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- EMDB-44359: Cryo-EM map of the E. coli cellulose synthase BcsAG3B6 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-44359
TitleCryo-EM map of the E. coli cellulose synthase BcsAG3B6 complex
Map dataComposite structure: Composite map reconstructed from focussed refinement of BcsAG3 and BcsB6
Sample
  • Complex: Bacterial cellulose synthase BcsA-BcsG3-BcsB6 complex
    • Protein or peptide: Cellulose synthase operon protein B
    • Protein or peptide: Cellulose biosynthesis protein BcsG
    • Protein or peptide: Cellulose synthase catalytic subunit [UDP-forming]
Keywordsphosphoethanolamine (pEtN) / catalytic BcsA-B / c-di-GMP binding protein / membrane proteins / 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 biosynthesis protein BcsG / Cellulose biosynthesis protein BcsG / Cellulose synthase, subunit A / : / Cellulose synthase, subunit B / Cellulose synthase BcsB, bacterial / Bacterial cellulose synthase subunit / PilZ domain / PilZ domain / Glycosyltransferase 2-like ...Cellulose biosynthesis protein BcsG / Cellulose biosynthesis protein BcsG / 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] / Cellulose biosynthesis protein BcsG
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsVerma P / Zimmer J
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM144130 United States
CitationJournal: Nat Commun / Year: 2024
Title: Insights into phosphoethanolamine cellulose synthesis and secretion across the Gram-negative cell envelope.
Authors: Preeti Verma / Ruoya Ho / Schuyler A Chambers / Lynette Cegelski / Jochen Zimmer /
Abstract: Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the ...Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the catalytically active BcsA enzyme, a hexameric semicircle of the periplasmic BcsB protein, and the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a periplasmic cellulase of unknown biological function. While cellulose synthesis and translocation by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three BcsG copies near the nascent cellulose polymer. Further, the semicircle's terminal BcsB subunit tethers the N-terminus of a single BcsC protein in a trans-envelope secretion system. BcsC's TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of translocation incompetent cellulose. Lastly, protein engineering introduces cellulose pEtN modification in orthogonal cellulose biosynthetic systems. These findings advance our understanding of pEtN cellulose modification and secretion.
History
DepositionApr 1, 2024-
Header (metadata) releaseFeb 19, 2025-
Map releaseFeb 19, 2025-
UpdateMay 21, 2025-
Current statusMay 21, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44359.png

Downloads & links

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Map

FileDownload / File: emd_44359.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite structure: Composite map reconstructed from focussed refinement of BcsAG3 and BcsB6
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.08 Å/pix.
x 360 pix.
= 388.8 Å		1.08 Å/pix.
x 360 pix.
= 388.8 Å		1.08 Å/pix.
x 360 pix.
= 388.8 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: 7.0
Minimum - Maximum-50.281219999999998 - 38.521790000000003
Average (Standard dev.)-0.009463032 (±1.1221722)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Sample components

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Entire : Bacterial cellulose synthase BcsA-BcsG3-BcsB6 complex

EntireName: Bacterial cellulose synthase BcsA-BcsG3-BcsB6 complex
Components
  • Complex: Bacterial cellulose synthase BcsA-BcsG3-BcsB6 complex
    • Protein or peptide: Cellulose synthase operon protein B
    • Protein or peptide: Cellulose biosynthesis protein BcsG
    • Protein or peptide: Cellulose synthase catalytic subunit [UDP-forming]

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Supramolecule #1: Bacterial cellulose synthase BcsA-BcsG3-BcsB6 complex

SupramoleculeName: Bacterial cellulose synthase BcsA-BcsG3-BcsB6 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Proteins: 1. Bacterial cellulose synthase subunit A (BcsA) 2. Bacterial cellulose synthase subunit B (BcsB) 3. Bacterial cellulose synthase subunit G (BcsG)
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Cellulose synthase operon protein B

MacromoleculeName: Cellulose synthase operon protein B / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 84.375984 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: AWSHPQFEKT PATQPLINAE PAVAAQTEQN PQVGQVMPGV QGADAPVVAQ NGPSRDVKLT FAQIAPPPGS MVLRGINPNG SIEFGMRSD EVVTKAMLNL EYTPSPSLLP VQSQLKVYLN DELMGVLPVT KEQLGKKTLA QMPINPLFIS DFNRVRLEFV G HYQDVCEK ...String:
AWSHPQFEKT PATQPLINAE PAVAAQTEQN PQVGQVMPGV QGADAPVVAQ NGPSRDVKLT FAQIAPPPGS MVLRGINPNG SIEFGMRSD EVVTKAMLNL EYTPSPSLLP VQSQLKVYLN DELMGVLPVT KEQLGKKTLA QMPINPLFIS DFNRVRLEFV G HYQDVCEK PASTTLWLDV GRSSGLDLTY QTLNVKNDLS HFPVPFFDPS DNRTNTLPMV FAGAPDVGLQ QASAIVASWF GS RSGWRGQ NFPVLYNQLP DRNAIVFATN DKRPDFLRDH PAVKAPVIEM INHPQNPYVK LLVVFGRDDK DLLQAAKGIA QGN ILFRGE SVVVNEVKPL LPRKPYDAPN WVRTDRPVTF GELKTYEEQL QSSGLEPAAI NVSLNLPPDL YLMRSTGIDM DINY RYTMP PVKDSSRMDI SLNNQFLQSF NLSSKQEANR LLLRIPVLQG LLDGKTDVSI PALKLGATNQ LRFDFEYMNP MPGGS VDNC ITFQPVQNHV VIGDDSTIDF SKYYHFIPMP DLRAFANAGF PFSRMADLSQ TITVMPKAPN EAQMETLLNT VGFIGA QTG FPAINLTVTD DGSTIQGKDA DIMIIGGIPD KLKDDKQIDL LVQATESWVK TPMRQTPFPG IVPDESDRAA ETRSTLT SS GAMAAVIGFQ SPYNDQRSVI ALLADSPRGY EMLNDAVNDS GKRATMFGSV AVIRESGINS LRVGDVYYVG HLPWFERV W YALANHPILL AVLAAISVIL LAWVLWRLLR IISRRRLNPD NE

