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- PDB-7l2z: Bacterial cellulose synthase BcsB hexamer -

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Basic information

Entry
Database: PDB / ID: 7l2z
TitleBacterial cellulose synthase BcsB hexamer
ComponentsCyclic di-GMP-binding protein
KeywordsBIOSYNTHETIC PROTEIN / bacterial cellulose / periplasmic / structural subunit
Function / homologyCellulose synthase, subunit B / Cellulose synthase BcsB, bacterial / Bacterial cellulose synthase subunit / cellulose biosynthetic process / UDP-alpha-D-glucose metabolic process / plasma membrane / Cyclic di-GMP-binding protein
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsAcheson, J.F. / Zimmer, J.
Funding support United States, 3items
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 24, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
B: Cyclic di-GMP-binding protein
A: Cyclic di-GMP-binding protein
C: Cyclic di-GMP-binding protein
D: Cyclic di-GMP-binding protein
E: Cyclic di-GMP-binding protein
F: Cyclic di-GMP-binding protein


Theoretical massNumber of molelcules
Total (without water)505,8296
Polymers505,8296
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Cyclic di-GMP-binding protein / Cellulose synthase regulatory subunit


Mass: 84304.906 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: bcsB, yhjN, b3532, JW3500 / Plasmid: petduet_BcsA_BcsB_AdrA / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): C43 / References: UniProt: P37652

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Bacterial cellulose synthase BcsB hexamer / Type: COMPLEX
Details: Periplasmic domain of BcsB refined using masked local refinement and signal subtraction and from E coli BCS inner-membrane complex projections.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.481 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Cellular location: periplasm innermembrane
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C43 / Plasmid: petduet_BcsA_BcsB_AdrA
Buffer solutionpH: 8
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.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
30.5 mMcellobioseC12H22O111
45 mMmagnesium chlorideMgCl21
50.003 %LMNGC47H88O221
60.0006 %DMNGC43H80O221
70.0006 %CHSC31H50O41
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 2 drops of amylamine were added to the chamber / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
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 imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: -2500 nm / Nominal defocus min: -1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 51 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2964 / Details: movie mode 40 frames
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 10 eV

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC2.15particle selectionTOPAZ wrapper
2LatitudeSimage acquisition
4cryoSPARC2.15CTF correctionPatch CTF
9cryoSPARCinitial Euler assignment
10cryoSPARC2.15final Euler assignment
13PHENIX1.18model refinementphenix.refine
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 409611
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115289 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 101.45 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: CC
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00230635
ELECTRON MICROSCOPYf_angle_d0.45741696
ELECTRON MICROSCOPYf_dihedral_angle_d14.0554147
ELECTRON MICROSCOPYf_chiral_restr0.0464680
ELECTRON MICROSCOPYf_plane_restr0.0035533

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