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- EMDB-10795: Negative stain map of S. cerevisiae Brf1 and TBP bound to the TFI... -

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Basic information

Entry
Database: EMDB / ID: EMD-10795
TitleNegative stain map of S. cerevisiae Brf1 and TBP bound to the TFIIIC-subcomplex tauA
Map dataRefined map
Sample
  • Complex: Complex between Brf1, TBP, and the tauA subcomplex of TFIIIC
    • Complex: Transcription factor tau 131 kDa subunit, Transcription factor tau 95 kDa subunit and Transcription factor tau 55 kDa subunit
      • Protein or peptide: Transcription factor tau 131 kDa subunit
      • Protein or peptide: Transcription factor tau 95 kDa subunit
      • Protein or peptide: Transcription factor tau 55 kDa subunit
    • Complex: Transcription factor IIIB 70 kDa subunit and TATA-box-binding protein
      • Protein or peptide: Transcription factor IIIB 70 kDa subunit
      • Protein or peptide: TATA-box-binding protein
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 32.36 Å
AuthorsVorlaender MK / Muller CW
CitationJournal: Nat Commun / Year: 2020
Title: Structure of the TFIIIC subcomplex τA provides insights into RNA polymerase III pre-initiation complex formation.
Authors: Matthias K Vorländer / Anna Jungblut / Kai Karius / Florence Baudin / Helga Grötsch / Jan Kosinski / Christoph W Müller /
Abstract: Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it ...Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex τA, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that τA binding to its promoter is auto-inhibited by a disordered acidic tail of subunit τ95. We further provide a negative-stain reconstruction of τA bound to the TFIIIB subunits Brf1 and TBP. This shows that a ruler element in τA achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.
History
DepositionMar 25, 2020-
Header (metadata) releaseApr 1, 2020-
Map releaseSep 23, 2020-
UpdateOct 14, 2020-
Current statusOct 14, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10795.png

Downloads & links

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Map

FileDownload / File: emd_10795.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefined map
Voxel sizeX=Y=Z: 2.292 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.06
Minimum - Maximum-0.063415006 - 0.20393701
Average (Standard dev.)-0.00061397516 (±0.009914577)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 458.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.2922.2922.292
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z458.400458.400458.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0630.204-0.001

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Supplemental data

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Half map: Half map 2

Fileemd_10795_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_10795_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex between Brf1, TBP, and the tauA subcomplex of TFIIIC

EntireName: Complex between Brf1, TBP, and the tauA subcomplex of TFIIIC
Components
  • Complex: Complex between Brf1, TBP, and the tauA subcomplex of TFIIIC
    • Complex: Transcription factor tau 131 kDa subunit, Transcription factor tau 95 kDa subunit and Transcription factor tau 55 kDa subunit
      • Protein or peptide: Transcription factor tau 131 kDa subunit
      • Protein or peptide: Transcription factor tau 95 kDa subunit
      • Protein or peptide: Transcription factor tau 55 kDa subunit
    • Complex: Transcription factor IIIB 70 kDa subunit and TATA-box-binding protein
      • Protein or peptide: Transcription factor IIIB 70 kDa subunit
      • Protein or peptide: TATA-box-binding protein

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Supramolecule #1: Complex between Brf1, TBP, and the tauA subcomplex of TFIIIC

SupramoleculeName: Complex between Brf1, TBP, and the tauA subcomplex of TFIIIC
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Crosslinked with Glutaraldehyde in absence of DNA
Molecular weightTheoretical: 300 KDa

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Supramolecule #2: Transcription factor tau 131 kDa subunit, Transcription factor ta...

SupramoleculeName: Transcription factor tau 131 kDa subunit, Transcription factor tau 95 kDa subunit and Transcription factor tau 55 kDa subunit
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Supramolecule #3: Transcription factor IIIB 70 kDa subunit and TATA-box-binding protein

SupramoleculeName: Transcription factor IIIB 70 kDa subunit and TATA-box-binding protein
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #4-#5
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Transcription factor tau 131 kDa subunit

