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- EMDB-26925: Staphylococcus epidermidis RP62a CRISPR short effector complex wi... -

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Basic information

Entry
Database: EMDB / ID: EMD-26925
TitleStaphylococcus epidermidis RP62a CRISPR short effector complex with self RNA target and ATP
Map data
Sample
  • Complex: Staphylococcus epidermidis RP62a CRISPR short effector complex with self target RNA and ATP
    • Protein or peptide: CRISPR system Cms endoribonuclease Csm3
    • Protein or peptide: CRISPR system Cms protein Csm5
    • Protein or peptide: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
    • RNA: RNA (37-MER)
    • Protein or peptide: CRISPR system Cms protein Csm4
    • Protein or peptide: CRISPR system Cms protein Csm2
    • RNA: RNA (37-MER)
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsType IIIA CRISPR / effector complex / RNA binding protein / HYDROLASE-RNA complex
Function / homology
Function and homology information


exonuclease activity / defense response to virus / endonuclease activity / RNA binding / ATP binding / metal ion binding
Similarity search - Function
: / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / Csm4, C-terminal / CRISPR Csm4 C-terminal domain / CRISPR-associated protein Csm5 / CRISPR-associated protein, Csm2 Type III-A / Csm2 Type III-A / : / CRISPR-associated RAMP Csm3 / CRISPR type III-associated RAMP protein Csm4 ...: / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / Csm4, C-terminal / CRISPR Csm4 C-terminal domain / CRISPR-associated protein Csm5 / CRISPR-associated protein, Csm2 Type III-A / Csm2 Type III-A / : / CRISPR-associated RAMP Csm3 / CRISPR type III-associated RAMP protein Csm4 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / Csm1, subunit domain B / Csm1 subunit domain B / Cas10/Cmr2, second palm domain / : / CRISPR type III-associated protein / RAMP superfamily / GGDEF domain profile. / GGDEF domain / HD domain / HD domain / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / CRISPR system Cms protein Csm2 / CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm4 / CRISPR system Cms protein Csm5
Similarity search - Component
Biological speciesStaphylococcus epidermidis (bacteria) / Staphylococcus epidermidis RP62A (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.67 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-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26925.png

Downloads & links

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Map

FileDownload / File: emd_26925.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
1.07 Å/pix.
x 416 pix.
= 444.704 Å		1.07 Å/pix.
x 416 pix.
= 444.704 Å		1.07 Å/pix.
x 416 pix.
= 444.704 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.069 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.145
Minimum - Maximum-0.45569092 - 1.286593
Average (Standard dev.)-0.0014437645 (±0.030193763)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 444.704 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26925_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_26925_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_26925_half_map_2.map
Projections & Slices
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Sample components

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Entire : Staphylococcus epidermidis RP62a CRISPR short effector complex wi...

EntireName: Staphylococcus epidermidis RP62a CRISPR short effector complex with self target RNA and ATP
Components
  • Complex: Staphylococcus epidermidis RP62a CRISPR short effector complex with self target RNA and ATP
    • Protein or peptide: CRISPR system Cms endoribonuclease Csm3
    • Protein or peptide: CRISPR system Cms protein Csm5
    • Protein or peptide: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
    • RNA: RNA (37-MER)
    • Protein or peptide: CRISPR system Cms protein Csm4
    • Protein or peptide: CRISPR system Cms protein Csm2
    • RNA: RNA (37-MER)
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Staphylococcus epidermidis RP62a CRISPR short effector complex wi...

SupramoleculeName: Staphylococcus epidermidis RP62a CRISPR short effector complex with self target RNA and ATP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Staphylococcus epidermidis (bacteria) / Strain: RP62A

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Macromolecule #1: CRISPR system Cms endoribonuclease Csm3

MacromoleculeName: CRISPR system Cms endoribonuclease Csm3 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria) / Strain: ATCC 35984 / RP62A
Molecular weightTheoretical: 24.033975 KDa
SequenceString: MYSKIKISGT IEVVTGLHIG GGGESSMIGA IDSPVVRDLQ TKLPIIPGSS IKGKMRNLLA KHFGLKMKQE SHNQDDERVL RLFGSSEKG NIQRARLQIS DAFFSEKTKE HFAQNDIAYT ETKFENTINR LTAVANPRQI ERVTRGSEFD FVFIYNVDEE S QVEDDFEN ...String:
MYSKIKISGT IEVVTGLHIG GGGESSMIGA IDSPVVRDLQ TKLPIIPGSS IKGKMRNLLA KHFGLKMKQE SHNQDDERVL RLFGSSEKG NIQRARLQIS DAFFSEKTKE HFAQNDIAYT ETKFENTINR LTAVANPRQI ERVTRGSEFD FVFIYNVDEE S QVEDDFEN IEKAIHLLEN DYLGGGGTRG NGRIQFKDTN IETVVGEYDS TNLKIK

