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- EMDB-39211: Cryo EM structure of Komagataella phaffii Rat1-Rai1-Rtt103 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-39211
TitleCryo EM structure of Komagataella phaffii Rat1-Rai1-Rtt103 complex
Map data
Sample
  • Complex: Komagataella phaffii Rat1-Rai1-Rtt103 complex
    • Protein or peptide: 5'-3' exoribonuclease
    • Protein or peptide: Decapping nuclease
    • Protein or peptide: Exonuclease Rat1p and Rai1p interacting protein
Keywordstranscription termination / RNA polymerase II / TRANSCRIPTION
Function / homology
Function and homology information


mRNA 5'-diphosphatase activity / NAD-cap decapping / 5'-3' RNA exonuclease activity / nuclease activity / mRNA 3'-end processing / exonuclease activity / RNA polymerase II C-terminal domain binding / nuclear-transcribed mRNA catabolic process / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / DNA-templated transcription termination ...mRNA 5'-diphosphatase activity / NAD-cap decapping / 5'-3' RNA exonuclease activity / nuclease activity / mRNA 3'-end processing / exonuclease activity / RNA polymerase II C-terminal domain binding / nuclear-transcribed mRNA catabolic process / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / DNA-templated transcription termination / mRNA processing / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / nucleotide binding / RNA binding / metal ion binding / nucleus / cytosol
Similarity search - Function
: / 5'-3' exoribonuclease type 2 / Xrn1, N-terminal / 5'-3' exoribonuclease / Xrn1, helical domain / XRN 5'-3' exonuclease N-terminus / Xrn1 helical domain / RAI1-like / RAI1-like family / RAI1 like PD-(D/E)XK nuclease ...: / 5'-3' exoribonuclease type 2 / Xrn1, N-terminal / 5'-3' exoribonuclease / Xrn1, helical domain / XRN 5'-3' exonuclease N-terminus / Xrn1 helical domain / RAI1-like / RAI1-like family / RAI1 like PD-(D/E)XK nuclease / CID domain / RPR / CID domain / CID domain profile. / ENTH/VHS
Similarity search - Domain/homology
Decapping nuclease / Exonuclease Rat1p and Rai1p interacting protein / 5'-3' exoribonuclease
Similarity search - Component
Biological speciesKomagataella phaffii (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.78 Å
AuthorsYanagisawa T / Murayama Y / Ehara H / Sekine SI
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP20H05690 Japan
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis of eukaryotic transcription termination by the Rat1 exonuclease complex.
Authors: Tatsuo Yanagisawa / Yuko Murayama / Haruhiko Ehara / Mie Goto / Mari Aoki / Shun-Ichi Sekine /
Abstract: The 5´-3´ exoribonuclease Rat1/Xrn2 is responsible for the termination of eukaryotic mRNA transcription by RNAPII. Rat1 forms a complex with its partner proteins, Rai1 and Rtt103, and acts as a ...The 5´-3´ exoribonuclease Rat1/Xrn2 is responsible for the termination of eukaryotic mRNA transcription by RNAPII. Rat1 forms a complex with its partner proteins, Rai1 and Rtt103, and acts as a "torpedo" to bind transcribing RNAPII and dissociate DNA/RNA from it. Here we report the cryo-electron microscopy structures of the Rat1-Rai1-Rtt103 complex and three Rat1-Rai1-associated RNAPII complexes (type-1, type-1b, and type-2) from the yeast, Komagataella phaffii. The Rat1-Rai1-Rtt103 structure revealed that Rat1 and Rai1 form a heterotetramer with a single Rtt103 bound between two Rai1 molecules. In the type-1 complex, Rat1-Rai1 forms a heterodimer and binds to the RNA exit site of RNAPII to extract RNA into the Rat1 exonuclease active site. This interaction changes the RNA path in favor of termination (the "pre-termination" state). The type-1b and type-2 complexes have no bound DNA/RNA, likely representing the "post-termination" states. These structures illustrate the termination mechanism of eukaryotic mRNA transcription.
History
DepositionFeb 24, 2024-
Header (metadata) releaseSep 25, 2024-
Map releaseSep 25, 2024-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_39211.png

