[English] 日本語
Yorodumi
- EMDB-0324: Negative-stain map of CPFcore -

+
Open data


ID or keywords:

Loading...

no data

-
Basic information

Entry
Database: EMDB / ID: 0324
TitleNegative-stain map of CPFcore
Map dataNegative-stain map of CPFcore
SampleCPFcore: a complex of Cft1-Pfs2-Yth1-Fip1-Pap1-Cft2-Ysh1-Mpe1:
Cft1 / Pfs2 / Yth1 / Fip1 / Pap1 / Cft2 / Ysh1 / Mpe1
SourceSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / negative staining / 25 Å resolution
AuthorsHill CH / Boreikaite V / Kumar A / Casanal A / Kubik P / Degliesposti G / Maslen S / Mariani A / von Loeffelholz O / Girbig M / Skehel M / Passmore LA
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 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.
DateDeposition: Oct 29, 2018 / Header (metadata) release: Feb 13, 2019 / Map release: Feb 13, 2019 / Last update: Feb 20, 2019

-
Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.065
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.065
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

-
Map

Fileemd_0324.map.gz (map file in CCP4 format, 8389 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
128 pix
3.98 Å/pix.
= 509.44 Å
128 pix
3.98 Å/pix.
= 509.44 Å
128 pix
3.98 Å/pix.
= 509.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.98 Å
Density
Contour Level:0.065 (by author), 0.065 (movie #1):
Minimum - Maximum-0.13506678 - 0.5055184
Average (Standard dev.)0.0005758424 (0.015362408)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions128128128
Origin0.00.00.0
Limit127.0127.0127.0
Spacing128128128
CellA=B=C: 509.44 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.983.983.98
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z509.440509.440509.440
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.1350.5060.001

-
Supplemental data

-
Sample components

+
Entire CPFcore: a complex of Cft1-Pfs2-Yth1-Fip1-Pap1-Cft2-Ysh1-Mpe1

EntireName: CPFcore: a complex of Cft1-Pfs2-Yth1-Fip1-Pap1-Cft2-Ysh1-Mpe1
Number of components: 9

+
Component #1: protein, CPFcore: a complex of Cft1-Pfs2-Yth1-Fip1-Pap1-Cft2-Ysh1-Mpe1

ProteinName: CPFcore: a complex of Cft1-Pfs2-Yth1-Fip1-Pap1-Cft2-Ysh1-Mpe1
Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: unidentified baculovirus

+
Component #2: protein, Cft1

ProteinName: Cft1 / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: unidentified baculovirus

+
Component #3: protein, Pfs2

ProteinName: Pfs2 / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: unidentified baculovirus

+
Component #4: protein, Yth1

ProteinName: Yth1 / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: unidentified baculovirus

+
Component #5: protein, Fip1

ProteinName: Fip1 / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: unidentified baculovirus

+
Component #6: protein, Pap1

ProteinName: Pap1 / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: unidentified baculovirus

+
Component #7: protein, Cft2

ProteinName: Cft2 / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: unidentified baculovirus

+
Component #8: protein, Ysh1

ProteinName: Ysh1 / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: unidentified baculovirus

+
Component #9: protein, Mpe1

ProteinName: Mpe1 / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: unidentified baculovirus

-
Experimental details

-
Sample preparation

SpecimenSpecimen state: particle / Method: negative staining
Sample solutionpH: 7.9
Support filmGrids were CF400-CU-UL from Electron Microscopy Sciences. Thin layer continuous carbon over 400-mesh copper. Manually inspected before use (with a light microscope)
StainingThree microliters of sample was applied to the support and allowed to adsorb for 60 s before wicking away with filter paper. Grids were then applied sequentially to two 30 ul drops of 2% w/v uranyl acetate, first to wash (quick) and then to stain (30 s). Excess stain was then wicked away with filter paper until dry.
VitrificationCryogen name: NONE

-
Electron microscopy imaging

Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI SPIRIT
Electron gunElectron source: TUNGSTEN HAIRPIN / Accelerating voltage: 120 kV / Electron dose: 4 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 26000.0 X (nominal) / Imaging mode: BRIGHT FIELD / Defocus: -600.0 nm
Specimen HolderModel: SIDE ENTRY, EUCENTRIC
CameraDetector: GATAN ULTRASCAN 1000 (2k x 2k)

-
Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 23969
3D reconstructionSoftware: RELION / Resolution: 25 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

-
Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 6EOJ, 3C66, 2I7X, 6I1D

+
About Yorodumi

-
News

-
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force. (see PDBe EMDB page)
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.: Q: What is "EMD"? / ID/Accession-code notation in Yorodumi/EM Navigator

External links: EMDB at PDBe / Contact to PDBj

-
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary. This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated. See below links for details.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software). Now, EM Navigator and Yorodumi are based on the updated data.

External links: wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

+
Jun 16, 2017. Omokage search with filter

Omokage search with filter

  • Result of Omokage search can be filtered by keywords and the database types

Related info.: Omokage search

+
Sep 15, 2016. EM Navigator & Yorodumi renewed

EM Navigator & Yorodumi renewed

  • New versions of EM Navigator and Yorodumi started

Related info.: Changes in new EM Navigator and Yorodumi

+
Aug 31, 2016. New EM Navigator & Yorodumi

New EM Navigator & Yorodumi

  • In 15th Sep 2016, the development versions of EM Navigator and Yorodumi will replace the official versions.
  • Current version will continue as 'legacy version' for some time.

Related info.: Changes in new EM Navigator and Yorodumi / EM Navigator / Yorodumi

Read more

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.

Related info.: EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more