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- EMDB-0324: Negative-stain map of CPFcore -

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Basic information

Entry
Database: EMDB / ID: EMD-0324
TitleNegative-stain map of CPFcore
Map dataNegative-stain map of CPFcore
Sample
  • Complex: CPFcore: a complex of Cft1-Pfs2-Yth1-Fip1-Pap1-Cft2-Ysh1-Mpe1
    • Protein or peptide: Cft1
    • Protein or peptide: Pfs2
    • Protein or peptide: Yth1
    • Protein or peptide: Fip1
    • Protein or peptide: Pap1
    • Protein or peptide: Cft2
    • Protein or peptide: Ysh1
    • Protein or peptide: Mpe1
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 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
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 29, 2018-
Header (metadata) releaseFeb 13, 2019-
Map releaseFeb 13, 2019-
UpdateApr 3, 2019-
Current statusApr 3, 2019Processing site: PDBe / Status: Released

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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

emd_0324.png

Downloads & links

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Map

FileDownload / File: emd_0324.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative-stain map of CPFcore
Voxel sizeX=Y=Z: 3.98 Å
Density
Contour LevelBy AUTHOR: 0.065 / Movie #1: 0.065
Minimum - Maximum-0.13506678 - 0.5055184
Average (Standard dev.)0.0005758424 (±0.015362408)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 509.44 Å
α=β=γ: 90.0 °

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-383-383-383
NX/NY/NZ768768768
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.1350.5060.001

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Supplemental data

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Sample components

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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
Components
  • Complex: CPFcore: a complex of Cft1-Pfs2-Yth1-Fip1-Pap1-Cft2-Ysh1-Mpe1
    • Protein or peptide: Cft1
    • Protein or peptide: Pfs2
    • Protein or peptide: Yth1
    • Protein or peptide: Fip1
    • Protein or peptide: Pap1
    • Protein or peptide: Cft2
    • Protein or peptide: Ysh1
    • Protein or peptide: Mpe1

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Supramolecule #1: CPFcore: a complex of Cft1-Pfs2-Yth1-Fip1-Pap1-Cft2-Ysh1-Mpe1

SupramoleculeName: CPFcore: a complex of Cft1-Pfs2-Yth1-Fip1-Pap1-Cft2-Ysh1-Mpe1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: unidentified baculovirus

