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- EMDB-20090: CryoEM structure of SigmaS-transcription initiation complex with ... -

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Basic information

Entry
Database: EMDB / ID: EMD-20090
TitleCryoEM structure of SigmaS-transcription initiation complex with activator Crl
Map dataE.coli RNA polymerase transcription initiation complex
Sample
  • Complex: CryoEM structure of SigmaS-transcription initiation complex with activator Crl
    • Protein or peptide: Crl
    • Protein or peptide: DNA-directed RNA polymerase subunit sigma-SPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit alphaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit betaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit beta'Polymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit omegaPolymerase
    • DNA: Template DNA strand
    • DNA: Non-template DNA strand
Function / homology
Function and homology information


positive regulation of cellulose biosynthetic process / DNA-templated transcription initiation => GO:0006352 / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / sigma factor activity / bacterial-type flagellum-dependent cell motility ...positive regulation of cellulose biosynthetic process / DNA-templated transcription initiation => GO:0006352 / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / sigma factor activity / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / DNA-directed RNA polymerase complex / nucleotidyltransferase activity / regulation of DNA-templated transcription elongation / transcription antitermination / cell motility / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / protein-containing complex assembly / intracellular iron ion homeostasis / protein dimerization activity / response to antibiotic / DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Sigma factor-binding transcriptional regulator Crl / Sigma factor-binding protein Crl superfamily / Sigma factor-binding transcriptional regulator Crl / RNA polymerase sigma factor RpoS / Sigma-70 factors family signature 1. / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. ...Sigma factor-binding transcriptional regulator Crl / Sigma factor-binding protein Crl superfamily / Sigma factor-binding transcriptional regulator Crl / RNA polymerase sigma factor RpoS / Sigma-70 factors family signature 1. / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6 / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
Sigma factor-binding protein Crl / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit beta / Sigma factor-binding protein Crl / RNA polymerase sigma factor RpoS
Similarity search - Component
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.26 Å
AuthorsJaramillo Cartagena A / Darst SA / Campbell EA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR35 GM118130 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Structural basis for transcription activation by Crl through tethering of σ and RNA polymerase.
Authors: Alexis Jaramillo Cartagena / Amy B Banta / Nikhil Sathyan / Wilma Ross / Richard L Gourse / Elizabeth A Campbell / Seth A Darst /
Abstract: In bacteria, a primary σ-factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ-factors are used to coordinate expression of additional ...In bacteria, a primary σ-factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ-factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative σ-factors are negatively regulated by anti-σ-factors. In , , and many other γ-proteobacteria, the transcription factor Crl positively regulates the alternative σ-regulon by promoting the association of σ with RNAP without interacting with promoter DNA. The molecular mechanism for Crl activity is unknown. Here, we determined a single-particle cryo-electron microscopy structure of Crl-σ-RNAP in an open promoter complex with a σ-regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of σ (σ), the structure, along with -benzoylphenylalanine cross-linking, reveals that Crl interacts with a structural element of the RNAP β'-subunit that we call the β'-clamp-toe (β'CT). Deletion of the β'CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-β'CT interaction. We conclude that Crl activates σ-dependent transcription in part through stabilizing σ-RNAP by tethering σ and the β'CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators.
History
DepositionApr 10, 2019-
Header (metadata) releaseAug 28, 2019-
Map releaseAug 28, 2019-
UpdateOct 2, 2019-
Current statusOct 2, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6omf
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20090.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationE.coli RNA polymerase transcription initiation complex
Voxel sizeX=Y=Z: 1.3 Å
Density
Contour LevelBy AUTHOR: 0.45 / Movie #1: 0.45
Minimum - Maximum-2.8030248 - 3.9349961
Average (Standard dev.)0.0013217092 (±0.09672034)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 332.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.31.31.3
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z332.800332.800332.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-31-35-52
NX/NY/NZ11798209
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-2.8033.9350.001

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Supplemental data

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Sample components

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Entire : CryoEM structure of SigmaS-transcription initiation complex with ...

