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- EMDB-11836: Cryo-EM density map corresponding to BcsRQAB subcomplex -

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Basic information

Entry
Database: EMDB / ID: EMD-11836
TitleCryo-EM density map corresponding to BcsRQAB subcomplex
Map data
Sample
  • Complex: Cryo-EM density map corresponding to BcsR2Q2AB subcomplex obtained after local refinement within the assembled Bcs macrocomplex(BcsRQABEF-BcsMacrocomplex.mrc).
    • Protein or peptide: Bacterial cellulose synthase regulator protein BcsB
    • Protein or peptide: Bacterial cellulose synthase protein BcsA
    • Protein or peptide: Bacterial cellulose synthase regulator protein BcsR
    • Protein or peptide: Bacterial cellulose synthase regulator protein BcsQ
Function / homology
Function and homology information


bacterial cellulose biosynthetic process / cell division / ATP binding / cytoplasm
Similarity search - Function
Cellulose synthase operon protein BcsQ / Protein YhjR / Cellulose biosynthesis protein BcsQ / Cellulose biosynthesis protein BcsR-like / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Cell division protein / Cellulose biosynthesis protein BcsR / Protein YhjR / Cellulose biosynthesis protein BcsQ
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsZouhir S
Funding support France, 4 items
OrganizationGrant numberCountry
ATIP-Avenir2016 France
European Research Council (ERC)Biomatrix 757507 France
Centre National de la Recherche Scientifique (CNRS) France
European Institute of Chemistry and Biology (IECB) France
CitationJournal: Sci Adv / Year: 2021
Title: Architecture and regulation of an enterobacterial cellulose secretion system.
Authors: Wiem Abidi / Samira Zouhir / Meryem Caleechurn / Stéphane Roche / Petya Violinova Krasteva /
Abstract: Many free-living and pathogenic enterobacteria secrete biofilm-promoting cellulose using a multicomponent, envelope-embedded Bcs secretion system under the control of intracellular second messenger c- ...Many free-living and pathogenic enterobacteria secrete biofilm-promoting cellulose using a multicomponent, envelope-embedded Bcs secretion system under the control of intracellular second messenger c-di-GMP. The molecular understanding of system assembly and cellulose secretion has been largely limited to the crystallographic studies of a distantly homologous BcsAB synthase tandem and a low-resolution reconstruction of an assembled macrocomplex that encompasses most of the inner membrane and cytosolic subunits and features an atypical layered architecture. Here, we present cryo-EM structures of the assembled Bcs macrocomplex, as well as multiple crystallographic snapshots of regulatory Bcs subcomplexes. The structural and functional data uncover the mechanism of asymmetric secretion system assembly and periplasmic crown polymerization and reveal unexpected subunit stoichiometry, multisite c-di-GMP recognition, and ATP-dependent regulation.
History
DepositionOct 13, 2020-
Header (metadata) releaseFeb 24, 2021-
Map releaseFeb 24, 2021-
UpdateFeb 24, 2021-
Current statusFeb 24, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 7
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11836.png

Downloads & links

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Map

FileDownload / File: emd_11836.map.gz / Format: CCP4 / Size: 262.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.05248 Å
Density
Contour LevelBy AUTHOR: 7.0 / Movie #1: 7
Minimum - Maximum-43.477592 - 123.02881
Average (Standard dev.)-1.1382667e-12 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions410410410
Spacing410410410
CellA=B=C: 431.51678 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.05248048780491.05248048780491.0524804878049
M x/y/z410410410
origin x/y/z0.0000.0000.000
length x/y/z431.517431.517431.517
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ410410410
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS410410410
D min/max/mean-43.478123.029-0.000

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Supplemental data

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Additional map: #1

Fileemd_11836_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #3

Fileemd_11836_additional_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #4

Fileemd_11836_additional_3.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #2

Fileemd_11836_additional_4.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM density map corresponding to BcsR2Q2AB subcomplex obtaine...

