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- EMDB-0325: Cryo-EM map of the Ysh1-Mpe1 nuclease complex -

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Basic information

Entry
Database: EMDB / ID: EMD-0325
TitleCryo-EM map of the Ysh1-Mpe1 nuclease complex
Map dataCryo-EM map of the Ysh1-Mpe1 complex
Sample
  • Complex: Ysh1-Mpe1 complex Highly anisotropic due to severe preferred orientation.
    • Protein or peptide: Ysh1
    • Protein or peptide: Mpe1
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.0 Å
AuthorsHill CH / Boreikaite V / Kumar A / Casanal A / Kubik P / Degliesposti G / Maslen S / Mariani A / von Loeffelholz O / Girbig M ...Hill CH / Boreikaite V / Kumar A / Casanal A / Kubik P / Degliesposti G / Maslen S / Mariani A / von Loeffelholz O / Girbig M / Skehel M / Passmore LA
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
European Research Council261151 United Kingdom
Medical Research Council (United Kingdom)MC_U105192715 United Kingdom
European Research Council725685 United Kingdom
CitationJournal: Mol Cell / Year: 2019
Title: Activation of the Endonuclease that Defines mRNA 3' Ends Requires Incorporation into an 8-Subunit Core Cleavage and Polyadenylation Factor Complex.
Authors: Chris H Hill / Vytautė Boreikaitė / Ananthanarayanan Kumar / Ana Casañal / Peter Kubík / Gianluca Degliesposti / Sarah Maslen / Angelica Mariani / Ottilie von Loeffelholz / Mathias ...Authors: Chris H Hill / Vytautė Boreikaitė / Ananthanarayanan Kumar / Ana Casañal / Peter Kubík / Gianluca Degliesposti / Sarah Maslen / Angelica Mariani / Ottilie von Loeffelholz / Mathias Girbig / Mark Skehel / Lori A Passmore /
Abstract: Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3' ends. CPF cleaves pre-mRNAs at a specific site and adds a poly(A) tail. The ...Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3' ends. CPF cleaves pre-mRNAs at a specific site and adds a poly(A) tail. The cleavage reaction defines the 3' end of the mature mRNA, and thus the activity of the endonuclease is highly regulated. Here, we show that reconstitution of specific pre-mRNA cleavage with recombinant yeast proteins requires incorporation of the Ysh1 endonuclease into an eight-subunit "CPF" complex. Cleavage also requires the accessory cleavage factors IA and IB, which bind substrate pre-mRNAs and CPF, likely facilitating assembly of an active complex. Using X-ray crystallography, electron microscopy, and mass spectrometry, we determine the structure of Ysh1 bound to Mpe1 and the arrangement of subunits within CPF. Together, our data suggest that the active mRNA 3' end processing machinery is a dynamic assembly that is licensed to cleave only when all protein factors come together at the polyadenylation site.
History
DepositionOct 30, 2018-
Header (metadata) releaseFeb 13, 2019-
Map releaseFeb 13, 2019-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0325.png

Downloads & links

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Map

FileDownload / File: emd_0325.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of the Ysh1-Mpe1 complex
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.047686033 - 0.079657555
Average (Standard dev.)0.000808997 (±0.005417154)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 139.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z139.520139.520139.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0480.0800.001

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Supplemental data

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Mask #1

Fileemd_0325_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map 1

Fileemd_0325_half_map_1.map
Annotationhalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map 2

Fileemd_0325_half_map_2.map
Annotationhalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ysh1-Mpe1 complex Highly anisotropic due to severe preferred orie...

EntireName: Ysh1-Mpe1 complex Highly anisotropic due to severe preferred orientation.
Components
  • Complex: Ysh1-Mpe1 complex Highly anisotropic due to severe preferred orientation.
    • Protein or peptide: Ysh1
    • Protein or peptide: Mpe1

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Supramolecule #1: Ysh1-Mpe1 complex Highly anisotropic due to severe preferred orie...

SupramoleculeName: Ysh1-Mpe1 complex Highly anisotropic due to severe preferred orientation.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The trimeric Ysh1-Mpe1-Yjr141w complex was analysed, however several regions are flexible/disordered. This map corresponds to the Ysh1 N-terminal nuclease domain in complex with the Mpe1 N- ...Details: The trimeric Ysh1-Mpe1-Yjr141w complex was analysed, however several regions are flexible/disordered. This map corresponds to the Ysh1 N-terminal nuclease domain in complex with the Mpe1 N-terminal ubiquitin-like domain. These are the only ordered, globular densities present in the above sample.
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: unidentified baculovirus / Recombinant cell: Sf9
Molecular weightTheoretical: 57 KDa