UniProtKB: Cyclic di-GMP-binding protein

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Macromolecule #2: Cellulose biosynthesis protein BcsG

MacromoleculeName: Cellulose biosynthesis protein BcsG / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 63.079508 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTQFTQNTAM PSSLWQYWRG LSGWNFYFLV KFGLLWAGYL NFHPLLNLVF AAFLLMPLPR YSLHRLRHWI ALPIGFALFW HDTWLPGPE SIMSQGSQVA GFSTDYLIDL VTRFINWQMI GAIFVLLVAW LFLSQWIRIT VFVVAILLWL NVLTLAGPSF S LWPAGQPT ...String:
MTQFTQNTAM PSSLWQYWRG LSGWNFYFLV KFGLLWAGYL NFHPLLNLVF AAFLLMPLPR YSLHRLRHWI ALPIGFALFW HDTWLPGPE SIMSQGSQVA GFSTDYLIDL VTRFINWQMI GAIFVLLVAW LFLSQWIRIT VFVVAILLWL NVLTLAGPSF S LWPAGQPT TTVTTTGGNA AATVAATGGA PVVGDMPAQT APPTTANLNA WLNNFYNAEA KRKSTFPSSL PADAQPFELL VI NICSLSW SDIEAAGLMS HPLWSHFDIE FKNFNSATSY SGPAAIRLLR ASCGQTSHTN LYQPANNDCY LFDNLSKLGF TQH LMMGHN GQFGGFLKEV RENGGMQSEL MDQTNLPVIL LGFDGSPVYD DTAVLNRWLD VTEKDKNSRS ATFYNTLPLH DGNH YPGVS KTADYKARAQ KFFDELDAFF TELEKSGRKV MVVVVPEHGG ALKGDRMQVS GLRDIPSPSI TDVPVGVKFF GMKAP HQGA PIVIEQPSSF LAISDLVVRV LDGKIFTEDN VDWKKLTSGL PQTAPVSENS NAVVIQYQDK PYVRLNGGDW VPYPQD YKD DDDK

UniProtKB: Cellulose biosynthesis protein BcsG

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Macromolecule #3: Cellulose synthase catalytic subunit [UDP-forming]

MacromoleculeName: Cellulose synthase catalytic subunit [UDP-forming] / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: cellulose synthase (UDP-forming)
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 101.862523 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSILTRWLL IPPVNARLIG RYRDYRRHGA SAFSATLGCF WMILAWIFIP LEHPRWQRIR AEHKNLYPHI NASRPRPLDP VRYLIQTCW LLIGASRKET PKPRRRAFSG LQNIRGRYHQ WMNELPERVS HKTQHLDEKK ELGHLSAGAR RLILGIIVTF S LILALICV ...String:
MGSILTRWLL IPPVNARLIG RYRDYRRHGA SAFSATLGCF WMILAWIFIP LEHPRWQRIR AEHKNLYPHI NASRPRPLDP VRYLIQTCW LLIGASRKET PKPRRRAFSG LQNIRGRYHQ WMNELPERVS HKTQHLDEKK ELGHLSAGAR RLILGIIVTF S LILALICV TQPFNPLAQF IFLMLLWGVA LIVRRMPGRF SALMLIVLSL TVSCRYIWWR YTSTLNWDDP VSLVCGLILL FA ETYAWIV LVLGYFQVVW PLNRQPVPLP KDMSLWPSVD IFVPTYNEDL NVVKNTIYAS LGIDWPKDKL NIWILDDGGR EEF RQFAQN VGVKYIARTT HEHAKAGNIN NALKYAKGEF VSIFDCDHVP TRSFLQMTMG WFLKEKQLAM MQTPHHFFSP DPFE RNLGR FRKTPNEGTL FYGLVQDGND MWDATFFCGS CAVIRRKPLD EIGGIAVETV TEDAHTSLRL HRRGYTSAYM RIPQA AGLA TESLSAHIGQ RIRWARGMVQ IFRLDNPLTG KGLKFAQRLC YVNAMFHFLS GIPRLIFLTA PLAFLLLHAY IIYAPA LMI ALFVLPHMIH ASLTNSKIQG KYRHSFWSEI YETVLAWYIA PPTLVALINP HKGKFNVTAK GGLVEEEYVD WVISRPY IF LVLLNLVGVA VGIWRYFYGP PTEMLTVVVS MVWVFYNLIV LGGAVAVSVE SKQVRRSHRV EMTMPAAIAR EDGHLFSC T VQDFSDGGLG IKINGQAQIL EGQKVNLLLK RGQQEYVFPT QVARVMGNEV GLKLMPLTTQ QHIDFVQCTF ARADTWALW QDSYPEDKPL ESLLDILKLG FRGYRHLAEF APSSVKGIFR VLTSLVSWVV SFIPRRPERS ETAQPSDQAL AQQHHHHHHL EHHHHHH

UniProtKB: Cellulose synthase catalytic subunit [UDP-forming]

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 51.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup model#0 - Type of model: INSILICO MODEL / #0 - In silico model: AlphaFold2 model of BcsA-BcsG3 / #1 - Type of model: PDB ENTRY
#1 - PDB model - PDB ID:

7l2z


#1 - Details: PDB: 7L2Z for BcsB hexamer
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 39522
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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