EntireName: CryoEM structure of SigmaS-transcription initiation complex with activator Crl
Components
  • Complex: CryoEM structure of SigmaS-transcription initiation complex with activator Crl
    • Protein or peptide: Crl
    • Protein or peptide: DNA-directed RNA polymerase subunit sigma-SPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit alphaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit betaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit beta'Polymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit omegaPolymerase
    • DNA: Template DNA strand
    • DNA: Non-template DNA strand

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Supramolecule #1: CryoEM structure of SigmaS-transcription initiation complex with ...

SupramoleculeName: CryoEM structure of SigmaS-transcription initiation complex with activator Crl
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 523.783 KDa

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Macromolecule #1: Crl

MacromoleculeName: Crl / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GPHMTLPSGH PKSRLIKKFT ALGPYIREGQ CEDNRFFFDC LAVCVNVKPA PEKREFWGWW MELEAQEKRF TYRYQFGLFD KEGNWTVVPI NETEVVERLE YTLREFHEKL RDLLISMELA LEPSDDFNDE PVKLSA

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Macromolecule #2: DNA-directed RNA polymerase subunit sigma-S

MacromoleculeName: DNA-directed RNA polymerase subunit sigma-S / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SMSQNTLKVH DLNEDAEFDE NGVEAFDEKA LSEEEPSDND LAEEELLSQG ATQRVLDATQ LYLGEIGYSP LLTAEEEVYF ARRALRGDVA SRRRMIESNL RLVVKIARRY GNRGLALLDL IEEGNLGLIR AVEKFDPERG FRFSTYATWW IRQTIERAIM NQTRTIRLPI ...String:
SMSQNTLKVH DLNEDAEFDE NGVEAFDEKA LSEEEPSDND LAEEELLSQG ATQRVLDATQ LYLGEIGYSP LLTAEEEVYF ARRALRGDVA SRRRMIESNL RLVVKIARRY GNRGLALLDL IEEGNLGLIR AVEKFDPERG FRFSTYATWW IRQTIERAIM NQTRTIRLPI HIVKELNVYL RTARELSHKL DHEPSAEEIA EQLDKPVDDV SRMLRLNERI TSVDTPLGGD SEKALLDILA DEKENGPEDT TQDDDMKQSI VKWLFELNAK QREVLARRFG LLGYEAATLE DVGREIGLTR ERVRQIQVEG LRRLREILQT QGLNIEALFR E

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Macromolecule #3: DNA-directed RNA polymerase subunit alpha

MacromoleculeName: DNA-directed RNA polymerase subunit alpha / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQGSVTEFLK PRLVDIEQVS STHAKVTLEP LERGFGHTLG NALRRILLSS MPGCAVTEVE IDGVLHEYST KEGVQEDILE ILLNLKGLAV RVQGKDEVIL TLNKSGIGPV TAADITHDGD VEIVKPQHVI CHLTDENASI SMRIKVQRGR GYVPASTRIH SEEDERPIGR ...String:
MQGSVTEFLK PRLVDIEQVS STHAKVTLEP LERGFGHTLG NALRRILLSS MPGCAVTEVE IDGVLHEYST KEGVQEDILE ILLNLKGLAV RVQGKDEVIL TLNKSGIGPV TAADITHDGD VEIVKPQHVI CHLTDENASI SMRIKVQRGR GYVPASTRIH SEEDERPIGR LLVDACYSPV ERIAYNVEAA RVEQRTDLDK LVIEMETNGT IDPEEAIRRA ATILAEQLEA FVDLEVLFQ

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Macromolecule #4: DNA-directed RNA polymerase subunit beta

