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- EMDB-14847: Cryo-EM structure of a CRISPR effector in complex with regulator -

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Basic information

Entry
Database: EMDB / ID: EMD-14847
TitleCryo-EM structure of a CRISPR effector in complex with regulator
Map data
Sample
  • Complex: Cas7-11 with TRP-CHAT-NTD
    • Protein or peptide: Cas7-11
    • RNA: gRNAGuide RNA
    • Protein or peptide: TPR-CHAT
  • Ligand: ZINC ION
Biological speciesDesulfonema magnum (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.03 Å
AuthorsBabatunde EE / Davide T / Bertrand B / Sergey N / Alexander M / Henning S / Dong CN
Funding support Switzerland, 2 items
OrganizationGrant numberCountry
Swiss National Science FoundationSNF Grants CRSII5_177195 Switzerland
Swiss National Science FoundationNCCR TransCure (185544) Switzerland
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Structural insights into the regulation of Cas7-11 by TPR-CHAT.
Authors: Babatunde Ekundayo / Davide Torre / Bertrand Beckert / Sergey Nazarov / Alexander Myasnikov / Henning Stahlberg / Dongchun Ni /
Abstract: The CRISPR-guided caspase (Craspase) complex is an assembly of the target-specific RNA nuclease known as Cas7-11 bound to CRISPR RNA (crRNA) and an ancillary protein known as TPR-CHAT ...The CRISPR-guided caspase (Craspase) complex is an assembly of the target-specific RNA nuclease known as Cas7-11 bound to CRISPR RNA (crRNA) and an ancillary protein known as TPR-CHAT (tetratricopeptide repeats (TPR) fused with a CHAT domain). The Craspase complex holds promise as a tool for gene therapy and biomedical research, but its regulation is poorly understood. TPR-CHAT regulates Cas7-11 nuclease activity via an unknown mechanism. In the present study, we use cryoelectron microscopy to determine structures of the Desulfonema magnum (Dm) Craspase complex to gain mechanistic insights into its regulation. We show that DmTPR-CHAT stabilizes crRNA-bound DmCas7-11 in a closed conformation via a network of interactions mediated by the DmTPR-CHAT N-terminal domain, the DmCas7-11 insertion finger and Cas11-like domain, resulting in reduced target RNA accessibility and cleavage.
History
DepositionApr 26, 2022-
Header (metadata) releaseNov 30, 2022-
Map releaseNov 30, 2022-
UpdateMar 1, 2023-
Current statusMar 1, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14847.png

Downloads & links

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Map

FileDownload / File: emd_14847.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.2769 Å
Density
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.93452775 - 1.7297311
Average (Standard dev.)-0.0002020467 (±0.04097234)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 331.99402 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #3

Fileemd_14847_additional_1.map
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Additional map: #2

Fileemd_14847_additional_2.map
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Additional map: #1

Fileemd_14847_additional_3.map
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Half map: #2

Fileemd_14847_half_map_1.map
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Half map: #1

Fileemd_14847_half_map_2.map
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Sample components

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Entire : Cas7-11 with TRP-CHAT-NTD

EntireName: Cas7-11 with TRP-CHAT-NTD
Components
  • Complex: Cas7-11 with TRP-CHAT-NTD
    • Protein or peptide: Cas7-11
    • RNA: gRNAGuide RNA
    • Protein or peptide: TPR-CHAT
  • Ligand: ZINC ION

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Supramolecule #1: Cas7-11 with TRP-CHAT-NTD

SupramoleculeName: Cas7-11 with TRP-CHAT-NTD / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Desulfonema magnum (bacteria)
Molecular weightTheoretical: 220 KDa

