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- EMDB-20859: Cryo-EM map of human CPSF73-CPSF100-Symplekin complex -

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Basic information

Entry
Database: EMDB / ID: EMD-20859
TitleCryo-EM map of human CPSF73-CPSF100-Symplekin complex
Map dataCryo-EM map of human CPSF73-CPSF100-Symplekin complex
Sample
  • Complex: The complex of human CPSF73-CPSF100-Symplekin
    • Protein or peptide: CPSF73
    • Protein or peptide: CPSF100
    • Protein or peptide: Symplekin
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.4 Å
AuthorsSun Y / Zhang Y / Walz T / Tong L
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR35GM118093 United States
CitationJournal: Mol Cell / Year: 2020
Title: Structural Insights into the Human Pre-mRNA 3'-End Processing Machinery.
Authors: Yixiao Zhang / Yadong Sun / Yongsheng Shi / Thomas Walz / Liang Tong /
Abstract: The mammalian pre-mRNA 3'-end-processing machinery consists of cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and other proteins, but the overall ...The mammalian pre-mRNA 3'-end-processing machinery consists of cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and other proteins, but the overall architecture of this machinery remains unclear. CPSF contains two functionally distinct modules: a cleavage factor (mCF) and a polyadenylation specificity factor (mPSF). Here, we have produced recombinant human CPSF and CstF and examined these factors by electron microscopy (EM). We find that mPSF is the organizational core of the machinery, while the conformations of mCF and CstF and the position of mCF relative to mPSF are highly variable. We have identified by cryo-EM a segment in CPSF100 that tethers mCF to mPSF, and we have named it the PSF interaction motif (PIM). Mutations in the PIM can abolish CPSF formation, indicating that it is a crucial contact in CPSF. We have also obtained reconstructions of mCF and CstF77 by cryo-EM, assembled around the mPSF core.
History
DepositionOct 23, 2019-
Header (metadata) releaseNov 27, 2019-
Map releaseNov 27, 2019-
UpdateMar 4, 2020-
Current statusMar 4, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.011
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.011
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20859.png

Downloads & links

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Map

FileDownload / File: emd_20859.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of human CPSF73-CPSF100-Symplekin complex
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.011 / Movie #1: 0.011
Minimum - Maximum-0.029016882 - 0.05099807
Average (Standard dev.)0.0000652628 (±0.002344527)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z273.920273.920273.920
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0290.0510.000

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Supplemental data

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Sample components

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Entire : The complex of human CPSF73-CPSF100-Symplekin

EntireName: The complex of human CPSF73-CPSF100-Symplekin
Components
  • Complex: The complex of human CPSF73-CPSF100-Symplekin
    • Protein or peptide: CPSF73
    • Protein or peptide: CPSF100
    • Protein or peptide: Symplekin

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Supramolecule #1: The complex of human CPSF73-CPSF100-Symplekin

SupramoleculeName: The complex of human CPSF73-CPSF100-Symplekin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #1: CPSF73

MacromoleculeName: CPSF73 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSAIPAEESD QLLIRPLGAG QEVGRSCIIL EFKGRKIMLD CGIHPGLEGM DALPYIDLID PAEIDLLLIS HFHLDHCGAL PWFLQKTSFK GRTFMTHATK AIYRWLLSDY VKVSNISADD MLYTETDLEE SMDKIETINF HEVKEVAGIK FWCYHAGHVL GAAMFMIEIA ...String:
MSAIPAEESD QLLIRPLGAG QEVGRSCIIL EFKGRKIMLD CGIHPGLEGM DALPYIDLID PAEIDLLLIS HFHLDHCGAL PWFLQKTSFK GRTFMTHATK AIYRWLLSDY VKVSNISADD MLYTETDLEE SMDKIETINF HEVKEVAGIK FWCYHAGHVL GAAMFMIEIA GVKLLYTGDF SRQEDRHLMA AEIPNIKPDI LIIESTYGTH IHEKREEREA RFCNTVHDIV NRGGRGLIPV FALGRAQELL LILDEYWQNH PELHDIPIYY ASSLAKKCMA VYQTYVNAMN DKIRKQININ NPFVFKHISN LKSMDHFDDI GPSVVMASPG MMQSGLSREL FESWCTDKRN GVIIAGYCVE GTLAKHIMSE PEEITTMSGQ KLPLKMSVDY ISFSAHTDYQ QTSEFIRALK PPHVILVHGE QNEMARLKAA LIREYEDNDE VHIEVHNPRN TEAVTLNFRG EKLAKVMGFL ADKKPEQGQR VSGILVKRN FNYHILSPCD LSNYTDLAMS TVKQTQAIPY TGPFNLLCYQ LQKLTGDVEE LEIQEKPALK VFKNITVIQE PGMVVLEWLA NPSNDMYADT VTTVILEVQS NPKIRKGAVQ KVSKKLEMHV YSKRLEIMLQ DIFGEDCVSV KDDSILSVTV DGKTANLNLE TRTVECEEGS EDDESLREMV ELAAQRLYEA LTPVH

