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- PDB-6i1d: Structure of the Ysh1-Mpe1 nuclease complex from S.cerevisiae -

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Basic information

Entry
Database: PDB / ID: 6i1d
TitleStructure of the Ysh1-Mpe1 nuclease complex from S.cerevisiae
Components
  • Endoribonuclease YSH1
  • Protein MPE1
KeywordsGENE REGULATION / pre-mRNA / mRNA / nuclease / endonuclease / cleavage / polyadenylation / polyA / CPF / metallo-beta-lactamase / 3' ends
Function / homology
Function and homology information


: / : / Processing of Intronless Pre-mRNAs / sno(s)RNA 3'-end processing / snoRNA splicing / mRNA cleavage and polyadenylation specificity factor complex / Antigen processing: Ubiquitination & Proteasome degradation / 5'-3' exonuclease activity / pre-mRNA binding / Hydrolases; Acting on ester bonds; Endoribonucleases producing 3'-phosphomonoesters ...: / : / Processing of Intronless Pre-mRNAs / sno(s)RNA 3'-end processing / snoRNA splicing / mRNA cleavage and polyadenylation specificity factor complex / Antigen processing: Ubiquitination & Proteasome degradation / 5'-3' exonuclease activity / pre-mRNA binding / Hydrolases; Acting on ester bonds; Endoribonucleases producing 3'-phosphomonoesters / : / termination of RNA polymerase II transcription / RNA endonuclease activity / RNA splicing / protein polyubiquitination / ubiquitin protein ligase activity / protein ubiquitination / RNA binding / zinc ion binding / metal ion binding / nucleus / cytosol
Similarity search - Function
E3 ubiquitin-protein ligase RBBP6 family / DWNN domain / DWNN domain / DWNN domain profile. / DWNN / Pre-mRNA 3'-end-processing endonuclease polyadenylation factor C-term / Pre-mRNA 3'-end-processing endonuclease polyadenylation factor C-term / CPSF73-100_C / Rossmann fold - #10890 / Beta-Casp domain ...E3 ubiquitin-protein ligase RBBP6 family / DWNN domain / DWNN domain / DWNN domain profile. / DWNN / Pre-mRNA 3'-end-processing endonuclease polyadenylation factor C-term / Pre-mRNA 3'-end-processing endonuclease polyadenylation factor C-term / CPSF73-100_C / Rossmann fold - #10890 / Beta-Casp domain / Beta-Casp domain / Beta-Casp domain / Zn-dependent metallo-hydrolase, RNA specificity domain / Zn-dependent metallo-hydrolase RNA specificity domain / Metallo-beta-lactamase superfamily / Metallo-beta-lactamase superfamily / Metallo-beta-lactamase / Ribonuclease Z/Hydroxyacylglutathione hydrolase-like / Metallo-beta-lactamase; Chain A / Ribonuclease Z/Hydroxyacylglutathione hydrolase-like / zinc finger / Zinc finger, CCHC-type superfamily / 4-Layer Sandwich / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Zinc finger, RING/FYVE/PHD-type / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Protein MPE1 / Endoribonuclease YSH1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.28 Å
AuthorsHill, C.H. / Boreikaite, V. / Kumar, A. / Casanal, A. / Kubik, P. / Degliesposti, G. / Maslen, S. / Mariani, A. / von Loeffelholz, O. / Girbig, M. ...Hill, C.H. / Boreikaite, V. / Kumar, A. / Casanal, A. / Kubik, P. / Degliesposti, G. / Maslen, S. / Mariani, A. / von Loeffelholz, O. / Girbig, M. / Skehel, M. / Passmore, L.A.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
European Research Council261151 United Kingdom
European Research Council725685 United Kingdom
Medical Research Council (United Kingdom)MC_U105192715 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 28, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 13, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 20, 2019Group: Data collection / Database references / Category: citation / citation_author / pdbx_database_proc
Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Apr 3, 2019Group: Data collection / Database references
Category: citation / database_PDB_rev ...citation / database_PDB_rev / database_PDB_rev_record / pdbx_database_proc
Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Endoribonuclease YSH1
B: Protein MPE1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)71,7575
Polymers71,5342
Non-polymers2233
Water3,585199
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, co-expressed together and co-purified by affinity, anion exchange and gel filtration, assay for oligomerization, Pull-down assays (see paper), mass spectrometry, HDX and XL- ...Evidence: gel filtration, co-expressed together and co-purified by affinity, anion exchange and gel filtration, assay for oligomerization, Pull-down assays (see paper), mass spectrometry, HDX and XL-MS experiments (see paper)
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2210 Å2
ΔGint-79 kcal/mol
Surface area23060 Å2
MethodPISA
Unit cell
Length a, b, c (Å)43.380, 124.270, 63.450
Angle α, β, γ (deg.)90.00, 103.21, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Endoribonuclease YSH1 / Yeast 73 kDa homolog 1 / mRNA 3'-end-processing protein YSH1


Mass: 53458.711 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: YSH1, BRR5, YLR277C / Cell line (production host): Sf9 / Production host: unidentified baculovirus
References: UniProt: Q06224, Hydrolases; Acting on ester bonds; Endoribonucleases producing 3'-phosphomonoesters
#2: Protein Protein MPE1


Mass: 18075.129 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MPE1, YKL059C, YKL316 / Cell line (production host): Sf9 / Production host: unidentified baculovirus / References: UniProt: P35728
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 199 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.33 Å3/Da / Density % sol: 47.3 % / Description: plates
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8.7
Details: 80 ul reservoir of 26 % w/v PEG 3000, 0.1 M CHES pH 8.7 The final drop of 400 nl comprised 200 nl protein and 200 nl crystallization buffer