MacromoleculeName: Transcription factor tau 131 kDa subunit / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MAAGKLKKEQ QNQSAERESA DTGKVNDEDE EHLYGNIDDY KHLIQDEEYD DEDVPHDLQL SEDEYNSER DSSLLAEFSD YGEISEDDEE DFMNAIREAS NFKVKKKKKN DKGKSYGRQR K ERVLDPEV AQLLSQANEA FVRNDLQVAE RLFNEVIKKD ARNFAAYETL ...String:
MAAGKLKKEQ QNQSAERESA DTGKVNDEDE EHLYGNIDDY KHLIQDEEYD DEDVPHDLQL SEDEYNSER DSSLLAEFSD YGEISEDDEE DFMNAIREAS NFKVKKKKKN DKGKSYGRQR K ERVLDPEV AQLLSQANEA FVRNDLQVAE RLFNEVIKKD ARNFAAYETL GDIYQLQGRL ND CCNSWFL AAHLNASDWE FWKIVAILSA DLDHVRQAIY CFSRVISLNP MEWESIYRRS MLY KKTGQL ARALDGFQRL YMYNPYDANI LRELAILYVD YDRIEDSIEL YMKVFNANVE RREA ILAAL ENALDSSDEE SAAEGEDADE KEPLEQDEDR QMFPDINWKK IDAKYKCIPF DWSSL NILA ELFLKLAVSE VDGIKTIKKC ARWIQRRESQ TFWDHVPDDS EFDNRRFKNS TFDSLL AAE KEKSYNIPID IRVRLGLLRL NTDNLVEALN HFQCLYDETF SDVADLYFEA ATALTRA EK YKEAIDFFTP LLSLEEWRTT DVFKPLARCY KEIESYETAK EFYELAIKSE PDDLDIRV S LAEVYYRLND PETFKHMLVD VVEMRKHQVD ETLHRISNEK SSNDTSDISS KPLLEDSKF RTFRKKKRTP YDAERERIER ERRITAKVVD KYEKMKKFEL NSGLNEAKQA SIWINTVSEL VDIFSSVKN FFMKSRSRKF VGILRRTKKF NTELDFQIER LSKLAEGDSV FEGPLMEERV T LTSATELR GLSYEQWFEL FMELSLVIAK YQSVEDGLSV VETAQEVNVF FQDPERVKMM KF VKLAIVL QMDDEEELAE NLRGLLNQFQ FNRKVLQVFM YSLCRGPSSL NILSSTIQQK FFL RQLKAF DSCRYNTEVN GQASITNKEV YNPNKKSSPY LYYIYAVLLY SSRGFLSALQ YLTR LEEDI PDDPMVNLLM GLSHIHRAMQ RLTAQRHFQI FHGLRYLYRY HKIRKSLYTD LEKQE ADYN LGRAFHLIGL VSIAIEYYNR VLENYDDGKL KKHAAYNSII IYQQSGNVEL ADHLME KYL SIRSGG

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Macromolecule #2: Transcription factor tau 95 kDa subunit

MacromoleculeName: Transcription factor tau 95 kDa subunit / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MHHHHHHENL YFQSMPVEEP LATLSSIPDS SADQAPPLIA DEFTLDLPRI PSLELPLNVS TKHSSIQKA IKMCGGIEKV KEAFKEHGPI ESQHGLQLYL NDDTDSDGSK SYFNEHPVIG K RVPFRDES VILKVTMPKG TLSKNNNSVK DSIKSLKDSN KLRVTPVSIV ...String:
MHHHHHHENL YFQSMPVEEP LATLSSIPDS SADQAPPLIA DEFTLDLPRI PSLELPLNVS TKHSSIQKA IKMCGGIEKV KEAFKEHGPI ESQHGLQLYL NDDTDSDGSK SYFNEHPVIG K RVPFRDES VILKVTMPKG TLSKNNNSVK DSIKSLKDSN KLRVTPVSIV DNTIKFREMS DF QIKLDNV PSAREFKSSF GSLEWNNFKS FVNSVPDNDS QPQENIGNLI LDRSVKIPST DFQ LPPPPK LSMVGFPLLY KYKANPFAKK KKNGVTEVKG TYIKNYQLFV HDLSDKTVIP SQAH EQVLY DFEVAKKTKV YPGTKSDSKF YESLEECLKI LRELFARRPI WVKRHLDGIV PKKIH HTMK IALALISYRF TMGPWRNTYI KFGIDPRSSV EYAQYQTEYF KIERKLLSSP IVKKNV PKP PPLVFESDTP GGIDSRFKFD GKRIPWYLML QIDLLIGEPN IAEVFHNVEY LDKANEL TG WFKELDLVKI RRIVKYELGC MVQGNYEYNK YKLKYFKTML FVKESMVPEN KNSEEGMG V NTNKDADGDI NMDAGSQMSS NAIEEDKGIA AGDDFDDNGA ITEEPDDAAL ENEEMDTDQ NLKVPASIDD DVDDVDADEE EQESFDVKTA SFQDIINKIA KLDPKTAETM KSELKGFVDE VDL