EntireName: Cryo-EM density map corresponding to BcsR2Q2AB subcomplex obtained after local refinement within the assembled Bcs macrocomplex(BcsRQABEF-BcsMacrocomplex.mrc).
Components
  • Complex: Cryo-EM density map corresponding to BcsR2Q2AB subcomplex obtained after local refinement within the assembled Bcs macrocomplex(BcsRQABEF-BcsMacrocomplex.mrc).
    • Protein or peptide: Bacterial cellulose synthase regulator protein BcsB
    • Protein or peptide: Bacterial cellulose synthase protein BcsA
    • Protein or peptide: Bacterial cellulose synthase regulator protein BcsR
    • Protein or peptide: Bacterial cellulose synthase regulator protein BcsQ

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Supramolecule #1: Cryo-EM density map corresponding to BcsR2Q2AB subcomplex obtaine...

SupramoleculeName: Cryo-EM density map corresponding to BcsR2Q2AB subcomplex obtained after local refinement within the assembled Bcs macrocomplex(BcsRQABEF-BcsMacrocomplex.mrc).
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Bcs macrocomplex was purified using recombinant co-expression of constructs pRSFDuet1-Bcs(Strep)E-F-G and pCDFDuet1-Bcs(His)R-Q-A(HA-FLAG)-B and affinity pull-down using an anti-FLAG M2 ...Details: Bcs macrocomplex was purified using recombinant co-expression of constructs pRSFDuet1-Bcs(Strep)E-F-G and pCDFDuet1-Bcs(His)R-Q-A(HA-FLAG)-B and affinity pull-down using an anti-FLAG M2 resin (Sigma). The proposed stoichiometry for the resultant assembly is BcsR2-Q2-E2-F2-A-B(5-6). Densities corresponding to a BcsR2Q2AB subcomplex were improved after particle subtraction and local refinement in cryoSPARC V2. BcsE and BcsF partake in the assembled Bcs macrocomplex, BcsG does not co-purify stably with the macrocomplex.
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094 / Location in cell: Inner membrane
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Recombinant plasmid: pRSFDuet1-Bcs(Strep)EFG and pCDFDuet1-Bcs(His)RQA(HA-FLAG)B
Molecular weightTheoretical: 257 KDa

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Macromolecule #1: Bacterial cellulose synthase regulator protein BcsB

MacromoleculeName: Bacterial cellulose synthase regulator protein BcsB / type: protein_or_peptide / ID: 1
Details: Genome-encoded BcsB harbors a signal sequence to be addressed to the periplam where according to the ServerP4.1 server prediction would result in a mature form starting with the residue #26 ...Details: Genome-encoded BcsB harbors a signal sequence to be addressed to the periplam where according to the ServerP4.1 server prediction would result in a mature form starting with the residue #26 TPATQ BcsB sequence was cloned in a pCDFDuet1 vector along Bcs(His)R, BcsQ and BcsA(HA-FLAG). 1 copy in locally refined BcsR2Q2AB map, up to 6 copies in the assembled Bcs macrocomplex.
Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: TPATQPLINA EPAVAAQTEQ NPQVGQVMPG VQGAD APVV AQNGPSRDVK LTFAQIAPPP GSMVLRGINP NGSIEFGMRS DEVVTKAMLN LEYTPS PSL LPVQSQLKVY LNDELMGVLP VTKEQLGKKT LAQMPINPLF ITDFNRVRLE FVGHYQD VC ENPASTTLWL ...String:
TPATQPLINA EPAVAAQTEQ NPQVGQVMPG VQGAD APVV AQNGPSRDVK LTFAQIAPPP GSMVLRGINP NGSIEFGMRS DEVVTKAMLN LEYTPS PSL LPVQSQLKVY LNDELMGVLP VTKEQLGKKT LAQMPINPLF ITDFNRVRLE FVGHYQD VC ENPASTTLWL DVGRSSGLDL TYQTLNVKND LSHFPVPFFD PRDNRTNTLP MVFAGAPD V GLQQASAIVA SWFGSRSGWR GQNFPVLYNQ LPDRNAIVFA TNDKRPDFLR DHPAVKAPV IEMINHPQNP YVKLLVVFGR DDKDLLQAAK GIAQGNILFR GESVVVNEVK PLLPRKPYDA PNWVRTDRP VTFGELKTYE EQLQSSGLEP AAINVSLNLP PDLYLMRSTG IDMDINYRYT M PPVKDSSR MDISLNNQFL QSFNLSSKQE ANRLLLRIPV LQGLLDGKTD VSIPALKLGA TN QLRFDFE YMNPMPGGSV DNCITFQPVQ NHVVIGDDST IDFSKYYHFI PMPDLRAFAN AGF PFSRMA DLSQTITVMP KAPNEAQMET LLNTVGFIGA QTGFPAINLT VTDDGSTIQG KDAD IMIIG GIPDKLKDDK QIDLLVQATE SWVKTPMRQT PFPGIVPDES DRAAETRSTL TSSGA MAAV IGFQSPYNDQ RSVIALLADS PRGYEMLNDA VNDSGKRATM FGSVAVIRES GINSLR VGD VYYVGHLPWF ERLWYALANH PILLAVLAAI SVILLAWVLW RLLRIISRRR LNPDNE