MacromoleculeName: DNA-directed RNA polymerase subunit beta / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVYSYTEKKR IRKDFGKRPQ VLDVPYLLSI QLDSFQKFIE QDPEGQYGLE AAFRSVFPIQ SYSGNSELQY VSYRLGEPVF DVQECQIRG VTYSAPLRVK LRLVIYEREA PEGTVKDIKE QEVYMGEIPL MTDNGTFVIN GTERVIVSQL HRSPGVFFDS D KGKTHSSG ...String:
MVYSYTEKKR IRKDFGKRPQ VLDVPYLLSI QLDSFQKFIE QDPEGQYGLE AAFRSVFPIQ SYSGNSELQY VSYRLGEPVF DVQECQIRG VTYSAPLRVK LRLVIYEREA PEGTVKDIKE QEVYMGEIPL MTDNGTFVIN GTERVIVSQL HRSPGVFFDS D KGKTHSSG KVLYNARIIP YRGSWLDFEF DPKDNLFVRI DRRRKLPATI ILRALNYTTE QILDLFFEKV IFEIRDNKLQ ME LVPERLR GETASFDIEA NGKVYVEKGR RITARHIRQL EKDDVKLIEV PVEYIAGKVV AKDYIDESTG ELICAANMEL SLD LLAKLS QSGHKRIETL FTNDLDHGPY ISETLRVDPT NDRLSALVEI YRMMRPGEPP TREAAESLFE NLFFSEDRYD LSAV GRMKF NRSLLREEIE GSGILSKDDI IDVMKKLIDI RNGKGEVDDI DHLGNRRIRS VGEMAENQFR VGLVRVERAV KERLS LGDL DTLMPQDMIN AKPISAAVKE FFGSSQLSQF MDQNNPLSEI THKRRISALG PGGLTRERAG FEVRDVHPTH YGRVCP IET PEGPNIGLIN SLSVYAQTNE YGFLETPYRK VTDGVVTDEI HYLSAIEEGN YVIAQANSNL DEEGHFVEDL VTCRSKG ES SLFSRDQVDY MDVSTQQVVS VGASLIPFLE HDDANRALMG ANMQRQAVPT LRADKPLVGT GMERAVAVDS GVTAVAKR G GVVQYVDASR IVIKVNEDEM YPGEAGIDIY NLTKYTRSNQ NTCINQMPCV SLGEPVERGD VLADGPSTDL GELALGQNM RVAFMPWNGY NFEDSILVSE RVVQEDRFTT IHIQELACVS RDTKLGPEEI TADIPNVGEA ALSKLDESGI VYIGAEVTGG DILVGKVTP KGETQLTPEE KLLRAIFGEK ASDVKDSSLR VPNGVSGTVI DVQVFTRDGV EKDKRALEIE EMQLKQAKKD L SEELQILE AGLFSRIRAV LVAGGVEAEK LDKLPRDRWL ELGLTDEEKQ NQLEQLAEQY DELKHEFEKK LEAKRRKITQ GD DLAPGVL KIVKVYLAVK RRIQPGDKMA GRHGNKGVIS KINPIEDMPY DENGTPVDIV LNPLGVPSRM NIGQILETHL GMA AKGIGD KINAMLKQQQ EVAKLREFIQ RAYDLGADVR QKVDLSTFSD EEVMRLAENL RKGMPIATPV FDGAKEAEIK ELLK LGDLP TSGQIRLYDG RTGEQFERPV TVGYMYMLKL NHLVDDKMHA RSTGSYSLVT QQPLGGKAQF GGQRFGEMEV WALEA YGAA YTLQEMLTVK SDDVNGRTKM YKNIVDGNHQ MEPGMPESFN VLLKEIRSLG INIELEDE

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Macromolecule #5: DNA-directed RNA polymerase subunit beta'