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04-1 / Wavelength: 0.9159 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Sep 9, 2017
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9159 Å / Relative weight: 1
ReflectionResolution: 2.28→62.13 Å / Num. obs: 29524 / % possible obs: 99.08 % / Redundancy: 3.39 % / CC1/2: 0.999 / Rmerge(I) obs: 0.065 / Rpim(I) all: 0.041 / Rrim(I) all: 0.077 / Net I/σ(I): 12.3
Reflection shellResolution: 2.28→2.32 Å / Redundancy: 3.4 % / Rmerge(I) obs: 1.13 / Mean I/σ(I) obs: 1.3 / Num. unique obs: 1442 / CC1/2: 0.578 / Rpim(I) all: 0.716 / Rrim(I) all: 1.346 / % possible all: 99.4

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Processing

Software
NameVersionClassification
PHENIX(1.12_2829: ???)refinement
XDSdata reduction
xia2data reduction
Cootmodel building
PHENIXmodel building
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2I7V, 2C7H

2i7v

2c7h


Resolution: 2.28→62.135 Å / SU ML: 0.29 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.49 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2219 1487 5.05 %
Rwork0.1726 --
obs0.175 29466 98.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.28→62.135 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4356 0 8 199 4563
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0034455
X-RAY DIFFRACTIONf_angle_d0.5456025
X-RAY DIFFRACTIONf_dihedral_angle_d11.3062665
X-RAY DIFFRACTIONf_chiral_restr0.046672
X-RAY DIFFRACTIONf_plane_restr0.003782
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.28-2.35360.3661440.32572510X-RAY DIFFRACTION99
2.3536-2.43770.32781430.2852562X-RAY DIFFRACTION99
2.4377-2.53530.31771450.26612529X-RAY DIFFRACTION99
2.5353-2.65070.2941410.23672534X-RAY DIFFRACTION100
2.6507-2.79050.28791320.22742501X-RAY DIFFRACTION98
2.7905-2.96530.26361430.19892563X-RAY DIFFRACTION99
2.9653-3.19420.26081290.19722567X-RAY DIFFRACTION100
3.1942-3.51570.23791340.17722532X-RAY DIFFRACTION99
3.5157-4.02430.2021310.15382533X-RAY DIFFRACTION98
4.0243-5.06980.18251060.12492565X-RAY DIFFRACTION99
5.0698-62.15810.15821390.14442583X-RAY DIFFRACTION98
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.20820.2428-0.56041.4502-0.02731.65470.0379-0.11380.2202-0.0196-0.0660.0686-0.092-0.05780.00850.30390.0406-0.01470.3441-0.03950.512.595185.84563.0794
24.85240.039-0.13121.3884-0.08122.48630.0863-0.16220.1787-0.0462-0.02080.3074-0.1509-0.1856-0.03430.37970.0130.00830.4623-0.04680.5891-5.442580.72868.5341
32.9030.1618-0.16671.46560.64842.4260.0381-1.0436-0.12260.3315-0.045-0.09920.12860.0525-0.03370.40630.0042-0.02150.70240.02260.45113.252677.896322.6136
43.6034-0.73460.89183.5928-1.02633.42440.11640.3195-0.6305-0.317-0.052-0.13740.23950.1964-0.07520.34520.0220.07530.4145-0.14410.553418.150571.114-7.3052
52.52022.08381.77761.77671.11723.88910.0238-0.08590.44550.54320.25520.6441-0.0619-0.401-0.22650.31520.04110.07780.30840.07660.79661.437106.7688-4.1818
65.42613.8916-0.30669.6998-2.45366.09950.17090.05120.37950.6923-0.26350.9128-0.4751-0.1947-0.00160.49820.10990.11720.48480.12860.8976.1318118.032-3.9352
74-0.29960.76538.4501-5.98357.27790.1177-0.04620.70720.9207-0.1253-1.2245-0.45620.29860.26950.4308-0.00860.02440.39470.01051.089614.5568107.876-1.4171
85.2704-1.09715.09152.7383-1.87369.42680.20120.18390.1499-0.4424-0.4-0.25270.04560.62860.1960.49670.07460.08360.34660.06640.854911.8261101.4666-9.1641
93.7187-2.98260.71317.118-2.60223.42370.36460.88910.2962-0.7298-0.3988-0.13450.1531-0.004-0.03520.41790.09510.13020.42070.07550.61998.6421106.9-12.8805
105.9749-2.99960.03012.9771-2.59975.24540.3086-0.01940.7118-0.65330.2363-1.823-0.12540.54340.27570.45110.00750.11330.58070.06091.155222.0181102.1131-4.6674
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 5 through 111 )
2X-RAY DIFFRACTION2chain 'A' and (resid 112 through 167 )
3X-RAY DIFFRACTION3chain 'A' and (resid 168 through 432 )
4X-RAY DIFFRACTION4chain 'A' and (resid 433 through 473 )
5X-RAY DIFFRACTION5chain 'B' and (resid 2 through 15 )
6X-RAY DIFFRACTION6chain 'B' and (resid 16 through 23 )
7X-RAY DIFFRACTION7chain 'B' and (resid 24 through 45 )
8X-RAY DIFFRACTION8chain 'B' and (resid 46 through 52 )
9X-RAY DIFFRACTION9chain 'B' and (resid 53 through 78 )
10X-RAY DIFFRACTION10chain 'B' and (resid 79 through 107 )

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