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- EMDB-8075: Structure of the cellulose synthase complex of Gluconacetobacter ... -

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Basic information

Entry
Database: EMDB / ID: EMD-8075
TitleStructure of the cellulose synthase complex of Gluconacetobacter hansenii at 23.4 Angstrom resolution
Map dataNone
Sample
  • Complex: membrane protein AcsABBiological membrane
    • Protein or peptide: AcsAB Cellulose Synthase of Gluconacetobacter hansenii
Biological speciesGluconacetobacter hansenii ATCC 23769 (bacteria) / Komagataeibacter hansenii (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 23.4 Å
AuthorsNixon BT / Du J
Funding support United States, 1 items
OrganizationGrant numberCountry
Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Basic Energy SciencesDE-SC0001090 United States
CitationJournal: PLoS One / Year: 2016
Title: Structure of the Cellulose Synthase Complex of Gluconacetobacter hansenii at 23.4 Å Resolution.
Authors: Juan Du / Venkata Vepachedu / Sung Hyun Cho / Manish Kumar / B Tracy Nixon /
Abstract: Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, ...Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsD in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 Å for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into β-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. The results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation components, and support the hypothesis that it is the extrusion mechanism and order in linearly arrayed TCs that enables production of crystalline cellulose.
History
DepositionFeb 19, 2016-
Header (metadata) releaseJun 8, 2016-
Map releaseJun 8, 2016-
UpdateNov 16, 2022-
Current statusNov 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.851
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.851
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8075.png

Downloads & links

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Map

FileDownload / File: emd_8075.map.gz / Format: CCP4 / Size: 34.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.45 Å
Density
Contour LevelBy AUTHOR: 0.851 / Movie #1: 0.851
Minimum - Maximum-1.0631679 - 2.9452677
Average (Standard dev.)0.0061345976 (±0.16795348)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-104-104-104
Dimensions208208208
Spacing208208208
CellA=B=C: 301.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.451.451.45
M x/y/z208208208
origin x/y/z0.0000.0000.000
length x/y/z301.600301.600301.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ129141209
MAP C/R/S123
start NC/NR/NS-104-104-104
NC/NR/NS208208208
D min/max/mean-1.0632.9450.006

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Supplemental data

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Sample components

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Entire : membrane protein AcsAB

EntireName: membrane protein AcsABBiological membrane
Components
  • Complex: membrane protein AcsABBiological membrane
    • Protein or peptide: AcsAB Cellulose Synthase of Gluconacetobacter hansenii

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Supramolecule #1: membrane protein AcsAB

SupramoleculeName: membrane protein AcsAB / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Gluconacetobacter hansenii ATCC 23769 (bacteria)
Recombinant expressionOrganism: Komagataeibacter hansenii ATCC 23769 (bacteria) / Recombinant plasmid: pUCD2CDHisAcsAB
Molecular weightTheoretical: 170 KDa

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Macromolecule #1: AcsAB Cellulose Synthase of Gluconacetobacter hansenii