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Macromolecule #2: CPSF100

MacromoleculeName: CPSF100 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MTSIIKLTTL SGVQEESALC YLLQVDEFRF LLDCGWDEHF SMDIIDSLRK HVHQIDAVLL SHPDPLHLGA LPYAVGKLGL NCAIYATIPV YKMGQMFMYD LYQSRHNTED FTLFTLDDVD AAFDKIQQLK FSQIVNLKGK GHGLSITPLP AGHMIGGTIW KIVKDGEEEI ...String:
MTSIIKLTTL SGVQEESALC YLLQVDEFRF LLDCGWDEHF SMDIIDSLRK HVHQIDAVLL SHPDPLHLGA LPYAVGKLGL NCAIYATIPV YKMGQMFMYD LYQSRHNTED FTLFTLDDVD AAFDKIQQLK FSQIVNLKGK GHGLSITPLP AGHMIGGTIW KIVKDGEEEI VYAVDFNHKR EIHLNGCSLE MLSRPSLLIT DSFNATYVQP RRKQRDEQLL TNVLETLRGD GNVLIAVDTA GRVLELAQLL DQIWRTKDAG LGVYSLALLN NVSYNVVEFS KSQVEWMSDK LMRCFEDKRN NPFQFRHLSL CHGLSDLARV PSPKVVLASQ PDLECGFSRD LFIQWCQDPK NSIILTYRTT PGTLARFLID NPSEKITEIE LRKRVKLEGK ELEEYLEKEK LKKEAAKKLE QSKEADIDSS DESDIEEDID QPSAHKTKHD LMMKGEGSRK GSFFKQAKKS YPMFPAPEER IKWDEYGEII KPEDFLVPEL QATEEEKSKL ESGLTNGDEP MDQDLSDVPT KCISTTESIE IKARVTYIDY EGRSDGDSIK KIINQMKPRQ LIIVHGPPEA SQDLAECCRA FGGKDIKVYM PKLHETVDAT SETHIYQVRL KDSLVSSLQF CKAKAELAWI DGVLDMRVSK VDTGVILEEG ELKDDGEDSE MQVEAPSDSS VIAQQKAMKS LFGDDEKETG EESEIIPTLE PLPPHEVPGH QSVFMNEPRL SDFKQVLLRE GIQAEFVGGV LVCNNQVAVR RTETGRIGLE GCLCQDFYRI RDLLYEQYAI V

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Macromolecule #3: Symplekin

MacromoleculeName: Symplekin / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: SDSTLKKMKL EPNLGEDDED KDLEPGPSGT SKASAQISGQ SDTDITAEFL QPLLTPDNVA NLVLISMVYL PEAMPASFQA IYTPVESAGT EAQIKHLARL MATQMTAAGL GPGVEQTKQC KEEPKEEKVV KTESVLIKRR LSAQGQAISV VGSLSSMSPL EEEAPQAKRR ...String:
SDSTLKKMKL EPNLGEDDED KDLEPGPSGT SKASAQISGQ SDTDITAEFL QPLLTPDNVA NLVLISMVYL PEAMPASFQA IYTPVESAGT EAQIKHLARL MATQMTAAGL GPGVEQTKQC KEEPKEEKVV KTESVLIKRR LSAQGQAISV VGSLSSMSPL EEEAPQAKRR PEPIIPVTQP RLAGAGGRKK IFRLSDVLKP LTDAQVEAMK LGAVKRILRA EKAVACSGAA QVRIKILASL VTQFNSGLKA EVLSFILEDV RARLDLAFAW LYQEYNAYLA AGASGSLDKY EDCLIRLLSG LQEKPDQKDG IFTKVVLEAP LITESALEVV RKYCEDESRT YLGMSTLRDL IFKRPSRQFQ YLHVLLDLSS HEKDKVRSQA LLFIKRMYEK EQLREYVEKF ALNYLQLLVH PNPPSVLFGA DKDTEVAAPW TEETVKQCLY LYLALLPQNH KLIHELAAVY TEAIADIKRT VLRVIEQPIR GMGMNSPELL LLVENCPKGA ETLVTRCLHS LTDKVPPSPE LVKRVRDLYH KRLPDVRFLI PVLNGLEKKE VIQALPKLIK LNPIVVKEVF NRLLGTQHGE GNSALSPLNP GELLIALHNI DSVKCDMKSI IKATNLCFAE RNVYTSEVLA VVMQQLMEQS PLPMLLMRTV IQSLTMYPRL GGFVMNILSR LIMKQVWKYP KVWEGFIKCC QRTKPQSFQV ILQLPPQQLG AVFDKCPELR EPLLAHVRSF TPHQQAHIPN SIMTILEASG KQEPEAKE

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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

Concentration0.25 mg/mL
BufferpH: 8 / Component - Concentration: 150.0 mM / Component - Formula: NaClSodium chloride / Component - Name: sodium chloride / Details: 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM DTT
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 2.8 µm / Calibrated defocus min: 0.9 µm / Calibrated magnification: 46729 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 225000
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: COUNTING / Average exposure time: 10.0 sec. / Average electron dose: 70.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 35040

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