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Macromolecule #2: Bacterial cellulose synthase protein BcsA

MacromoleculeName: Bacterial cellulose synthase protein BcsA / type: protein_or_peptide / ID: 2
Details: BcsA sequence was cloned in pCDFDuet1 vector along with Bcs(His)R, BcsQ and BcsB. BcsA harbours a C-terminus HA-Flag Tag. 1 copy in both the locally refined BcsR2Q2AB complex and the assembled Bcs macrocomplex.
Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSILTRWLLI PPVNARLIGR YRDYRRHGAS AFSATLGCFW MILAWIFIPL EHPRWQRIRA EHKNLYPHIN ASRPRPLDPV RYLIQTCWLL IGASRKETPK PRRRAFSGLQ NIRGRYHQWM NELPERVSHK TQHLDEKKEL GHLSAGARRL ILGIIVTFSL ILALICVTQP ...String:
MSILTRWLLI PPVNARLIGR YRDYRRHGAS AFSATLGCFW MILAWIFIPL EHPRWQRIRA EHKNLYPHIN ASRPRPLDPV RYLIQTCWLL IGASRKETPK PRRRAFSGLQ NIRGRYHQWM NELPERVSHK TQHLDEKKEL GHLSAGARRL ILGIIVTFSL ILALICVTQP FNPLAQFIFL MLLWGVALIV RRMPGRFSAL MLIVLSLTVS CRYIWWRYTS TLNWDDPVSL VCGLILLFAE TYAWIVLVLG YFQVVWPLNR QPVPLPKDMS LWPSVDIFVP TYNEDLNVVK NTIYASLGID WPKDKLNIWI LDDGGREEFR QFAQNVGVKY IARTTHEHAK AGNINNALKY AKGEFVSIFD CDHVPTRSFL QMTMGWFLKE KQLAMMQTPH HFFSPDPFER NLGRFRKTPN EGTLFYGLVQ DGNDMWDATF FCGSCAVIRR KPLDEIGGIA VETVTEDAHT SLRLHRRGYT SAYMRIPQAA GLATESLSAH IGQRIRWARG MVQIFRLDNP LTGKGLKFAQ RLCYVNAMFH FLSGIPRLIF LTAPLAFLLL HAYIIYAPAL MIALFVLPHM IHASLTNSKI QGKYRHSFWS EIYETVLAWY IAPPTLVALI NPHKGKFNVT AKGGLVEEEY VDWVISRPYI FLVLLNLVGV AVGIWRYFYG PPTEMLTVVV SMVWVFYNLI VLGGAVAVSV ESKQVRRSHR VEMTMPAAIA REDGHLFSCT VQDFSDGGLG IKINGQAQIL EGQKVNLLLK RGQQEYVFPT QVARVMGNEV GLKLMPLTTQ QHIDFVQCTF ARADTWALWQ DSYPEDKPLE SLLDILKLGF RGYRHLAEFA PSSVKGIFRV LTSLVSWVVS FIPRRPERSE TAQPSDQALA QQGSARSSGR TGLEFEEFYP YDVPDYAADY KDDDDKRS

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Macromolecule #3: Bacterial cellulose synthase regulator protein BcsR

