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- EMDB-26926: Staphylococcus epidermidis RP62A CRISPR effector complex with non... -

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Basic information

Entry
Database: EMDB / ID: EMD-26926
TitleStaphylococcus epidermidis RP62A CRISPR effector complex with non-self target RNA 1
Map data
Sample
  • Complex: Staphylococcus epidermidis RP62a CRISPR effector complex with non-self target RNA
    • Protein or peptide: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
    • Protein or peptide: CRISPR system Cms protein Csm2
    • Protein or peptide: CRISPR system Cms endoribonuclease Csm3
    • Protein or peptide: CRISPR system Cms protein Csm4
    • RNA: RNA (37-MER)
    • RNA: RNA (40-MER)
Biological speciesStaphylococcus epidermidis (bacteria) / Staphylococcus epidermidis RP62A (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.06 Å
AuthorsSmith EM / Ferrell SH / Tokars VL / Mondragon A
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM118108 United States
American Cancer Society134255-PF-20-041-01-DMC United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM129541 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM129539 United States
CitationJournal: Structure / Year: 2022
Title: Structures of an active type III-A CRISPR effector complex.
Authors: Eric M Smith / Sé Ferrell / Valerie L Tokars / Alfonso Mondragón /
Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) provide many prokaryotes with an adaptive immune system against invading genetic material. ...Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) provide many prokaryotes with an adaptive immune system against invading genetic material. Type III CRISPR systems are unique in that they can degrade both RNA and DNA. In response to invading nucleic acids, they produce cyclic oligoadenylates that act as secondary messengers, activating cellular nucleases that aid in the immune response. Here, we present seven single-particle cryo-EM structures of the type III-A Staphylococcus epidermidis CRISPR effector complex. The structures reveal the intact S. epidermidis effector complex in an apo, ATP-bound, cognate target RNA-bound, and non-cognate target RNA-bound states and illustrate how the effector complex binds and presents crRNA. The complexes bound to target RNA capture the type III-A effector complex in a post-RNA cleavage state. The ATP-bound structures give details about how ATP binds to Cas10 to facilitate cyclic oligoadenylate production.
History
DepositionMay 9, 2022-
Header (metadata) releaseJul 6, 2022-
Map releaseJul 6, 2022-
UpdateAug 17, 2022-
Current statusAug 17, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26926.png

Downloads & links

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Map

FileDownload / File: emd_26926.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.17
Minimum - Maximum-0.43728274 - 1.4034503
Average (Standard dev.)-0.00048015686 (±0.031459466)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 462.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26926_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_26926_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: #2

Fileemd_26926_half_map_2.map
Projections & Slices
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Sample components

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Entire : Staphylococcus epidermidis RP62a CRISPR effector complex with non...

EntireName: Staphylococcus epidermidis RP62a CRISPR effector complex with non-self target RNA
Components
  • Complex: Staphylococcus epidermidis RP62a CRISPR effector complex with non-self target RNA
    • Protein or peptide: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
    • Protein or peptide: CRISPR system Cms protein Csm2
    • Protein or peptide: CRISPR system Cms endoribonuclease Csm3
    • Protein or peptide: CRISPR system Cms protein Csm4
    • RNA: RNA (37-MER)
    • RNA: RNA (40-MER)

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Supramolecule #1: Staphylococcus epidermidis RP62a CRISPR effector complex with non...

SupramoleculeName: Staphylococcus epidermidis RP62a CRISPR effector complex with non-self target RNA
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Staphylococcus epidermidis (bacteria) / Strain: RP62A

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Macromolecule #1: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1...

MacromoleculeName: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria)
SequenceString: MNKKNILMYG SLLHDIGKII YRSGDHTFSR GTHSKLGHQF LSQFSEFKDN EVLDNVAYHH YKELAKANL DNDNTAYITY IADNIASGID RRDIIEEGDE EYEKQLFNFD KYTPLYSVFN I VNSEKLKQ TNGKFKFSNE SNIEYPKTEN IQYSSGNYTT LMKDMSHDLE ...String:
MNKKNILMYG SLLHDIGKII YRSGDHTFSR GTHSKLGHQF LSQFSEFKDN EVLDNVAYHH YKELAKANL DNDNTAYITY IADNIASGID RRDIIEEGDE EYEKQLFNFD KYTPLYSVFN I VNSEKLKQ TNGKFKFSNE SNIEYPKTEN IQYSSGNYTT LMKDMSHDLE HKLSIKEGTF PS LLQWTES LWQYVPSSTN KNQLIDISLY DHSRITCAIA SCIFDYLNEN NIHNYKDELF SKY ENTKSF YQKEAFLLLS MDMSGIQDFI YNISGSKALK SLRSRSFYLE LMLEVIVDQL LERL ELARA NLLYTGGGHA YLLVSNTDKV KKKITQFNNE LKKWFMSEFT TDLSLSMAFE KCSGD DLMN TSGNYRTIWR NVSSKLSDIK AHKYSAEDIL KLNHFHSYGD RECKECLRSD IDINDD GLC SICEGIINIS NDLRDKSFFV LSETGKLKMP FNKFISVIDY EEAEMLVQNN NQVRIYS KN KPYIGIGIST NLWMCDYDYA SQNQDMREKG IGSYVDREEG VKRLGVVRAD IDNLGATF I SGIPEKYNSI SRTATLSRQL SLFFKYELNH LLENYQITAI YSGGDDLFLI GAWDDIIEA SIYINDKFKE FTLDKLTLSA GVGMFSGKYP VSKMAFETGR LEEAAKTGEK NQISLWLQEK VYNWDEFKK NILEEKLLVL QQGFSQTDEH GKAFIYKMLA LLRNNEAINI ARLAYLLARS K MNEDFTSK IFNWAQNDKD KNQLITALEY YIYQIREAD

