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- EMDB-23267: Bacterial cellulose synthase BcsB with polyalanine BcsA model -

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Basic information

Entry
Database: EMDB / ID: EMD-23267
TitleBacterial cellulose synthase BcsB with polyalanine BcsA model
Map dataBacterial cellulose synthase BcsB with polyalanine BcsA model
Sample
  • Complex: Bacterial cellulose synthase BcsB with BcsA
    • Protein or peptide: Cellulose synthase catalytic subunit [UDP-forming]
    • Protein or peptide: Cyclic di-GMP-binding protein
Keywordsbacterial cellulose / synthase / structural subunit / BIOSYNTHETIC PROTEIN / TRANSFERASE
Function / homology
Function and homology information


bacterial cellulose biosynthetic process / cellulose synthase (UDP-forming) / cellulose synthase (UDP-forming) activity / cellulose biosynthetic process / UDP-alpha-D-glucose metabolic process / hexosyltransferase activity / cyclic-di-GMP binding / plasma membrane
Similarity search - Function
Cellulose synthase, subunit A / : / Cellulose synthase, subunit B / Cellulose synthase BcsB, bacterial / Bacterial cellulose synthase subunit / PilZ domain / PilZ domain / Glycosyltransferase 2-like / Glycosyl transferase family 2 / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
Cyclic di-GMP-binding protein / Cellulose synthase catalytic subunit [UDP-forming]
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 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
Citation
Journal: 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.
#1: Journal: Nat.Struct.Mol.Biol. / Year: 2021
Title: Molecular Organization of the E. coli Cellulose Synthase Macrocomplex
Authors: Acheson JF / Ho R / Goularte NF / Cegelski L / Zimmer J
History
DepositionJan 9, 2021-
Header (metadata) releaseMar 24, 2021-
Map releaseMar 24, 2021-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.235
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.235
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7lby
  • Surface level: 0.27
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7lby
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23267.png

Downloads & links

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Map

FileDownload / File: emd_23267.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBacterial cellulose synthase BcsB with polyalanine BcsA model
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 400 pix.
= 432. Å		1.08 Å/pix.
x 400 pix.
= 432. Å		1.08 Å/pix.
x 400 pix.
= 432. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.235 / Movie #1: 0.235
Minimum - Maximum-1.783147 - 3.8408334
Average (Standard dev.)-0.0010064957 (±0.041981425)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 432.00003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z432.000432.000432.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-1.7833.841-0.001

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Supplemental data

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Mask #1

Fileemd_23267_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 with BcsA

EntireName: Bacterial cellulose synthase BcsB with BcsA
Components
  • Complex: Bacterial cellulose synthase BcsB with BcsA
    • Protein or peptide: Cellulose synthase catalytic subunit [UDP-forming]
    • Protein or peptide: Cyclic di-GMP-binding protein

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Supramolecule #1: Bacterial cellulose synthase BcsB with BcsA

SupramoleculeName: Bacterial cellulose synthase BcsB with BcsA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: BcsB monomer with BcsA refined using masked local refinement and signal subtraction from E coli BCS inner-membrane complex projections.
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Location in cell: inner membrane
Molecular weightTheoretical: 180 KDa

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Macromolecule #1: Cellulose synthase catalytic subunit [UDP-forming]