Downloads & links

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Map

FileDownload / File: emd_39211.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.49 Å/pix.
x 200 pix.
= 298.8 Å		1.49 Å/pix.
x 200 pix.
= 298.8 Å		1.49 Å/pix.
x 200 pix.
= 298.8 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.494 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0144
Minimum - Maximum-0.04789768 - 0.122454494
Average (Standard dev.)0.00004000738 (±0.0039564962)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 298.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_39211_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_39211_half_map_2.map
Projections & Slices
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Sample components

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Entire : Komagataella phaffii Rat1-Rai1-Rtt103 complex

EntireName: Komagataella phaffii Rat1-Rai1-Rtt103 complex
Components
  • Complex: Komagataella phaffii Rat1-Rai1-Rtt103 complex
    • Protein or peptide: 5'-3' exoribonuclease
    • Protein or peptide: Decapping nuclease
    • Protein or peptide: Exonuclease Rat1p and Rai1p interacting protein

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Supramolecule #1: Komagataella phaffii Rat1-Rai1-Rtt103 complex

SupramoleculeName: Komagataella phaffii Rat1-Rai1-Rtt103 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Komagataella phaffii (fungus)

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Macromolecule #1: 5'-3' exoribonuclease

MacromoleculeName: 5'-3' exoribonuclease / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters
Source (natural)Organism: Komagataella phaffii (fungus)
Molecular weightTheoretical: 115.314461 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGVPALFRWL SRKYPKIISP VIQDEDVDID GESRPTRYED PNPNGELDNL YLDMNGIVHP CSHPEHKPVP ETEDEMMLDV FAYTENVIM MARPRKVIYI AVDGVAPRAK MNQQRSRRFR SAQDAKDANE KKAAELKEME KKGEIIDDAI KNKKTWDSNA I TPGTPFMH ...String:
MGVPALFRWL SRKYPKIISP VIQDEDVDID GESRPTRYED PNPNGELDNL YLDMNGIVHP CSHPEHKPVP ETEDEMMLDV FAYTENVIM MARPRKVIYI AVDGVAPRAK MNQQRSRRFR SAQDAKDANE KKAAELKEME KKGEIIDDAI KNKKTWDSNA I TPGTPFMH RLADSLRYWA AYKLTTDPGW SGIEVIISDA SVPGEGEHKI MSYVRSLRSS PKHDPNTTHC IYGLAAALIF LG LATHEPH FKILREDVFA QDKKSYSLQD QLRMTDIERQ ELKDKKTPFL WLHLNILREY LQIELNVPGL SFPFDLEKSI DDW VFICFF CGNDFLPHLP SLDVRDNSIT TLVTIWKQIL PTMKGYLTTD GYLNLPAVER LLAELAKKED YIFRKRYEDE KRSL ENQKR RKLAQEQSSA RSQNAPNIST GKDKAPLTPN QNIPLYTTSG ESVGIKMTDS EMVNNSALIT KANEANKSIA ELLKQ NLQN EINKKRKISN EEQEVVKESV EEVVEEEDDV LVTSDPEDSS TEILIPKNEE IRLWEPGYRK RYYETKFHTK DPQKVK KIA RNMVQKYIEG VSWVLLYYYQ GCPSWNWYYP YHYAPFAADF VNLSELKIEF VEGTPFRPYE QLMSVLPAAS SHNLPDV FR SLMSDANSEI IDFYPEEFPL DMNGKKVIWQ AIPLLPFIDE NRLLKAVQSK YDQLTEDEKF RNTNRSEILV LGRSHSHY P TLVKELYEEG KDSYEFQVDS SGVSGVAIKL QSFDRSGVLR LPVKQLEGYR HYPDISNRDF LMVEFKQLPK SHAKSMILS GLIPHLRRLT QEDKDSILYG GTNFYGRNRF SPEENADFKQ YIGPHGKSQY LPRQGGYKAF IQIHSDEAKG HRHGIYHGGS HTETEFRRG GGYHQHGNRG GRGGYQGNQG YQANSGGYQN SYQGSYQGGY RGGYQGGSQG RYQAGYQSGY QGGYQGEYKN G YQGGYQGN QGNQGYNRQT YNASKSGTLP MKRRHNSGPS SGLEVLFQ

