39859.png

Downloads & links

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Map

FileDownload / File: emd_39859.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.95 Å/pix.
x 300 pix.
= 285. Å		0.95 Å/pix.
x 300 pix.
= 285. Å		0.95 Å/pix.
x 300 pix.
= 285. Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.95 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-1.0245827 - 2.1544547
Average (Standard dev.)0.0017444444 (±0.04852937)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 285.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_39859_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_39859_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : protein structure

EntireName: protein structure
Components
  • Complex: protein structure
    • Protein or peptide: Protein structure
    • Protein or peptide: Protein structure
    • Protein or peptide: Protein structure
    • RNA: RNA (48-MER)
    • RNA: RNA (41-MER)
  • Ligand: ZINC ION

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Supramolecule #1: protein structure

SupramoleculeName: protein structure / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: metagenome (others)

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Macromolecule #1: Protein structure

MacromoleculeName: Protein structure / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 22.255604 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAKTMKKIYV TMKTLSPLYT GEVRREDKEA AQKRVNFPVR KTATNKVLIP FKGALRSALE IMLKAKGENV CDTGESRARP CGRCVTCSL FGSMGRAGRA SVDFLISNDT KEQIVRESTH LRIERQTKSA SDTFKGEEVI EGATFTATIT ISNPQEKDLS L IQSALKFI ...String:
MAKTMKKIYV TMKTLSPLYT GEVRREDKEA AQKRVNFPVR KTATNKVLIP FKGALRSALE IMLKAKGENV CDTGESRARP CGRCVTCSL FGSMGRAGRA SVDFLISNDT KEQIVRESTH LRIERQTKSA SDTFKGEEVI EGATFTATIT ISNPQEKDLS L IQSALKFI EENGIGGWLN KGYGRVSFEV KSEDVATDRF LK

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Macromolecule #2: Protein structure

MacromoleculeName: Protein structure / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 27.139533 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKEIKGILES ITGFSIPLDN GEYALYPAGR HLRGAIGYIA FNLDLPISSK FLDFDFDDII FRDLLPISKC GKIFYPEKNS NSLKCPSCN EIYGSSVLRN IMARGLSYKE VIEGKKYRLS IIVKDEKYLN EMEAIIRYIL SYGIYLGNKV SKGYGKFKIK E YSIVDILP ...String:
MKEIKGILES ITGFSIPLDN GEYALYPAGR HLRGAIGYIA FNLDLPISSK FLDFDFDDII FRDLLPISKC GKIFYPEKNS NSLKCPSCN EIYGSSVLRN IMARGLSYKE VIEGKKYRLS IIVKDEKYLN EMEAIIRYIL SYGIYLGNKV SKGYGKFKIK E YSIVDILP VKDSEVLLLS DAIIDNGEKD IVFSKKEISS SKFEIIRKRG KAKGDIIRDN NHNGFYIGKY GGLGFGEIIS LK

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Macromolecule #3: Protein structure

MacromoleculeName: Protein structure / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 70.468898 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIKFIGGASK VTGSAFLLET GNAKILIDCG IEQEKGIEKD NNEIIEKKIN EIGKADICIL THAHLAASGL VPLLVKKRKV NKIISTPAT KELCRLLFND FQRIQEENND IPLYSYDDIE SSFEIWDEID DRNTIELFDT KITFYNNSHI IGSVSVFIET H NGNYLFSG ...String:
MIKFIGGASK VTGSAFLLET GNAKILIDCG IEQEKGIEKD NNEIIEKKIN EIGKADICIL THAHLAASGL VPLLVKKRKV NKIISTPAT KELCRLLFND FQRIQEENND IPLYSYDDIE SSFEIWDEID DRNTIELFDT KITFYNNSHI IGSVSVFIET H NGNYLFSG DIGSKLQQLM DYPPDMPDGN VDYLILESTY GNKSHDSSDR DRLLEIAKTT CENGGKVLIP SFAIGRLQEV LY TFSNYNF NFPVYIDSPM GSKVTNLIKE YNIYLKKKLR RLSITDDLFN NKYIAINTSN QSKELSNSKE PAVIISASGM LEG GRILNH LEQIKNDENS TLIFVGYQAQ NTRGRKILDG EEKVRCRIEK LNSFSAHADQ DELIDYIERL KYTPYKVFLV HGEK EQREI LAKRIISKKI RVELPENYSQ GKEILIEKKV VLNINTDNMC NFASYRLMPF SGFIVEKDDR IEINDKNWFD MIWNE EYNK MRSQIVAEDF STDQNEDSMA LPDMSHDKII ENIEYLFNIK ILSKNRIKEF WEEFCKGQKA AIKYITQVHR KNPNTG RRN WNPPEGDFTD NEIEKLYETA YNTLLSLIKY DKNKVYNILI NFNPKL

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Macromolecule #4: RNA (48-MER)

MacromoleculeName: RNA (48-MER) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 19.142277 KDa
SequenceString:
GGUUAAAACU CUUCUCAUGC UGGAUUCGAA AUUAGGUGCG CUUCGCGUUU AAGUCCCAUA

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Macromolecule #5: RNA (41-MER)

MacromoleculeName: RNA (41-MER) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 17.386514 KDa
SequenceString:
CAGAAGAACA CCUAAACGCG AAGCGCACCU AAUUUCGAAU CCAGCAUGAG AAGC

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 9 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 223443
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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