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- EMDB-34484: Cryo-EM structure of a riboendonuclease -

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Basic information

Entry
Database: EMDB / ID: EMD-34484
TitleCryo-EM structure of a riboendonuclease
Map data
Sample
  • Complex: Apo-form riboendonuclease
    • Protein or peptide: CRISPR-associated endonuclease Cas9
Keywordsriboendonuclease / RNA
Function / homologyUncharacterized protein
Function and homology information
Biological speciesThermoclostridium caenicola (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsLi Z / Wang F
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: J Mol Biol / Year: 2023
Title: Structural Basis for the Ribonuclease Activity of a Thermostable CRISPR-Cas13a from Thermoclostridium caenicola.
Authors: Feng Wang / Chendi Zhang / Haijiang Xu / Wanting Zeng / Lixin Ma / Zhuang Li /
Abstract: The RNA-targeting type VI CRISPR-Cas effector complexes are widely used in biotechnology applications such as gene knockdown, RNA editing, and molecular diagnostics. Compared with Cas13a from ...The RNA-targeting type VI CRISPR-Cas effector complexes are widely used in biotechnology applications such as gene knockdown, RNA editing, and molecular diagnostics. Compared with Cas13a from mesophilic organisms, a newly discovered Cas13a from thermophilic bacteria Thermoclostridium caenicola (TccCas13a) shows low sequence similarity, high thermostability, and lacks pre-crRNA processing activity. The thermostability of TccCas13a has been harnessed to make a sensitive and robust tool for nucleic acid detection. Here we present the structures of TccCas13a-crRNA binary complex at 2.8 Å, and TccCas13a at 3.5 Å. Although TccCas13a shares a similarly bilobed architecture with other mesophilic organism-derived Cas13a proteins, TccCas13a displayed distinct structure features. Specifically, it holds a long crRNA 5'-flank, forming extensive polar contacts with Helical-1 and HEPN2 domains. The detailed analysis of the interaction between crRNA 5'-flank and TccCas13a suggested lack of suitable nucleophile to attack the 2'-OH of crRNA 5'-flank may explain why TccCas13a fails to cleave pre-crRNA. The stem-loop segment of crRNA spacer toggles between double-stranded and single-stranded conformational states, suggesting a potential safeguard mechanism for target recognition. Superimposition of the structures of TccCas13a and TccCas13a-crRNA revealed several conformational changes required for crRNA loading, including dramatic movement of Helical-2 domain. Collectively, these structural insights expand our understanding into type VI CRISPR-Cas effectors, and would facilitate the development of TccCas13a-based applications.
History
DepositionOct 11, 2022-
Header (metadata) releaseAug 30, 2023-
Map releaseAug 30, 2023-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_34484.png

Downloads & links

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Map

FileDownload / File: emd_34484.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.0168
Minimum - Maximum-0.060347807 - 0.106377564
Average (Standard dev.)0.00020577939 (±0.0030788495)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 187.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_34484_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34484_half_map_2.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 : Apo-form riboendonuclease

EntireName: Apo-form riboendonuclease
Components
  • Complex: Apo-form riboendonuclease
    • Protein or peptide: CRISPR-associated endonuclease Cas9

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Supramolecule #1: Apo-form riboendonuclease

SupramoleculeName: Apo-form riboendonuclease / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thermoclostridium caenicola (bacteria)

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Macromolecule #1: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermoclostridium caenicola (bacteria)
Molecular weightTheoretical: 143.582641 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKITKRKWGE HHPPLYFYRD EDSGRLLAQN DRKQDYTDTL FNDIAQDTFE RSLRNRLLKT PEKGDKRFYS NEIVKLVEKL CQGADVAEI MKSMERNEKL RPKNEKEIKN LKKQLDGTLS EYGKRYTAPE GAMTLNDALF YLVEGNPLKQ AMAKAELGKI R EALIKEKE ...String:
MKITKRKWGE HHPPLYFYRD EDSGRLLAQN DRKQDYTDTL FNDIAQDTFE RSLRNRLLKT PEKGDKRFYS NEIVKLVEKL CQGADVAEI MKSMERNEKL RPKNEKEIKN LKKQLDGTLS EYGKRYTAPE GAMTLNDALF YLVEGNPLKQ AMAKAELGKI R EALIKEKE NRINRVRYSI KNNKIPLRIQ EDGGITPNND RAAWLLGLMK PADPAKGITD CYPLLGELEE VFDFDKLSKT LH EKISRCQ GRPRSIAMAV DEALKQYLRE LWEKSPSRQQ DLKYYFQAVQ EYFKDNFPIR TKRMGARLRQ ELLKDKTSLS RLL EPKHMA NAVRRRLINQ STQMHILYGK LYAYCCGEDG RLLVNSETLQ RIQVHEAVKK QAMTAVLWSI SRLRYFYQFE DGDI LSNKN PIKDFRDKFL RDTNKYTHED VEACKEKLQD FFPLKELQEK IKEDAKGLQE TDNKQADTTD FKAIGHIVRD DRKLC NQLL AECVSCIGEL RHHIFHYKNV TLIQALKRIA DKVKPEDLSV LRAIYLLDRR NLKKAFAKRI SSMNLPLYYR EDLLSR IFK KEGTAFFLYS AKIQMTPSFQ RVYERGKNLR REFECERMKA EASNGQNGQD GDRLKWFRQL AAGDSADTHF NWAVEAY AE SAADVENNVE FDTDVDAQRA LRNLLLLIYR HHFLPEVQKD ETLVTGKIHK VLERNRQLSE GQGPNQGKAH GYSVIEEL Y HEGMPLSDLM KQLQRRISET ERESRELAQE KTDYAQRFIL DIFAEAFNDF LEAHYGEEYL EIMSPRKDAE AAKKWVKES KTVDLKTSID EKEPEGHLLV LYPVLRLLDE RELGELQQQM IRYRTSLASW QGESNFSEEI RIAGQIEELT ELVKLTEPEP QFAEEVWGK RAKEAFEDFI EGNMKNYEAF YLQSDNNTPV YRRNMSRLLR SGLMGVYQKV LASHKQALKR DYLLWSEKHW N VKDENGAD ISSAEQAQCL LQRLHRKYAE SPSRFTEEDC KLYEKVLRRL EDYNQAVKNL SFSSLYEICV LNLEILSRWV GF VQDWERD MYFLLLAWVR QGKLDGIKEE DVRDIFSEGN IIRNLVDTLK GENMNAFESV YFPENKGSKY LGVRNDVAHL DLM RKNGWR LEAGKTCSVM EDYINRLRFL LSYDQKRMNA VTKTLQQIFD RHKVKIRFTV EKGGMLKIED VTADKIVHLK GSRL SGIEI PSHGERFIDT LKALMVYPRG

UniProtKB: Uncharacterized protein

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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

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 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: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 134153
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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