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- EMDB-33040: Cryo-EM structure of SbCas7-11 in complex with crRNA and target RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-33040
TitleCryo-EM structure of SbCas7-11 in complex with crRNA and target RNA
Map data
Sample
  • Complex: binary complex
    • Protein or peptide: RAMP superfamily protein
    • RNA: RNA (33-MER)
  • Ligand: ZINC ION
Function / homologyCRISPR type III-associated protein / RAMP superfamily / defense response to virus / RAMP superfamily protein
Function and homology information
Biological speciesCandidatus Scalindua brodae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsYu G / Wang X / Deng Z / Zhang H
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32071218 China
CitationJournal: Nat Microbiol / Year: 2022
Title: Structure and function of a bacterial type III-E CRISPR-Cas7-11 complex.
Authors: Guimei Yu / Xiaoshen Wang / Yi Zhang / Qiyin An / Yanan Wen / Xuzichao Li / Hang Yin / Zengqin Deng / Heng Zhang /
Abstract: The type III-E CRISPR-Cas system uses a single multidomain effector called Cas7-11 (also named gRAMP) to cleave RNA and associate with a caspase-like protease Csx29, showing promising potential for ...The type III-E CRISPR-Cas system uses a single multidomain effector called Cas7-11 (also named gRAMP) to cleave RNA and associate with a caspase-like protease Csx29, showing promising potential for RNA-targeting applications. The structural and molecular mechanisms of the type III-E CRISPR-Cas system remain poorly understood. Here we report four cryo-electron microscopy structures of Cas7-11 at different functional states. Cas7-11 has four Cas7-like domains, which assemble into a helical filament to accommodate CRISPR RNA (crRNA), and a Cas11-like domain facilitating crRNA-target RNA duplex formation. The Cas7.1 domain is critical for crRNA maturation, whereas Cas7.2 and Cas7.3 are responsible for target RNA cleavage. Target RNA binding induces the structural arrangements of Csx29, potentially exposing the catalytic site of Csx29. These results delineate the molecular mechanisms underlying pre-crRNA processing, target RNA recognition and cleavage for Cas7-11, and provide a structural framework to understand the role of Csx29 in type III-E CRISPR system.
History
DepositionMar 9, 2022-
Header (metadata) releaseNov 16, 2022-
Map releaseNov 16, 2022-
UpdateDec 14, 2022-
Current statusDec 14, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33040.png

Downloads & links

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Map

FileDownload / File: emd_33040.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.95 Å
Density
Contour LevelBy AUTHOR: 5.7
Minimum - Maximum-30.716549 - 47.438374
Average (Standard dev.)-9.753904e-12 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 284.9997 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: unmasked

Fileemd_33040_half_map_1.map
Annotationunmasked
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unmasked

Fileemd_33040_half_map_2.map
Annotationunmasked
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : binary complex

EntireName: binary complex
Components
  • Complex: binary complex
    • Protein or peptide: RAMP superfamily protein
    • RNA: RNA (33-MER)
  • Ligand: ZINC ION

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Supramolecule #1: binary complex

SupramoleculeName: binary complex / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)

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Macromolecule #1: RAMP superfamily protein