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Macromolecule #1: Cft1

MacromoleculeName: Cft1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MNVYDDVLDA TVVSHSLATH FTTSDYEELL VVRTNILSVY RPTRDGKLYL TDEFKFHGLI TDIGLIPQK DSPLSCLLLC TGVAKISILK FNTLTNSIDT LSLHYYEGKF KGKSLVELAK I STLRMDPG SSCALLFNND IIAFLPFHVN KNDDDEEEED EDENIDDSEL ...String:
MNVYDDVLDA TVVSHSLATH FTTSDYEELL VVRTNILSVY RPTRDGKLYL TDEFKFHGLI TDIGLIPQK DSPLSCLLLC TGVAKISILK FNTLTNSIDT LSLHYYEGKF KGKSLVELAK I STLRMDPG SSCALLFNND IIAFLPFHVN KNDDDEEEED EDENIDDSEL IHSMNQKSQG TN TFNKRKR TKLGDKFTAP SVVLVASELY EGAKNIIDIQ FLKNFTKPTI ALLYQPKLVW AGN TTISKL PTQYVILTLN IQPAESATKI ESTTIAFVKE LPWDLHTIVP VSNGAIIVGT NELA FLDNT GVLQSTVLLN SFADKELQKT KIINNSSLEI MFREKNTTSI WIPSSKSKNG GSNND ETLL LMDLKSNIYY IQMEAEGRLL IKFDIFKLPI VNDLLKENSN PKCITRLNAT NSNKNM DLF IGFGSGNALV LRLNNLKSTI ETREAHNPSS GTNSLMDIND DDDEEMDDLY ADEAPEN GL TTNDSKGTVE TVQPFDIELL SSLRNVGPIT SLTVGKVSSI DDVVKGLPNP NKNEYSLV A TSGNGSGSHL TVIQTSVQPE IELALKFISI TQIWNLKIKG RDRYLITTDS TKSRSDIYE SDNNFKLHKG GRLRRDATTV YISMFGEEKR IIQVTTNHLY LYDTHFRRLT TIKFDYEVIH VSVMDPYIL VTVSRGDIKI FELEEKNKRK LLKVDLPEIL NEMVITSGLI LKSNMCNEFL I GLSKSQEE QLLFTFVTAD NQIIFFTKDH NDRIFQLNGV DQLNESLYIS TYQLGDEIVP DP SIKQVMI NKLGHDNKEE YLTILTFGGE IYQYRKLPQR RSRFYRNVTR NDLAITGAPD NAY AKGVSS IERIMHYFPD YNGYSVIFVT GSVPYILIKE DDSTPKIFKF GNIPLVSVTP WSER SVMCV DDIKNARVYT LTTDNMYYGN KLPLKQIKIS NVLDDYKTLQ KLVYHERAQL FLVSY CKRV PYEALGEDGE KVIGYDENVP HAEGFQSGIL LINPKSWKVI DKIDFPKNSV VNEMRS SMI QINSKTKRKR EYIIAGVANA TTEDTPPTGA FHIYDVIEVV PEPGKPDTNY KLKEIFQ EE VSGTVSTVCE VSGRFMISQS QKVLVRDIQE DNSVIPVAFL DIPVFVTDSK SFGNLLII G DAMQGFQFIG FDAEPYRMIS LGRSMSKFQT MSLEFLVNGG DMYFAATDAD RNVHVLKYA PDEPNSLSGQ RLVHCSSFTL HSTNSCMMLL PRNEEFGSPQ VPSFQNVGGQ VDGSVFKIVP LSEEKYRRL YVIQQQIIDR ELQLGGLNPR MERLANDFYQ MGHSMRPMLD FNVIRRFCGL A IDRRKSIA QKAGRHAHFE AWRDIINIEF SMRSLCQGK

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Macromolecule #2: Pfs2

MacromoleculeName: Pfs2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MDGHNQNQYQ NQNQIQQSQQ PPLKKYVTQR RSVDVSSPYI NLYYNRRHGL PNLVVEPETS YTIDIMPPN AYRGRDRVIN LPSKFTHLSS NKVKHVIPAI QWTPEGRRLV VATYSGEFSL W NASSFTFE TLMQAHDSAV TTMKYSHDSD WMISGDADGM IKIWQPNFSM ...String:
MDGHNQNQYQ NQNQIQQSQQ PPLKKYVTQR RSVDVSSPYI NLYYNRRHGL PNLVVEPETS YTIDIMPPN AYRGRDRVIN LPSKFTHLSS NKVKHVIPAI QWTPEGRRLV VATYSGEFSL W NASSFTFE TLMQAHDSAV TTMKYSHDSD WMISGDADGM IKIWQPNFSM VKEIDAAHTE SI RDMAFSS NDSKFVTCSD DNILKIWNFS NGKQERVLSG HHWDVKSCDW HPEMGLIASA SKD NLVKLW DPRSGNCISS ILKFKHTVLK TRFQPTKGNL LMAISKDKSC RVFDIRYSMK ELMC VRDET DYMTLEWHPI NESMFTLACY DGSLKHFDLL QNLNEPILTI PYAHDKCITS LSYNP VGHI FATAAKDRTI RFWTRARPID PNAYDDPTYN NKKINGWFFG INNDINAVRE KSEFGA APP PPATLEPHAL PNMNGFINKK PRQEIPGIDS NIKSSTLPGL SI