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Macromolecule #1: Ysh1

MacromoleculeName: Ysh1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MERTNTTTFK FFSLGGSNEV GRSCHILQYK GKTVMLDAGI HPAYQGLASL PFYDEFDLSK VDILLISHF HLDHAASLPY VMQRTNFQGR VFMTHPTKAI YRWLLRDFVR VTSIGSSSSS M GTKDEGLF SDEDLVDSFD KIETVDYHST VDVNGIKFTA FHAGHVLGAA ...String:
MERTNTTTFK FFSLGGSNEV GRSCHILQYK GKTVMLDAGI HPAYQGLASL PFYDEFDLSK VDILLISHF HLDHAASLPY VMQRTNFQGR VFMTHPTKAI YRWLLRDFVR VTSIGSSSSS M GTKDEGLF SDEDLVDSFD KIETVDYHST VDVNGIKFTA FHAGHVLGAA MFQIEIAGLR VL FTGDYSR EVDRHLNSAE VPPLSSNVLI VESTFGTATH EPRLNRERKL TQLIHSTVMR GGR VLLPVF ALGRAQEIML ILDEYWSQHA DELGGGQVPI FYASNLAKKC MSVFQTYVNM MNDD IRKKF RDSQTNPFIF KNISYLRNLE DFQDFGPSVM LASPGMLQSG LSRDLLERWC PEDKN LVLI TGYSIEGTMA KFIMLEPDTI PSINNPEITI PRRCQVEEIS FAAHVDFQEN LEFIEK ISA PNIILVHGEA NPMGRLKSAL LSNFASLKGT DNEVHVFNPR NCVEVDLEFQ GVKVAKA VG NIVNEIYKEE NVEIKEEIAA KIEPIKEENE DNLDSQAEKG LVDEEEHKDI VVSGILVS D DKNFELDFLS LSDLREHHPD LSTTILRERQ SVRVNCKKEL IYWHILQMFG EAEVLQDDD RVTNQEPKVK EESKDNLTNT GKLILQIMGD IKLTIVNTLA VVEWTQDLMN DTVADSIIAI LMNVDSAPA SVKLSSHSCD DHDHNNVQSN AQGKIDEVER VKQISRLFKE QFGDCFTLFL N KDEYASNK EETITGVVTI GKSTAKIDFN NMKILECNSN PLKGRVESLL NIGGNLVTPL C

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Macromolecule #2: Mpe1

MacromoleculeName: Mpe1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MSSTIFYRFK SQRNTSRILF DGTGLTVFDL KREIIQENKL GDGTDFQLKI YNPDTEEEYD DDAFVIPRS TSVIVKRSPA IKSFSVHSRL KGNVGAAALG NATRYVTGRP RVLQKRQHTA T TTANVSGT TEEERIASMF ATQENQWEQT QEEMSAATPV FFKSQTNKNS ...String:
MSSTIFYRFK SQRNTSRILF DGTGLTVFDL KREIIQENKL GDGTDFQLKI YNPDTEEEYD DDAFVIPRS TSVIVKRSPA IKSFSVHSRL KGNVGAAALG NATRYVTGRP RVLQKRQHTA T TTANVSGT TEEERIASMF ATQENQWEQT QEEMSAATPV FFKSQTNKNS AQENEGPPPP GY MCYRCGG RDHWIKNCPT NSDPNFEGKR IRRTTGIPKK FLKSIEIDPE TMTPEEMAQR KIM ITDEGK FVVQVEDKQS WEDYQRKREN RQIDGDETIW RKGHFKDLPD DLKCPLTGGL LRQP VKTSK CCNIDFSKEA LENALVESDF VCPNCETRDI LLDSLVPDQD KEKEVETFLK KQEEL HGSS KDGNQPETKK MKLMDPTGTA GLNNNTSLPT SVNNGGTPVP PVPLPFGIPP FPMFPM PFM PPTATITNPH QADASPKK

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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: 7.9
Component:
ConcentrationName
150.0 mMsodium chloride
10.0 mMHEPES
GridModel: Quantifoil, UltrAuFoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blotted for 10 s.
Details350 nM complex was used.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Specialist opticsPhase plate: VOLTA PHASE PLATE / Spherical aberration corrector: hexapole Cs-corrector / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-42 / Number grids imaged: 1 / Number real images: 994 / Average exposure time: 9.0 sec. / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 509298
Details: Initially using Gaussian-blob based picking, then after clean-up and enrichment of rare views, conducted template-based autopick to obtain 429703 better centred particles.
CTF correctionSoftware - Name: Gctf
Details: Gctf was also used to fit and refine the phase shift per image created by the Volta phase plate
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6i1d


Details: The pdb file was converted to an EM volume and low-pass filtered to 20 angstroms resolution prior to any refinement
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0)
Details: Even though the gold-standard FSC cutoff indicated a resolution of 4.8A, the map was filtered to 6A in post-processing. This dataset had a severe preferred orientation leading to highly ...Details: Even though the gold-standard FSC cutoff indicated a resolution of 4.8A, the map was filtered to 6A in post-processing. This dataset had a severe preferred orientation leading to highly anisotropic reconstructions. The main purpose of the 3D refinement was to compare with the crystal structure (truncated proteins) to see if any more ordered domains were visible when imaging full-length proteins.
Number images used: 43308
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6i1d

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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