UniProtKB: CRISPR system Cms endoribonuclease Csm3

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Macromolecule #2: CRISPR system Cms protein Csm5

MacromoleculeName: CRISPR system Cms protein Csm5 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria) / Strain: ATCC 35984 / RP62A
Molecular weightTheoretical: 39.449125 KDa
SequenceString: MTIKNYEVVI KTLGPIHIGS GQVMKKQDYI YDFYNSKVYM INGNKLVKFL KRKNLLYTYQ NFLRYPPKNP RENGLKDYLD AQNVKQSEW EAFVSYSEKV NQGKKYGNTR PKPLNDLHLM VRDGQNKVYL PGSSIKGAIK TTLVSKYNNE KNKDIYSKIK V SDSKPIDE ...String:
MTIKNYEVVI KTLGPIHIGS GQVMKKQDYI YDFYNSKVYM INGNKLVKFL KRKNLLYTYQ NFLRYPPKNP RENGLKDYLD AQNVKQSEW EAFVSYSEKV NQGKKYGNTR PKPLNDLHLM VRDGQNKVYL PGSSIKGAIK TTLVSKYNNE KNKDIYSKIK V SDSKPIDE SNLAIYQKID INKSEKSMPL YRECIDVNTE IKFKLTIEDE IYSINEIEQS IQDFYKNYYD KWLVGFKETK GG RRFALEG GIPDVLNQNI LFLGAGTGFV SKTTHYQLKN RKQAKQDSFE ILTKKFRGTY GKMKEIPSNV PVALKGTTNQ SRH TSYQQG MCKVSFQELN NEVL

UniProtKB: CRISPR system Cms protein Csm5

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Macromolecule #3: 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: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria) / Strain: ATCC 35984 / RP62A
Molecular weightTheoretical: 87.685781 KDa
SequenceString: MNKKNILMYG SLLHDIGKII YRSGDHTFSR GTHSKLGHQF LSQFSEFKDN EVLDNVAYHH YKELAKANLD NDNTAYITYI ADNIASGID RRDIIEEGDE EYEKQLFNFD KYTPLYSVFN IVNSEKLKQT NGKFKFSNES NIEYPKTENI QYSSGNYTTL M KDMSHDLE ...String:
MNKKNILMYG SLLHDIGKII YRSGDHTFSR GTHSKLGHQF LSQFSEFKDN EVLDNVAYHH YKELAKANLD NDNTAYITYI ADNIASGID RRDIIEEGDE EYEKQLFNFD KYTPLYSVFN IVNSEKLKQT NGKFKFSNES NIEYPKTENI QYSSGNYTTL M KDMSHDLE HKLSIKEGTF PSLLQWTESL WQYVPSSTNK NQLIDISLYD HSRITCAIAS CIFDYLNENN IHNYKDELFS KY ENTKSFY QKEAFLLLSM DMSGIQDFIY NISGSKALKS LRSRSFYLEL MLEVIVDQLL ERLELARANL LYTGGGHAYL LVS NTDKVK KKITQFNNEL KKWFMSEFTT DLSLSMAFEK CSGDDLMNTS GNYRTIWRNV SSKLSDIKAH KYSAEDILKL NHFH SYGDR ECKECLRSDI DINDDGLCSI CEGIINISND LRDKSFFVLS ETGKLKMPFN KFISVIDYEE AEMLVQNNNQ VRIYS KNKP YIGIGISTNL WMCDYDYASQ NQDMREKGIG SYVDREEGVK RLGVVRADID NLGATFISGI PEKYNSISRT ATLSRQ LSL FFKYELNHLL ENYQITAIYS GGDDLFLIGA WDDIIEASIY INDKFKEFTL DKLTLSAGVG MFSGKYPVSK MAFETGR LE EAAKTGEKNQ ISLWLQEKVY NWDEFKKNIL EEKLLVLQQG FSQTDEHGKA FIYKMLALLR NNEAINIARL AYLLARSK M NEDFTSKIFN WAQNDKDKNQ LITALEYYIY QIREAD