MacromoleculeName: RAMP superfamily protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 197.823797 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKSNDMNITV ELTFFEPYRL VEWFDWDARK KSHSAMRGQA FAQWTWKGKG RTAGKSFITG TLVRSAVIKA VEELLSLNNG KWEGVPCCN GSFQTDESKG KKPSFLRKRH TLQWQANNKN ICDKEEACPF CILLGRFDNA GKVHERNKDY DIHFSNFDLD H KQEKNDLR ...String:
MKSNDMNITV ELTFFEPYRL VEWFDWDARK KSHSAMRGQA FAQWTWKGKG RTAGKSFITG TLVRSAVIKA VEELLSLNNG KWEGVPCCN GSFQTDESKG KKPSFLRKRH TLQWQANNKN ICDKEEACPF CILLGRFDNA GKVHERNKDY DIHFSNFDLD H KQEKNDLR LVDIASGRIL NRVDFDTGKA KDYFRTWEAD YETYGTYTGR ITLRNEHAKK LLLASLGFVD KLCGALCRIE VI KKSESPL PSDTKEQSYT KDDTVEVLSE DHNDELRKQA EVIVEAFKQN DKLEKIRILA DAIRTLRLHG EGVIEKDELP DGK EERDKG HHLWDIKVQG TALRTKLKEL WQSNKDIGWR KFTEMLGSNL YLIYKKETGG VSTRFRILGD TEYYSKAHDS EGSD LFIPV TPPEGIETKE WIIVGRLKAA TPFYFGVQQP SDSIPGKEKK SEDSLVINEH TSFNILLDKE NRYRIPRSAL RGALR RDLR TAFGSGCNVS LGGQILCNCK VCIEMRRITL KDSVSDFSEP PEIRYRIAKN PGTATVEDGS LFDIEVGPEG LTFPFV LRY RGHKFPEQLS SVIRYWEEND GKNGMAWLGG LDSTGKGRFA LKDIKIFEWD LNQKINEYIK ERGMRGKEKE LLEMGES SL PDGLIPYKFF EERECLFPYK ENLKPQWSEV QYTIEVGSPL LTADTISALT EPGNRDAIAY KKRVYNDGNN AIEPEPRF A VKSETHRGIF RTAVGRRTGD LGKEDHEDCT CDMCIIFGNE HESSKIRFED LELINGNEFE KLEKHIDHVA IDRFTGGAL DKAKFDTYPL AGSPKKPLKL KGRFWIKKGF SGDHKLLITT ALSDIRDGLY PLGSKGGVGY GWVAGISIDD NVPDDFKEMI NKTEMPLPE EVEESNNGPI NNDYVHPGHQ SPKQDHKNKN IYYPHYFLDS GSKVYREKDI ITHEEFTEEL LSGKINCKLE T LTPLIIPD TSDENGLKLQ GNKPGHKNYK FFNINGELMI PGSELRGMLR THFEALTKSC FAIFGEDSTL SWRMNADEKD YK IDSNSIR KMESQRNPKY RIPDELQKEL RNSGNGLFNR LYTSERRFWS DVSNKFENSI DYKREILRCA GRPKNYKGGI IRQ RKDSLM AEELKVHRLP LYDNFDIPDS AYKANDHCRK SATCSTSRGC RERFTCGIKV RDKNRVFLNA ANNNRQYLNN IKKS NHDLY LQYLKGEKKI RFNSKVITGS ERSPIDVIAE LNERGRQTGF IKLSGLNNSN KSQGNTGTTF NSGWDRFELN ILLDD LETR PSKSDYPRPR LLFTKDQYEY NITKRCERVF EIDKGNKTGY PVDDQIKKNY EDILDSYDGI KDQEVAERFD TFTRGS KLK VGDLVYFHID GDNKIDSLIP VRISRKCASK TLGGKLDKAL HPCTGLSDGL CPGCHLFGTT DYKGRVKFGF AKYENGP EW LITRGNNPER SLTLGVLESP RPAFSIPDDE SEIPGRKFYL HHNGWRIIRQ KQLEIRETVQ PERNVTTEVM DKGNVFSF D VRFENLREWE LGLLLQSLDP GKNIAHKLGK GKPYGFGSVK IKIDSLHTFK INSNNDKIKR VPQSDIREYI NKGYQKLIE WSGNNSIQKG NVLPQWHVIP HIDKLYKLLW VPFLNDSKLE PDVRYPVLNE ESKGYIEGSD YTYKKLGDKD NLPYKTRVKG LTTPWSPWN PFQVIAEHEE QEVNVTGSRP SVTDKIERDG KMV

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Macromolecule #2: RNA (33-MER)

MacromoleculeName: RNA (33-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 10.404171 KDa
SequenceString:
GACUUAAUGU CACGGUACCC AAUUUUCUGC CCC

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / 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

Concentration0.6 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
DetailsNo further treatment.

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Electron microscopy

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

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Image processing

Particle selectionNumber selected: 2817147
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 225876
FSC plot (resolution estimation)

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