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- PDB-6o1o: Cryo-EM structure of the T. thermophilus Csm complex bound to tar... -

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Basic information

Entry
Database: PDB / ID: 6o1o
TitleCryo-EM structure of the T. thermophilus Csm complex bound to target ssRNA
Components
  • Csm1
  • Csm2
  • Csm3
  • Csm4
  • RNA (30-MER)
  • RNA (36-MER)
KeywordsRNA BINDING PROTEIN/RNA / Csm / RNA substrate / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Biological speciesThermus thermophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsLiu, J.J. / Liu, T.Y.
Funding support United States, 4items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01GM051487 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM122334 United States
Life Sciences Research Foundation United States
CitationJournal: Nat Commun / Year: 2019
Title: Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble.
Authors: Tina Y Liu / Jun-Jie Liu / Abhishek J Aditham / Eva Nogales / Jennifer A Doudna /
Abstract: Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and ...Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.
Validation Report
SummaryFull reportAbout validation report
History
DepositionFeb 21, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 10, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: Csm1
B: Csm4
C: Csm3
D: Csm3
E: Csm3
F: Csm3
G: Csm4
H: Csm4
I: Csm2
J: Csm2
K: Csm2
L: Csm2
M: RNA (36-MER)
N: RNA (30-MER)


Theoretical massNumber of molelcules
Total (without water)278,29714
Polymers278,29714
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 12 molecules ABGHCDEFIJKL

#1: Protein Csm1 / /


Mass: 60953.055 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (bacteria) / Production host: Escherichia coli (E. coli)
#2: Protein Csm4


Mass: 23762.168 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (bacteria) / Production host: Escherichia coli (E. coli)
#3: Protein
Csm3


Mass: 18570.826 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (bacteria) / Production host: Escherichia coli (E. coli)
#4: Protein
Csm2


Mass: 10060.393 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (bacteria) / Production host: Escherichia coli (E. coli)

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RNA chain , 2 types, 2 molecules MN

#5: RNA chain RNA (36-MER)


Mass: 15537.359 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermus thermophilus (bacteria)
#6: RNA chain RNA (30-MER)


Mass: 15995.450 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermus thermophilus (bacteria)

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Details

Sequence detailsThe complete sequence of Csm1 is MASMLGDGLSVALAGLLHDVGKLYSRARWGERDDQVPDRTHTAYTAHFVREHAGLFRGAG ...The complete sequence of Csm1 is MASMLGDGLSVALAGLLHDVGKLYSRARWGERDDQVPDRTHTAYTAHFVREHAGLFRGAG LDPDWLARTASRHHEGWRDRPQYRPETPEEWCVALADTYASKEREEGEGGGSPPEVPLSP PFRRLLLGGEEGREGGYSPVGAGGRVGLEAGGLYPEERPNVSKDVYKRLLERLEGRLEEM ARFSLGKEALVLNLALALQETLSLVPSDTQSEPDVSLYDHLRLTAAIAHALWLFHGGSPS AQNLRQDGEKFLLVVGDMGGIQGHIYRIAGAEAGVGGIAKRLRARSLEVSLAAEAMALGL LWRLGLTSLNRILGAGGKFYLLLPNTEEARAALEGTREAWGRWALKRGGSLVPHLAWVAF RGQDFRDFAALLKRLHEALAREKLRPFAFLASTGGVLGAPLRPCAACGLEPARRDEPGSL CPDCEREAALGARLPRSDRVGFFLEEAPRPYLDFPGLKVGLGGPLEGAFHLFRARLDFAP WPDPSEAKPLLGHLPRVEHALKAKGWSLEAYRAWAEEEGLLEDEEPHPEKVLTFAELAAL SEGAPYLGALMLDADRMGEAFATGFRREGRDLATPSRLAALSRTLEVFFTTEVLTLLEEP RRYRERLGWDDLEAQRKEARYPLLYSVYSGGDDLFLLGPWDALLDFALDLERLYRLFTRH PRLTLSGGFLLVPPSLPVPELARLLGEAEKRAKAEGRERLFLFGQAVPWETLRGLRAWAE GLRQDLRAERVSRAQVYRWLLLWRRFSPLEDPGERMRYKPLLAYALRRVRERDEEAWKRY LKLLDHQDPAWAYLPVWVQWALYRERRV The complete sequence of Csm4 is MRATLFRLYFQGPLKALPRAPTLLGHLFWWYRYTHGREALEELLERFRQEPPFRLSSVYP EGWLPRPKLPPVQVEETTLRKALKSLSLVRLDTFQALAERGEEALLEAPEVQGKARPPEM RRLRRTRVGVDRAAGTARPGVLFTQEYLFPDPRTPYALYVLGEAPFDLGEALAFVGEMGY GGQASLGLGRFRVEGPFAVELPEAKEPNAYATLAPGPLEEALYYEVEPYWGRLGGAYVGA RPFKRPYLRAKEGSVYRGPTHRLLEVTPTEPPEAGARVWEALVVFPLGVRV The complete sequence of Csm3 is MKLKKVIRIRSVLLAKTGLRIGMSRDQMAIGDLDNPVVRNPLTDEPYIPGSSLKGKLRYL LEWSLGGDYILKAKERQVYASPDPKDPVARIFGLAPENDERSLAVARERGPTRLLVRDAY LTEDAKEALERTSARGGLYTEIKQEVFIPRLGGNANPRTTERVPAGARFRVEMTYRVLDD LDEEYFGKYLLRALELLELDGLGGHISRGYGQVYFLHPERLTEDQEGWPLKERLKVEEVV L The complete sequence of Csm2 is MPALEFFKDKERGVLDPKVFERAREVAEGLARGKLKSSQFRNYFAELRALENRFERERRK EGEELAFARLVPQLELLRAKLFYNTRSQGPLRDAKEFVEFMEEALEAGKRSPKDFEAMMK YVEAVLAYFYALAK

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Thermus thermophilus Csm complex bound to target ssRNA
Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Thermus thermophilus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.9
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 87321 / Symmetry type: POINT

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