MacromoleculeName: Cellulose synthase catalytic subunit [UDP-forming] / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: cellulose synthase (UDP-forming)
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 101.67125 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSILTRWLLI PPVNARLIGR YRDYRRHGAS AFSATLGCFW MILAWIFIPL EHPRWQRIRA EHKNLYPHIN ASRPRPLDPV RYLIQTCWL LIGASRKETP KPRRRAFSGL QNIRGRYHQW MNELPERVSH KTQHLDEKKE LGHLSAGARR LILGIIVTFS L ILALICVT ...String:
GSILTRWLLI PPVNARLIGR YRDYRRHGAS AFSATLGCFW MILAWIFIPL EHPRWQRIRA EHKNLYPHIN ASRPRPLDPV RYLIQTCWL LIGASRKETP KPRRRAFSGL QNIRGRYHQW MNELPERVSH KTQHLDEKKE LGHLSAGARR LILGIIVTFS L ILALICVT QPFNPLAQFI FLMLLWGVAL IVRRMPGRFS ALMLIVLSLT VSCRYIWWRY TSTLNWDDPV SLVCGLILLF AE TYAWIVL VLGYFQVVWP LNRQPVPLPK DMSLWPSVDI FVPTYNEDLN VVKNTIYASL GIDWPKDKLN IWILDDGGRE EFR QFAQNV GVKYIARTTH EHAKAGNINN ALKYAKGEFV SIFDCDHVPT RSFLQMTMGW FLKEKQLAMM QTPHHFFSPD PFER NLGRF RKTPNEGTLF YGLVQDGNDM WDATFFCGSC AVIRRKPLDE IGGIAVETVT EDAHTSLRLH RRGYTSAYMR IPQAA GLAT ESLSAHIGQR IRWARGMVQI FRLDNPLTGK GLKFAQRLCY VNAMFHFLSG IPRLIFLTAP LAFLLLHAYI IYAPAL MIA LFVLPHMIHA SLTNSKIQGK YRHSFWSEIY ETVLAWYIAP PTLVALINPH KGKFNVTAKG GLVEEEYVDW VISRPYI FL VLLNLVGVAV GIWRYFYGPP TEMLTVVVSM VWVFYNLIVL GGAVAVSVES KQVRRSHRVE MTMPAAIARE DGHLFSCT V QDFSDGGLGI KINGQAQILE GQKVNLLLKR GQQEYVFPTQ VARVMGNEVG LKLMPLTTQQ HIDFVQCTFA RADTWALWQ DSYPEDKPLE SLLDILKLGF RGYRHLAEFA PSSVKGIFRV LTSLVSWVVS FIPPRPERSE TAQPSDQALA QQHHHHHHLE HHHHHH

UniProtKB: Cellulose synthase catalytic subunit [UDP-forming]

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Macromolecule #2: Cyclic di-GMP-binding protein

MacromoleculeName: Cyclic di-GMP-binding protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 86.101289 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVL RGINPNGSIE FGMRSDEVVT KAMLNLEYTP SPSLLPVQSQ LKVYLNDELM GVLPVTKEQL GKKTLAQMPI N PLFISDFN ...String:
MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVL RGINPNGSIE FGMRSDEVVT KAMLNLEYTP SPSLLPVQSQ LKVYLNDELM GVLPVTKEQL GKKTLAQMPI N PLFISDFN RVRLEFVGHY QDVCEKPAST TLWLDVGRSS GLDLTYQTLN VKNDLSHFPV PFFDPSDNRT NTLPMVFAGA PD VGLQQAS AIVASWFGSR SGWRGQNFPV LYNQLPDRNA IVFATNDKRP DFLRDHPAVK APVIEMINHP QNPYVKLLVV FGR DDKDLL QAAKGIAQGN ILFRGESVVV NEVKPLLPRK PYDAPNWVRT DRPVTFGELK TYEEQLQSSG LEPAAINVSL NLPP DLYLM RSTGIDMDIN YRYTMPPVKD SSRMDISLNN QFLQSFNLSS KQEANRLLLR IPVLQGLLDG KTDVSIPALK LGATN QLRF DFEYMNPMPG GSVDNCITFQ PVQNHVVIGD DSTIDFSKYY HFIPMPDLRA FANAGFPFSR MADLSQTITV MPKAPN EAQ METLLNTVGF IGAQTGFPAI NLTVTDDGST IQGKDADIMI IGGIPDKLKD DKQIDLLVQA TESWVKTPMR QTPFPGI VP DESDRAAETR STLTSSGAMA AVIGFQSPYN DQRSVIALLA DSPRGYEMLN DAVNDSGKRA TMFGSVAVIR ESGINSLR V GDVYYVGHLP WFERVWYALA NHPILLAVLA AISVILLAWV LWRLLRIISR RRLNPDNE

UniProtKB: Cyclic di-GMP-binding protein

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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 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 / Pretreatment - Atmosphere: OTHER / Details: 2 drops amylamine 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
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -2.5 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 409611
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 193152
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 2.15)
Final 3D classificationNumber classes: 1
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-7lby:
Bacterial cellulose synthase BcsB with polyalanine BcsA model

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