MacromoleculeName: DNA-directed RNA polymerase subunit beta' / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: VKDLLKFLKA QTKTEEFDAI KIALASPDMI RSWSFGEVKK PETINYRTFK PERDGLFCAR IFGPVKDYEC LCGKYKRLKH RGVICEKCGV EVTQTKVRRE RMGHIELASP TAHIWFLKSL PSRIGLLLDM PLRDIERVLY FESYVVIEGG MTNLERQQIL TEEQYLDALE ...String:
VKDLLKFLKA QTKTEEFDAI KIALASPDMI RSWSFGEVKK PETINYRTFK PERDGLFCAR IFGPVKDYEC LCGKYKRLKH RGVICEKCGV EVTQTKVRRE RMGHIELASP TAHIWFLKSL PSRIGLLLDM PLRDIERVLY FESYVVIEGG MTNLERQQIL TEEQYLDALE EFGDEFDAKM GAEAIQALLK SMDLEQECEQ LREELNETNS ETKRKKLTKR IKLLEAFVQS GNKPEWMILT VLPVLPPDLR PLVPLDGGRF ATSDLNDLYR RVINRNNRLK RLLDLAAPDI IVRNEKRMLQ EAVDALLDNG RRGRAITGSN KRPLKSLADM IKGKQGRFRQ NLLGKRVDYS GRSVITVGPY LRLHQCGLPK KMALELFKPF IYGKLELRGL ATTIKAAKKM VEREEAVVWD ILDEVIREHP VLLNRAPTLH RLGIQAFEPV LIEGKAIQLH PLVCAAYNAD FDGDQMAVHV PLTLEAQLEA RALMMSTNNI LSPANGEPII VPSQDVVLGL YYMTRDCVNA KGEGMVLTGP KEAERLYRSG LASLHARVKV RITEYEKDAN GELVAKTSLK DTTVGRAILW MIVPKGLPYS IVNQALGKKA ISKMLNTCYR ILGLKPTVIF ADQIMYTGFA YAARSGASVG IDDMVIPEKK HEIISEAEAE VAEIQEQFQS GLVTAGERYN KVIDIWAAAN DRVSKAMMDN LQTETVINRD GQEEKQVSFN SIYMMADSGA RGSAAQIRQL AGMRGLMAKP DGSIIETPIT ANFREGLNVL QYFISTHGAR KGLADTALKT ANSGYLTRRL VDVAQDLVVT EDDCGTHEGI MMTPVIEGGD VKEPLRDRVL GRVTAEDVLK PGTADILVPR NTLLHEQWCD LLEENSVDAV KVRSVVSCDT DFGVCAHCYG RDLARGHIIN KGEAIGVIAA QSIGEPGTQL TMRTFHIGGA ASRAAAESSI QVKNKGSIKL SNVKSVVNSS GKLVITSRNT ELKLIDEFGR TKESYKVPYG AVLAKGDGEQ VAGGETVANW DPHTMPVITE VSGFVRFTDM IDGQTITRQT DELTGLSSLV VLDSAERTAG GKDLRPALKI VDAQGNDVLI PGTDMPAQYF LPGKAIVQLE DGVQISSGDT LARIPQESGG TKDITGGLPR VADLFEARRP KEPAILAEIS GIVSFGKETK GKRRLVITPV DGSDPYEEMI PKWRQLNVFE GERVERGDVI SDGPEAPHDI LRLRGVHAVT RYIVNEVQDV YRLQGVKIND KHIEVIVRQM LRKATIVNAG SSDFLEGEQV EYSRVKIANR ELEANGKVGA TYSRDLLGIT KASLATESFI SAASFQETTR VLTEAAVAGK RDELRGLKEN VIVGRLIPAG TGYAYHQDRM RRRAAGEAPA APQVTAEDAS ASLAELLNAG LGGSDNELE

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Macromolecule #6: DNA-directed RNA polymerase subunit omega

MacromoleculeName: DNA-directed RNA polymerase subunit omega / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MARVTVQDAV EKIGNRFDLV LVAARRARQM QVGGKDPLVP EENDKTTVIA LREIEEGLIN NQILDVRERQ EQQEQEAAEL QAVTAIAEG RR

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Macromolecule #7: Template DNA strand

MacromoleculeName: Template DNA strand / type: dna / ID: 7 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString:
GCTATGTCCC AGTAATTAAC GAGTAATAGT ATAGCACCGG CTATGTGTTC CGCTATTCTG AATTCG

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Macromolecule #8: Non-template DNA strand

MacromoleculeName: Non-template DNA strand / type: dna / ID: 8 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString:
CGAATTCAGA ATAGCGGAAC ACATAGCCGG TGCTATACTT AATCTCGTTA ATTACTGGGA CATAGC

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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

Concentration5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMK-GlutamatePotassium glutamate
50.0 mMTris-HClTristris(hydroxymethyl)aminomethane
10.0 mMMgCl2Magnesium chloride
10.0 mMDTTDescriptionDithiothreitol
GridModel: C-flat-1.2/1.3 4C / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298.15 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 opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.42 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 658000
CTF correctionSoftware - Name: RELION (ver. 3)
Startup modelType of model: INSILICO MODEL
In silico model: Alpha, beta, beta', and omega subunits from PDB: 5VT0. SigmaS subunit from 5IPL. Crl from 3RPJ. Mostly de novo built DNA.
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 292000

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-6omf:
CryoEM structure of SigmaS-transcription initiation complex with activator Crl

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