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- EMDB-23146: Bacterial cellulose synthase BcsB hexamer -

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Basic information

Entry
Database: EMDB / ID: EMD-23146
TitleBacterial cellulose synthase BcsB hexamer
Map data
Sample
  • Complex: Bacterial cellulose synthase BcsB hexamer
    • Protein or peptide: Cyclic di-GMP-binding protein
Function / homologyCellulose synthase, subunit B / Cellulose synthase BcsB, bacterial / Bacterial cellulose synthase subunit / cellulose biosynthetic process / UDP-glucose metabolic process / plasma membrane / Cyclic di-GMP-binding protein
Function and homology information
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsAcheson JF / Zimmer J
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM101001 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24-GM116790 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1F32GM126647-01 United States
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: Molecular organization of the E. coli cellulose synthase macrocomplex.
Authors: Justin F Acheson / Ruoya Ho / Nicolette F Goularte / Lynette Cegelski / Jochen Zimmer /
Abstract: Cellulose is frequently found in communities of sessile bacteria called biofilms. Escherichia coli and other enterobacteriaceae modify cellulose with phosphoethanolamine (pEtN) to promote host tissue ...Cellulose is frequently found in communities of sessile bacteria called biofilms. Escherichia coli and other enterobacteriaceae modify cellulose with phosphoethanolamine (pEtN) to promote host tissue adhesion. The E. coli pEtN cellulose biosynthesis machinery contains the catalytic BcsA-B complex that synthesizes and secretes cellulose, in addition to five other subunits. The membrane-anchored periplasmic BcsG subunit catalyzes pEtN modification. Here we present the structure of the roughly 1 MDa E. coli Bcs complex, consisting of one BcsA enzyme associated with six copies of BcsB, determined by single-particle cryo-electron microscopy. BcsB homo-oligomerizes primarily through interactions between its carbohydrate-binding domains as well as intermolecular beta-sheet formation. The BcsB hexamer creates a half spiral whose open side accommodates two BcsG subunits, directly adjacent to BcsA's periplasmic channel exit. The cytosolic BcsE and BcsQ subunits associate with BcsA's regulatory PilZ domain. The macrocomplex is a fascinating example of cellulose synthase specification.
History
DepositionDec 17, 2020-
Header (metadata) releaseMar 24, 2021-
Map releaseMar 24, 2021-
UpdateMar 24, 2021-
Current statusMar 24, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7l2z
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23146.png

Downloads & links

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Map

FileDownload / File: emd_23146.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.4
Minimum - Maximum-0.96168786 - 2.351905
Average (Standard dev.)0.0015793882 (±0.056786753)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 388.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z388.800388.800388.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.9622.3520.002

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Supplemental data

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Mask #1

Fileemd_23146_msk_1.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 : Bacterial cellulose synthase BcsB hexamer

EntireName: Bacterial cellulose synthase BcsB hexamer
Components
  • Complex: Bacterial cellulose synthase BcsB hexamer
    • Protein or peptide: Cyclic di-GMP-binding protein

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Supramolecule #1: Bacterial cellulose synthase BcsB hexamer

SupramoleculeName: Bacterial cellulose synthase BcsB hexamer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Periplasmic domain of BcsB refined using masked local refinement and signal subtraction and from E coli BCS inner-membrane complex projections.
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Location in cell: periplasm innermembrane
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: C43 / Recombinant plasmid: petduet_BcsA_BcsB_AdrA
Molecular weightTheoretical: 481 KDa

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Macromolecule #1: Cyclic di-GMP-binding protein

MacromoleculeName: Cyclic di-GMP-binding protein / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 84.304906 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: WSHPQFEKTP ATQPLINAEP AVAAQTEQNP QVGQVMPGVQ GADAPVVAQN GPSRDVKLTF AQIAPPPGSM VLRGINPNGS IEFGMRSDE VVTKAMLNLE YTPSPSLLPV QSQLKVYLND ELMGVLPVTK EQLGKKTLAQ MPINPLFISD FNRVRLEFVG H YQDVCEKP ...String:
WSHPQFEKTP ATQPLINAEP AVAAQTEQNP QVGQVMPGVQ GADAPVVAQN GPSRDVKLTF AQIAPPPGSM VLRGINPNGS IEFGMRSDE VVTKAMLNLE YTPSPSLLPV QSQLKVYLND ELMGVLPVTK EQLGKKTLAQ MPINPLFISD FNRVRLEFVG H YQDVCEKP ASTTLWLDVG RSSGLDLTYQ TLNVKNDLSH FPVPFFDPSD NRTNTLPMVF AGAPDVGLQQ ASAIVASWFG SR SGWRGQN FPVLYNQLPD RNAIVFATND KRPDFLRDHP AVKAPVIEMI NHPQNPYVKL LVVFGRDDKD LLQAAKGIAQ GNI LFRGES VVVNEVKPLL PRKPYDAPNW VRTDRPVTFG ELKTYEEQLQ SSGLEPAAIN VSLNLPPDLY LMRSTGIDMD INYR YTMPP VKDSSRMDIS LNNQFLQSFN LSSKQEANRL LLRIPVLQGL LDGKTDVSIP ALKLGATNQL RFDFEYMNPM PGGSV DNCI TFQPVQNHVV IGDDSTIDFS KYYHFIPMPD LRAFANAGFP FSRMADLSQT ITVMPKAPNE AQMETLLNTV GFIGAQ TGF PAINLTVTDD GSTIQGKDAD IMIIGGIPDK LKDDKQIDLL VQATESWVKT PMRQTPFPGI VPDESDRAAE TRSTLTS SG AMAAVIGFQS PYNDQRSVIA LLADSPRGYE MLNDAVNDSG KRATMFGSVA VIRESGINSL RVGDVYYVGH LPWFERVW Y ALANHPILLA VLAAISVILL AWVLWRLLRI ISRRRLNPDN E

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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

Concentration0.4 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride
0.5 mMC12H22O11cellobiose
5.0 mMMgCl2magnesium chloride
0.003 %C47H88O22LMNG
0.0006 %C43H80O22DMNG
0.0006 %C31H50O4CHS

Details: All buffers made fresh from concentrated stock. A 10% LMNG 2% DMNG 2% CHS solution was made made in milliQ water without buffer. After rocking for one day at room temp the solution was ...Details: All buffers made fresh from concentrated stock. A 10% LMNG 2% DMNG 2% CHS solution was made made in milliQ water without buffer. After rocking for one day at room temp the solution was briefly sonicated to completely dissolve the CHS.
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Details: 2 drops of amylamine were added to the chamber
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 3.5 uL applied to c-flat 1.2/1.3 300 mesh grids incubated for 30s then blotted using force 6 for 12 seconds.

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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 / Cs: 2.7 mm / Nominal defocus max: -2.5 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 2964 / Average electron dose: 51.0 e/Å2 / Details: movie mode 40 frames
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 409611
CTF correctionSoftware - Name: cryoSPARC (ver. 2.15) / Software - details: Patch CTF
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 1
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 2.15)
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 115289
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 101.45 / Target criteria: CC
Output model

PDB-7l2z:
Bacterial cellulose synthase BcsB hexamer

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