UniProtKB: 5'-3' exoribonuclease

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Macromolecule #2: Decapping nuclease

MacromoleculeName: Decapping nuclease / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides
Source (natural)Organism: Komagataella phaffii (fungus)
Molecular weightTheoretical: 44.595996 KDa
Recombinant expressionOrganism: Escherichia coli KRX (bacteria)
SequenceString: GPGMSKEKIL PLAARSKKAM LRQPKQVAYF SRDLNYKTHP DRSNLSYYYL PDGDIDNSID LSVGSKHFLL GDSVELSKLD PILLALKEI EKESGAKTKD RIITWRGIMR KLLTLPYDSE EDFVLDVVSF DGQLFIQFNV PYLKSKDVQK QGDTEFHKKL Q FSGYKFEK ...String:
GPGMSKEKIL PLAARSKKAM LRQPKQVAYF SRDLNYKTHP DRSNLSYYYL PDGDIDNSID LSVGSKHFLL GDSVELSKLD PILLALKEI EKESGAKTKD RIITWRGIMR KLLTLPYDSE EDFVLDVVSF DGQLFIQFNV PYLKSKDVQK QGDTEFHKKL Q FSGYKFEK MATLPKPWPE CTRKEIDSRA KSKCNNIEQY GAIVRTGISR IKILIGGAVA CTADYYDEND PLSRYIELKT TR TINQYKD MIAFEKKLFR TWAQCFLLGI PKIIYGFRDD NCILRTVEEF STNDIPLMVK NNPLNEQPKK ENCYMSSINF YGA VVEWLN ESVKDDQVWK LSYAKRNRQY LVLKEVTDEN EKQQIVDSAI PAWFKEWRSE LRNSEGNI

UniProtKB: Decapping nuclease

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Macromolecule #3: Exonuclease Rat1p and Rai1p interacting protein

MacromoleculeName: Exonuclease Rat1p and Rai1p interacting protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Komagataella phaffii (fungus)
Molecular weightTheoretical: 42.27293 KDa
Recombinant expressionOrganism: Escherichia coli KRX (bacteria)
SequenceString: MSYSKDIVLE KLASLEETQI SIQSIGQWCL FHHRHAPETV AIWSEFVGST QTKKLAGLYL ANEIIQQSRA KRKTTFLDEF AKVLPSTLE QIYPSMISTH QAKLKRIIDV WSQRKIFDSN LIHRLYQSIQ DQKAYNGLSS SSSNSPPINS GDLAPEISSL S TLFNKIST ...String:
MSYSKDIVLE KLASLEETQI SIQSIGQWCL FHHRHAPETV AIWSEFVGST QTKKLAGLYL ANEIIQQSRA KRKTTFLDEF AKVLPSTLE QIYPSMISTH QAKLKRIIDV WSQRKIFDSN LIHRLYQSIQ DQKAYNGLSS SSSNSPPINS GDLAPEISSL S TLFNKIST LKSSTSLVVN QINEQYSSLF DSETLPGTDI YLKQLGDLST LITSARSKSQ ETQELRESII NELKKLIQVQ ES WITKDAE SSGSLDEKLA TVQQKESELK EFINDVEEED GVPQYAASSD EEGDENVSKK RKMESPPTET VDSGEQPTNP VHP QLSSIL ESLSRTTGLT PEPASTSDES ATATQKQDET PVSSSINPAL ASLLSKLNGL EVLFQ

UniProtKB: Exonuclease Rat1p and Rai1p interacting 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
Component:
ConcentrationFormulaName
20.0 mMHEPES2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic acid
100.0 mMNaClSodium chloride
10.0 mMKClpotassium chloride
1.0 mMMgCl2magnesium chloride
1.0 mMTCEPTris(2-carboxyethyl)phosphine
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 75 % / Chamber temperature: 283 K / Instrument: LEICA EM GP / Details: EMGP2.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 61.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: alphafold2 model
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 77924
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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