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Macromolecule #2: CRISPR system Cms protein Csm2

MacromoleculeName: CRISPR system Cms protein Csm2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria)
SequenceString:
MTFAHEVVKS NVKNVKDRKG KEKQVLFNGL TTSKLRNLME QVNRLYTIAF NSNEDQLNEE FIDELEYLK IKFYYEAGRE KSVDEFLKKT LMFPIIDRVI KKESKKFFLD YCKYFEALVA Y AKYYQKED

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Macromolecule #3: CRISPR system Cms endoribonuclease Csm3

MacromoleculeName: CRISPR system Cms endoribonuclease Csm3 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria)
SequenceString: MYSKIKISGT IEVVTGLHIG GGGESSMIGA IDSPVVRDLQ TKLPIIPGSS IKGKMRNLLA KHFGLKMKQ ESHNQDDERV LRLFGSSEKG NIQRARLQIS DAFFSEKTKE HFAQNDIAYT E TKFENTIN RLTAVANPRQ IERVTRGSEF DFVFIYNVDE ESQVEDDFEN ...String:
MYSKIKISGT IEVVTGLHIG GGGESSMIGA IDSPVVRDLQ TKLPIIPGSS IKGKMRNLLA KHFGLKMKQ ESHNQDDERV LRLFGSSEKG NIQRARLQIS DAFFSEKTKE HFAQNDIAYT E TKFENTIN RLTAVANPRQ IERVTRGSEF DFVFIYNVDE ESQVEDDFEN IEKAIHLLEN DY LGGGGTR GNGRIQFKDT NIETVVGEYD STNLKIK

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Macromolecule #4: CRISPR system Cms protein Csm4

MacromoleculeName: CRISPR system Cms protein Csm4 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria)
SequenceString: MTLATKVFKL SFKTPVHFGK KRLSDGEMTI TADTLFSALF IETLQLGKDT DWLLNDLIIS DTFPYENEL YYLPKPLIKI DSKEEDNHKA FKKLKYVPVH HYNQYLNGEL SAEDATDLND I FNIGYFSL QTKVSLIAQE TDSSADSEPY SVGTFTFEPE AGLYFIAKGS ...String:
MTLATKVFKL SFKTPVHFGK KRLSDGEMTI TADTLFSALF IETLQLGKDT DWLLNDLIIS DTFPYENEL YYLPKPLIKI DSKEEDNHKA FKKLKYVPVH HYNQYLNGEL SAEDATDLND I FNIGYFSL QTKVSLIAQE TDSSADSEPY SVGTFTFEPE AGLYFIAKGS EETLDHLNNI MT ALQYSGL GGKRNAGYGQ FEYEIINNQQ LSKLLNQNGK HSILLSTAMA KKEEIESALK EAR YILTKR SGFVQSTNYS EMLVKKSDFY SFSSGSVFKN IFNGDIFNVG HNGKHPVYRY AKPL WLEV

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Macromolecule #5: RNA (37-MER)

MacromoleculeName: RNA (37-MER) / type: rna / ID: 5
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria)
SequenceString:
ACGAGAACUA GUAAUAAUUG UCAUUUGCAU ACGUUAC

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Macromolecule #6: RNA (40-MER)

MacromoleculeName: RNA (40-MER) / type: rna / ID: 6
Source (natural)Organism: Staphylococcus epidermidis (bacteria) / Strain: RP62A
SequenceString:
AGCCUGGUAA CGUAUGCAAA UGACAAUUAU UACUAUCCAG

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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

Concentration3.2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC4H11NO32-Amino-2-(hydroxymethyl)propane-1,3-diol
250.0 mMNaClNaCl
1.0 mMC4H10O2S2Dithiothreitol
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
Details: cryo-EM grids were prepared by glow discharging for 10 seconds at 15 mA in a Pelco easiGlow glow discharger.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 3 ul of the protein-RNA complex were added to the grid and allowed to incubate for 10 seconds. After incubation the grid was blotted in a Vitrobot Mark IV (FEI Thermo Fischer) (humidity 95% ...Details: 3 ul of the protein-RNA complex were added to the grid and allowed to incubate for 10 seconds. After incubation the grid was blotted in a Vitrobot Mark IV (FEI Thermo Fischer) (humidity 95% and temperature 4 C) for 3 seconds with a force of 0 before plunge freezing in liquid ethane..

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 2455 / Average electron dose: 49.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 44454 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2368249
CTF correctionSoftware - Name: cryoSPARC
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.06 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 53034
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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