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Macromolecule #1: Cas7-11

MacromoleculeName: Cas7-11 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Desulfonema magnum (bacteria)
Molecular weightTheoretical: 207.402859 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SQDPMQHIIP LTITFLESFR VIEWHKSSDR NSLRFLRGYA YARWHRSLKN NKGRPYITGT LVRSAIIRAA EELLWLNDG NYKGAICCPG EFNGSNAKVF REGKARRLRR RQTLTWPTKC ACHEKEPCPF CLLLGRYEKS SKTSKSPVLN N VNFSNFNV ...String:
MGSSHHHHHH SQDPMQHIIP LTITFLESFR VIEWHKSSDR NSLRFLRGYA YARWHRSLKN NKGRPYITGT LVRSAIIRAA EELLWLNDG NYKGAICCPG EFNGSNAKVF REGKARRLRR RQTLTWPTKC ACHEKEPCPF CLLLGRYEKS SKTSKSPVLN N VNFSNFNV LGKEKEFLNI EDVADMRVVN RVDQQSGKAE DFFNIWEITD GAWKIFRGEI QVSDKGWSDE ENFSKFLTLL KG ASVLVDK ISGGLCYLTL DKPELAELPA VKTEKDVLEP GDDASVLEIS RPPYWNNMLN LAGTISEAFE REDKLVHLRL FAD TVRELR RSDIETLDLP KGHADRLGKP SDHFIWDIEI NKKVKLRNWL FWIFNEFRDT YAYFDWRTFC EALGQALYLE AKKQ VPNQF SSERPVGATP AMEVKTPEHD PGRAAQGPRY EWLIKGELVS QTPFFFGWST EADNREHTNL KLLAARDGRL RLPLS VLRG ILRRDIKVVL DNKCRAELAM KQPCSCPVCN LMKKITIRDS FSSNYAEPPK IRHKIRLDPK SGTVAKGALF DSEVGP RGV VFPFELRLRS ADDTLPQALK TVFSWWQQGT VSFSGDAGTG KGIFCLRNLK SIRWDLKTEM DKYAATLGGR KTPVGKW DK CHIPDDKTYP WVKETVEISV CSPFITKDPV NSLIDSAGYD AICYTTVDLE KSENINSLPE SEISLLFEMF DLQYPMSY L FPNKDIYLLK GESFRGMLRT AVGRGENLLL REHEDCSCTL CRIFGNEHNA GKIRVEDFII QGEPRTKLVD RVAIDRFTA GAKDKFKFDA APIVGTPTNK LKFRGNIWIH RDLDGLACES LKLALEDIEN GLYPFGGLGN AGFGWVNYNP LSHPAQEENK ADFSLTKKM ELNWSMKELS TDKIYWPHYF LPFGKKVLRE KTPPSHACID ENEDSELYSG KIVCTLETRT PLIIPDSEFQ G EEHKSYDF FNLNGELCIP GSEIRGMISS VFEALTNSCM RIFDEKKRLS WRMNPNKKDK KNNNRRELDD FIPGRVTNDR KM EEMKEYR YPFYDQAITA NDKQNKYFDQ WEATIELTDE SLEKLKAEKI LQSVLDALRP LTKKKEKYKN TETFVFDLKK FIG KIDDNQ QMISEALERG KVRLTGNSLK QISKTKKIPR QILNQLKGLK DNTYENREQF ISVLKTTVRG INDEQISLIL DNID EDVRL TDLSLTKIRK AKVPQTLVDM LADLKKDEPY KNEKEFLSEF KKKMGEIIGL ILKHAAKTGG DVPRYNHPTP TDKML LSLA AYNRNHKHEN GKAEYRIVKP KHNLKVDFMF AVTPFENPFK GYNPAAVVEE PVGGYLKVSG PNKIEKVKKV NPNSVS VRD DKNQEIIHNG VYLRKITVAN AKSKNKLRER LVPEFAWYDK DSEAAYAMTK RCERVFVEIG AKPIPIQPSA REKFKIL TQ EYQKNAKQQK TPEAFQTILP KDGELRPGDL VYFREDKKTN TVTDIIPVRI SRTVDDEVLA RKIPDDVGDV RPCVREIL D KEKQKEIADA GVKEVFQHHP DGLCPACSLF GTTFYKGRIA FGFAFHKDKD PELANNGKHI TLPLLERPRP TWSMPKKES RVPGRKFYVH HQGWERVIKH SNLDESDPNA TKQTVNNRSV QAIKEEQKFQ FEVRFENLRE WELGLLVYVL QLEPQFAHKL GMGKALGFG SVRIRVGEIH SGPKELDESR LVSSAMKKME EIWDRDKNVL EKLFRLLYFN ESKDIKVRYP KLQKEKEEEE E ESGYMELA KEEYQPEQRR NKLTNPWEGW GNILKKPAIL I

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Macromolecule #3: TPR-CHAT

MacromoleculeName: TPR-CHAT / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Desulfonema magnum (bacteria)
Molecular weightTheoretical: 17.361045 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSSAFSGLKI PELSVDPAEV FKSDNPQLVS VLLDEFELQE QRPFFSGLIP EKQINIALKK SPQLKKLACH LLEAYEINGR RWKHADRRR VLEKAIRLLE KVSNELKGDI QKLENNVKES GKDSEELNKT REKHGEILAD MGRAYLHRAK II

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Macromolecule #2: gRNA

MacromoleculeName: gRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Desulfonema magnum (bacteria)
Molecular weightTheoretical: 12.344251 KDa
SequenceString:
UAUGUGAUGG AACCUCUCCG GAUAAUUCUA UCUCUUCUG

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 4 / 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

Concentration0.9 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1719283
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 53969
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 43
Output model

PDB-7zol:
Cryo-EM structure of a CRISPR effector in complex with regulator

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