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Macromolecule #3: Yth1

MacromoleculeName: Yth1 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MSLIHPDTAK YPFKFEPFLR QEYSFSLDPD RPICEFYNSR EGPKSCPRGP LCPKKHVLPI FQNKIVCRH WLRGLCKKND QCEYLHEYNL RKMPECVFFS KNGYCTQSPD CQYLHIDPAS K IPKCENYE MGFCPLGSSC PRRHIKKVFC QRYMTGFCPL GKDECDMEHP ...String:
MSLIHPDTAK YPFKFEPFLR QEYSFSLDPD RPICEFYNSR EGPKSCPRGP LCPKKHVLPI FQNKIVCRH WLRGLCKKND QCEYLHEYNL RKMPECVFFS KNGYCTQSPD CQYLHIDPAS K IPKCENYE MGFCPLGSSC PRRHIKKVFC QRYMTGFCPL GKDECDMEHP QFIIPDEGSK LR IKRDDEI NTRKMDEEKE RRLNAIINGE V

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Macromolecule #4: Fip1

MacromoleculeName: Fip1 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MSSSEDEDDK FLYGSDSELA LPSSKRSRDD EADAGASSNP DIVKRQKFDS PVEETPATAR DDRSDEDIY SDSSDDDSDS DLEVIISLGP DPTRLDAKLL DSYSTAATSS SKDVISVATD V SNTITKTS DERLITEGEA NQGVTATTVK ATESDGNVPK AMTGSIDLDK ...String:
MSSSEDEDDK FLYGSDSELA LPSSKRSRDD EADAGASSNP DIVKRQKFDS PVEETPATAR DDRSDEDIY SDSSDDDSDS DLEVIISLGP DPTRLDAKLL DSYSTAATSS SKDVISVATD V SNTITKTS DERLITEGEA NQGVTATTVK ATESDGNVPK AMTGSIDLDK EGIFDSVGIT TI DPEVLKE KPWRQPGANL SDYFNYGFNE FTWMEYLHRQ EKLQQDYNPR RILMGLLSLQ QQG KLNSAN DTDSNLGNII DNNNNVNNAN MSNLNSNMGN SMSGTPNPPA PPMHPSFPPL PMFG SFPPF PMPGMMPPMN QQPNQNQNQN SK

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Macromolecule #5: Pap1

MacromoleculeName: Pap1 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MSSQKVFGIT GPVSTVGATA AENKLNDSLI QELKKEGSFE TEQETANRVQ VLKILQELAQ RFVYEVSKK KNMSDGMARD AGGKIFTYGS YRLGVHGPGS DIDTLVVVPK HVTREDFFTV F DSLLRERK ELDEIAPVPD AFVPIIKIKF SGISIDLICA RLDQPQVPLS ...String:
MSSQKVFGIT GPVSTVGATA AENKLNDSLI QELKKEGSFE TEQETANRVQ VLKILQELAQ RFVYEVSKK KNMSDGMARD AGGKIFTYGS YRLGVHGPGS DIDTLVVVPK HVTREDFFTV F DSLLRERK ELDEIAPVPD AFVPIIKIKF SGISIDLICA RLDQPQVPLS LTLSDKNLLR NL DEKDLRA LNGTRVTDEI LELVPKPNVF RIALRAIKLW AQRRAVYANI FGFPGGVAWA MLV ARICQL YPNACSAVIL NRFFIILSEW NWPQPVILKP IEDGPLQVRV WNPKIYAQDR SHRM PVITP AYPSMCATHN ITESTKKVIL QEFVRGVQIT NDIFSNKKSW ANLFEKNDFF FRYKF YLEI TAYTRGSDEQ HLKWSGLVES KVRLLVMKLE VLAGIKIAHP FTKPFESSYC CPTEDD YEM IQDKYGSHKT ETALNALKLV TDENKEEESI KDAPKAYLST MYIGLDFNIE NKKEKVD IH IPCTEFVNLC RSFNEDYGDH KVFNLALRFV KGYDLPDEVF DENEKRPSKK SKRKNLDA R HETVKRSKSD AASGDNINGT TAAVDVN