UniProtKB: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)

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Macromolecule #5: CRISPR system Cms protein Csm4

MacromoleculeName: CRISPR system Cms protein Csm4 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria) / Strain: ATCC 35984 / RP62A
Molecular weightTheoretical: 34.551938 KDa
SequenceString: MTLATKVFKL SFKTPVHFGK KRLSDGEMTI TADTLFSALF IETLQLGKDT DWLLNDLIIS DTFPYENELY YLPKPLIKID SKEEDNHKA FKKLKYVPVH HYNQYLNGEL SAEDATDLND IFNIGYFSLQ TKVSLIAQET DSSADSEPYS VGTFTFEPEA G LYFIAKGS ...String:
MTLATKVFKL SFKTPVHFGK KRLSDGEMTI TADTLFSALF IETLQLGKDT DWLLNDLIIS DTFPYENELY YLPKPLIKID SKEEDNHKA FKKLKYVPVH HYNQYLNGEL SAEDATDLND IFNIGYFSLQ TKVSLIAQET DSSADSEPYS VGTFTFEPEA G LYFIAKGS EETLDHLNNI MTALQYSGLG GKRNAGYGQF EYEIINNQQL SKLLNQNGKH SILLSTAMAK KEEIESALKE AR YILTKRS GFVQSTNYSE MLVKKSDFYS FSSGSVFKNI FNGDIFNVGH NGKHPVYRYA KPLWLEV

UniProtKB: CRISPR system Cms protein Csm4

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Macromolecule #6: CRISPR system Cms protein Csm2

MacromoleculeName: CRISPR system Cms protein Csm2 / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria) / Strain: ATCC 35984 / RP62A
Molecular weightTheoretical: 15.436794 KDa
SequenceString:
MTFAHEVVKS NVKNVKDRKG KEKQVLFNGL TTSKLRNLME QVNRLYTIAF NSNEDQLNEE FIDELEYLKI KFYYEAGREK SVDEFLKKT LMFPIIDRVI KKESKKFFLD YCKYFEALVA YAKYYQKED

UniProtKB: CRISPR system Cms protein Csm2

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Macromolecule #4: RNA (37-MER)

MacromoleculeName: RNA (37-MER) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria) / Strain: ATCC 35984 / RP62A
Molecular weightTheoretical: 11.895168 KDa
SequenceString:
ACGAGAACAC GUAUGCCGAA GUAUAUAAAU CAUCAGU

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Macromolecule #7: RNA (37-MER)

MacromoleculeName: RNA (37-MER) / type: rna / ID: 7 / Number of copies: 1
Source (natural)Organism: Staphylococcus epidermidis (bacteria)
Molecular weightTheoretical: 11.733888 KDa
SequenceString:
ACUGAUGAUU UAUAUACUUC GGCAUACGUG UUCUCGU

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Macromolecule #8: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 8 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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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 / Pretreatment - Time: 10 sec.
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
DetailsThe purified complex was mixed with 1 mM ATP. This complex was allowed to incubate for 10 minutes ice before being dialyzed (Slide-A-Lyzer MINI Dialysis Devices, Thermo Fisher) overnight in 50 mM Tris pH 8.0, 250 mM NaCl, 1 mM DTT at 4 C. Prior to vitrification the complex was mixed in a 1:10 molar ratio with a 37 nt RNA that was completely complementary to the 37 nt version of crRNA 1. Additional ATP was added to the incubated ribonucleoprotein complex to a final concentration of 2 mM and allowed to incubate on ice for 15 minutes.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 7527 / Average electron dose: 52.79 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 46772 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 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: 1790337
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.67 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 130430
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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Atomic model buiding 1

Initial model
PDB IDChain

6nbt

chain_id: A, source_name: PDB, initial_model_type: experimental model

6nbt

chain_id: B, source_name: PDB, initial_model_type: experimental model

6nbt

chain_id: C, source_name: PDB, initial_model_type: experimental model

6nbu

chain_id: I, source_name: PDB, initial_model_type: experimental model

6nbu

chain_id: K, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-7v01:
Staphylococcus epidermidis RP62a CRISPR short effector complex with self RNA target and ATP

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