MacromoleculeName: Bacterial cellulose synthase regulator protein BcsR / type: protein_or_peptide / ID: 3
Details: BcsR sequence was cloned in pCDFDuet1 vector along with BcsQ, BcsA(HA-FLAG) and BcsB. BcsR harbours a N-terminal octahistidine tag (N-His8). Likely two copies in the assembled secretion ...Details: BcsR sequence was cloned in pCDFDuet1 vector along with BcsQ, BcsA(HA-FLAG) and BcsB. BcsR harbours a N-terminal octahistidine tag (N-His8). Likely two copies in the assembled secretion complexes, however the local resolution does not allow backbone tracing. High-resolution structures solved by X-ray crystallography.
Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MGSSHHHHHH HHAAGSMNNN EPDTLPDPAI GYIFQNDIVA LKQAFSLPDI DYADISQREQ LAAALKRWPL LAEFAQQK

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Macromolecule #4: Bacterial cellulose synthase regulator protein BcsQ

MacromoleculeName: Bacterial cellulose synthase regulator protein BcsQ / type: protein_or_peptide / ID: 4
Details: BcsQ sequence was cloned in pCDFDuet1 vector along with Bcs(His)R, BcsA(HA-FLAG) and BcsB
Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAVLGLQGVR GGVGTTTITA ALAWSLQMLG ENVLVVDACP DNLLRLSFNV DFTHRQGWAR AMLDGQDWRD AGLRYTSQLD LLPFGQLSIE EQENPQHWQT RLSDICSGLQ QLKASGRYQW ILIDLPRDAS QITHQLLSLC DHSLAIVNVD ANCHIRLHQQ ALPDGAHILI ...String:
MAVLGLQGVR GGVGTTTITA ALAWSLQMLG ENVLVVDACP DNLLRLSFNV DFTHRQGWAR AMLDGQDWRD AGLRYTSQLD LLPFGQLSIE EQENPQHWQT RLSDICSGLQ QLKASGRYQW ILIDLPRDAS QITHQLLSLC DHSLAIVNVD ANCHIRLHQQ ALPDGAHILI NNFRIGSQVQ DDIYQLWLQS QRRLLPMLIH RDEAMAECLA AKQPVGEYRS DALAAEEILT LANWCLLNYS GLKTPVGSKS

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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

Concentration0.8 mg/mL
BufferpH: 8
Component:
ConcentrationFormula
0.2 MHEPES
0.12 MNaClSodium chloride
0.005 MMgCl2
2e-06 MAppCp
2e-06 Mc-di-GMPCyclic di-GMP
0.008 %LM.NPG
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Details: Elmo Glow Discharge system
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 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.75 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionDetails: Particle picking using cryoSPARC's template picker and 2D classes generated from an initial dataset collected on the Elsa the Talos Arctica at the IECB Bordeaux.
CTF correctionSoftware - Name: Gctf / Details: Gctf through the cryoSPARC v2 interface
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model generation in cryosparc V2
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v2)
Details: All 2D classification, particle curation, ab initio model generation, particle subtraction and local refinement were performed in cryosparc v2
Final 3D classificationSoftware - Name: cryoSPARC (ver. v2) / Details: 3D variability analysis
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2)
Details: Two rounds of local refinement of the BcsR2Q2AB assembly were performed using different fulcrum placements. The resulting maps gave FSC values of ~3.9 and 4.1A, respectively, as determined ...Details: Two rounds of local refinement of the BcsR2Q2AB assembly were performed using different fulcrum placements. The resulting maps gave FSC values of ~3.9 and 4.1A, respectively, as determined by cryoSPARC using the 0.143 cut-off. The unsharpened maps were combined and conservatively sharpened to 4.5 A resolution. The sharpened map was used for building the C-terminal tail-anchor of BcsB and flexible fitting of a Robetta-derived homology model of BcsA. A BcsR2Q2 dimer solved by X-ray crystallography was rigid-body fitted in the apical density.
Number images used: 173294
DetailsData collection at the CM01 line in ESRF Grenoble (Titan Krion, GATAN K2 Summit DED and Quantum LS Imaging filter).

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