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Macromolecule #6: Cft2

MacromoleculeName: Cft2 / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MTYKYNCCDD GSGTTVGSVV RFDNVTLLID PGWNPSKVSY EQCIKYWEKV IPEIDVIILS QPTIECLGA HSLLYYNFTS HFISRIQVYA TLPVINLGRV STIDSYASAG VIGPYDTNKL D LEDIEISF DHIVPLKYSQ LVDLRSRYDG LTLLAYNAGV CPGGSIWCIS ...String:
MTYKYNCCDD GSGTTVGSVV RFDNVTLLID PGWNPSKVSY EQCIKYWEKV IPEIDVIILS QPTIECLGA HSLLYYNFTS HFISRIQVYA TLPVINLGRV STIDSYASAG VIGPYDTNKL D LEDIEISF DHIVPLKYSQ LVDLRSRYDG LTLLAYNAGV CPGGSIWCIS TYSEKLVYAK RW NHTRDNI LNAASILDAT GKPLSTLMRP SAIITTLDRF GSSQPFKKRS KIFKDTLKKG LSS DGSVII PVDMSGKFLD LFTQVHELLF ESTKINAHTQ VPVLILSYAR GRTLTYAKSM LEWL SPSLL KTWENRNNTS PFEIGSRIKI IAPNELSKYP GSKICFVSEV GALINEVIIK VGNSE KTTL ILTKPSFECA SSLDKILEIV EQDERNWKTF PEDGKSFLCD NYISIDTIKE EPLSKE ETE AFKVQLKEKK RDRNKKILLV KRESKKLANG NAIIDDTNGE RAMRNQDILV ENVNGVP PI DHIMGGDEDD DEEEENDNLL NLLKDNSEKS AAKKNTEVPV DIIIQPSAAS KHKMFPFN P AKIKKDDYGT VVDFTMFLPD DSDNVNQNSR KRPLKDGAKT TSPVNEEDNK NEEEDGYNM SDPISKRSKH RASRYSGFSG TGEAENFDNL DYLKIDKTLS KRTISTVNVQ LKCSVVILNL QSLVDQRSA SIIWPSLKSR KIVLSAPKQI QNEEITAKLI KKNIEVVNMP LNKIVEFSTT I KTLDISID SNLDNLLKWQ RISDSYTVAT VVGRLVKESL PQVNNHQKTA SRSKLVLKPL HG SSRSHKT GALSIGDVRL AQLKKLLTEK NYIAEFKGEG TLVINEKVAV RKINDAETII DGT PSELFD TVKKLVTDML AKI

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Macromolecule #7: Ysh1

MacromoleculeName: Ysh1 / type: protein_or_peptide / ID: 7 / 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 #8: Mpe1

MacromoleculeName: Mpe1 / type: protein_or_peptide / ID: 8 / 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

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.9
Component:
ConcentrationName
10.0 mMHEPES
150.0 mMsodium chloride
0.5 mMmagnesium acetate
1.0 mMTCEP
StainingType: NEGATIVE / Material: Uranyl Acetate
Details: Three 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 ...Details: Three 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.
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 4.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Details: Grids were CF400-CU-UL from Electron Microscopy Sciences. Thin layer continuous carbon over 400-mesh copper. Manually inspected before use (with a light microscope)
Details250 nM complex was used

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -0.6 µm / Nominal defocus min: -0.6 µm / Nominal magnification: 26000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Image recordingFilm or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Average exposure time: 2.0 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 274806
Details: Several rounds of reference-free 2D classification were used clean bad particles from the dataset, and to discard particles classified on the basis of stain thickness, leaving a final subset ...Details: Several rounds of reference-free 2D classification were used clean bad particles from the dataset, and to discard particles classified on the basis of stain thickness, leaving a final subset of 107,817 particles in equivalently thick stain.
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 2.0)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 2.0)
Details: One round of 3D classification (4 classes) was used to separate conformational heterogeneity, prior to a final 3D refinement
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 2.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 23969
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

6eoj

,

3c66

,

2i7x

,

6i1d

)
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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