MacromoleculeName: AcsAB Cellulose Synthase of Gluconacetobacter hansenii
type: protein_or_peptide / ID: 1
Details: Heterodimer of AcsA and AcsB; membrane protein expressed as a fusion protein and then proteolytically processed.
Enantiomer: LEVO
Source (natural)Organism: Komagataeibacter hansenii (bacteria)
Recombinant expressionOrganism: Komagataeibacter hansenii (bacteria)
SequenceString: MPEVRSSTQS ESGMSQWMGK ILSIRGAGLI IGVFGLCALI AATSVTLPPE QQLIVAFVCV VIFFIVGHKP SRRSQIFLEV LSGLVSLRYL TWRLTETLSF DTWLQGLLGT MLLVAELYAL MMLFLSYFQT IAPLHRAPLP LPPNPDEWPT VDIFVPTYNE ELSIVRLTVL ...String:
MPEVRSSTQS ESGMSQWMGK ILSIRGAGLI IGVFGLCALI AATSVTLPPE QQLIVAFVCV VIFFIVGHKP SRRSQIFLEV LSGLVSLRYL TWRLTETLSF DTWLQGLLGT MLLVAELYAL MMLFLSYFQT IAPLHRAPLP LPPNPDEWPT VDIFVPTYNE ELSIVRLTVL GSLGIDWPPE KVRVHILDDG RRPEFAAFAA ECGANYIARP TNEHAKAGNL NYAIGHTDGD YILIFDCDHV PTRAFLQLTM GWMVEDPKIA LMQTPHHFYS PDPFQRNLSA GYRTPPEGNL FYGVVQDGND FWDATFFCGS CAILRRTAIE QIGGFATQTV TEDAHTALKM QRLGWSTAYL RIPLAGGLAT ERLILHIGQR VRWARGMLQI FRIDNPLFGR GLSWGQRLCY LSAMTSFLFA VPRVIFLSSP LAFLFFGQNI IAASPLALLA YAIPHMFHAV GTASKINKGW RYSFWSEVYE TTMALFLVRV TIVTLLSPSR GKFNVTDKGG LLEKGYFDLG AVYPNIILGL IMFGGLARGV YELSFGHLDQ IAERAYLLNS AWAMLSLIII LAAIAVGRET QQKRNSHRIP ATIPVEVANA DGSIIVTGVT EDLSMGGAAV KMSWPAKLSG PTPVYIRTVL DGEELILPAR IIRAGNGRGI FIWTIDNLQQ EFSVIRLVFG RADAWVDWGN YKADRPLLSL MDMVLSVKGL FRSSGDIVHR SSPTKPSAGN ALSDDTNNPS RKERVLKGTV KMVSLLALLT FASSAQAASA PRAVAAKAPA HQPEASDLPP LPALLPATSG AAQAGSGDAG ANGPGSPTGQ PLAADSADAL VENAENTSDT ATVHNYTLKD LGAAGSITMR GLAPLQGIEF GIPSDQLVTS ARLVLSGSMS PNLRPETNSV TMTLNEQYIG TLRPDPAHPT FGPMSFEINP IFFVSGNRLN FNFASGSKGC SDITNDTLWA TISQNSQLQI TTIALPPRRL LSRLPQPFYD KNVRQHVTVP MVLAQTYDPQ ILKSAGILAS WFGKQTDFLG VTFPVSSTIP QSGNAILIGV ADELPTSLGR PQVNGPAVLE LPNPSDANAT ILVVTGRDRD EVITASKGIA FASAPLPTDS HMDVAPVDIA PRKPNDAPSF IAMDHPVRFG DLVTASKLQG TGFTSGVLSV PFRIPPDLYT WRNRPYKMQV RFRSPAGEAK DVEKSRLDVG INEVYLHSYP LRETHGLIGA VLQGVGLARP ASGMQVHDLD VPPWTVFGQD QLNFYFDAMP LARGICQSGA ANNAFHLGLD PDSTIDFSRA HHIAQMPNLA YMATVGFPFT TYADLSQTAV VLPEHPNAAT VGAYLDLMGF MGAATWYPVA GVDIVSADHV SDVADRNLLV ISTLATSGEI APLLSRSSYE VADGHLRTVS HASALDNAIK AVDDPLTAFR DRDSKPQDVD TPLTGGVGAM IEAESPLTAG RTVLALLSSD GAGLNNLLQM LGERKKQANI QGDLVVAHGE DLSSYRTSPV YTIGTLPLWL WPDWYMHNRP VRVLLVGLLG CILIVSVLAR ALARHATRRF KQLEDERRKS

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.2
StainingType: NEGATIVE / Material: uranyl formate

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI EAGLE (4k x 4k) / Average electron dose: 20.0 e/Å2
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 23.4 Å / Resolution method: FSC 0.5 CUT-OFF / Number images used: 3168

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