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Macromolecule #3: Transcription factor tau 55 kDa subunit

MacromoleculeName: Transcription factor tau 55 kDa subunit / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MVVNTIYIAR HGYRSNWLPE GPYPDPLTGI DSDVPLAEHG VQQAKELAHY LLSLDNQPEA AFASPFYRC LETVQPIAKL LEIPVYLERG IGEWYRPDRK PVIPVPAGYE ILSKFFPGVI S QEWDSTLT PNEKGETEQE MYMRFKKFWP LFIERVEKEY PNVECILLVT ...String:
MVVNTIYIAR HGYRSNWLPE GPYPDPLTGI DSDVPLAEHG VQQAKELAHY LLSLDNQPEA AFASPFYRC LETVQPIAKL LEIPVYLERG IGEWYRPDRK PVIPVPAGYE ILSKFFPGVI S QEWDSTLT PNEKGETEQE MYMRFKKFWP LFIERVEKEY PNVECILLVT HAASKIALGM SL LGYDNPR MSLNENGDKI RSGSCSLDKY EILKKSYDTI DETDDQTSFT YIPFSDRKWV LTM NGNTEF LSSGEEMNWN FDCVAEAGSD ADIKKRQMTK KTSSPIPEAD DQTEVETVYI SVDI PSGNY KERTEIAKSA ILQYSGLETD APLFRIGNRL YEGSWERLVG TELAFPNAAH VHKKT AGLL SPTEENETTN AGQSKGSSTA NDPNIQIQEE DVGLPDSTNT SRDHTGDKEE VQSEKI YRI KERIVLSNVR PM

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Macromolecule #4: Transcription factor IIIB 70 kDa subunit

MacromoleculeName: Transcription factor IIIB 70 kDa subunit / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSALESREAT LNNARRKLRA VSYALHIPEY ITDAAFQWYK LALANNFVQG R RSQNVIAS CLYVACRKEK THHMLIDFSS RLQVSVYSIG ATFLKMVKKL HITELPLADP SL FIQHFAE KLDLADKKIK VVKDAVKLAQ RMSKDWMFEG RRPAGIAGAC ILLACRMNNL RRT ...String:
MSALESREAT LNNARRKLRA VSYALHIPEY ITDAAFQWYK LALANNFVQG R RSQNVIAS CLYVACRKEK THHMLIDFSS RLQVSVYSIG ATFLKMVKKL HITELPLADP SL FIQHFAE KLDLADKKIK VVKDAVKLAQ RMSKDWMFEG RRPAGIAGAC ILLACRMNNL RRT HTEIVA VSHVAEETLQ QRLNEFKNTK AAKLSVQKFR ENDVEDGEAR PPSFVKNRKK ERKI KDSLD KEEMFQTSEE ALNKNPILTQ VLGEQELSSK EVLFYLKQFS ERRARVVERI KATNG IDGE NIYHEGSENE TRKRKLSEVS IQNEHVEGED KETEGTEEKV KKVKTKTSEE KKENES GHF QDAIDGYSLE TDPYCPRNLH LLPTTDTYLS KVSDDPDNLE DVDDEELNAH LLNEEAS KL KERIWIGLNA DFLLEQESKR LKQEADIATG NTSVKKKRTR RRNNTRSDEP TKTVDAAA A IGLMSDLQDK SGLHAALKAA EESGDFTTAD SVKNMLQKAS FSKKINYDAI DGLFRLEHHH HHH

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Macromolecule #5: TATA-box-binding protein

MacromoleculeName: TATA-box-binding protein / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SGIVPTLQNI VATVTLGCRL DLKTVALHAR NAEYNPKRFA AVIMRIREPK TTALIFASGK MVVTGAKSE DDSKLASRKY ARIIQKIGFA AKFTDFKIQN IVGSCDVKFP IRLEGLAFSH G TFSSYEPE LFPGLIYRMV KPKIVLLIFV SGKIVLTGAK QREEIYQAFE AIYPVLSEFR KM

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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: 7.5
StainingType: NEGATIVE / Material: Uranyl Acetate
DetailsCrosslinked sample

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 20.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model generated in CryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 32.36 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 10731
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT / Target criteria: Envelope score

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