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- PDB-7znr: Inactive D62N mutant of BT1760 Endo-acting levanase from Bacteroi... -

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Basic information

Entry
Database: PDB / ID: 7znr
TitleInactive D62N mutant of BT1760 Endo-acting levanase from Bacteroides thetaiotaomicron VPI-5482
ComponentsSucrose-6-phosphate hydrolase
KeywordsHYDROLASE / Levan / fructan / endo levanase / gut microbiota
Function / homology
Function and homology information


beta-fructofuranosidase activity / beta-fructofuranosidase / carbohydrate metabolic process
Similarity search - Function
GH32, BT1760-like, C-terminal domain / GH32, BT1760-like, C-terminal domain / Glycoside hydrolase, family 32 / Glycosyl hydrolase family 32, N-terminal / Glycosyl hydrolases family 32 N-terminal domain / Glycosyl hydrolases family 32 / Glycosyl hydrolase, five-bladed beta-propellor domain superfamily / Prokaryotic membrane lipoprotein lipid attachment site profile. / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Sucrose-6-phosphate hydrolase
Similarity search - Component
Biological speciesBacteroides thetaiotaomicron (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.65 Å
AuthorsBasle, A. / Bolam, D.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/F014163/1 United Kingdom
CitationJournal: Nature / Year: 2023
Title: Outer membrane utilisomes mediate glycan uptake in gut Bacteroidetes.
Authors: Joshua B R White / Augustinas Silale / Matthew Feasey / Tiaan Heunis / Yiling Zhu / Hong Zheng / Akshada Gajbhiye / Susan Firbank / Arnaud Baslé / Matthias Trost / David N Bolam / Bert van ...Authors: Joshua B R White / Augustinas Silale / Matthew Feasey / Tiaan Heunis / Yiling Zhu / Hong Zheng / Akshada Gajbhiye / Susan Firbank / Arnaud Baslé / Matthias Trost / David N Bolam / Bert van den Berg / Neil A Ranson /
Abstract: Bacteroidetes are abundant members of the human microbiota, utilizing a myriad of diet- and host-derived glycans in the distal gut. Glycan uptake across the bacterial outer membrane of these bacteria ...Bacteroidetes are abundant members of the human microbiota, utilizing a myriad of diet- and host-derived glycans in the distal gut. Glycan uptake across the bacterial outer membrane of these bacteria is mediated by SusCD protein complexes, comprising a membrane-embedded barrel and a lipoprotein lid, which is thought to open and close to facilitate substrate binding and transport. However, surface-exposed glycan-binding proteins and glycoside hydrolases also play critical roles in the capture, processing and transport of large glycan chains. The interactions between these components in the outer membrane are poorly understood, despite being crucial for nutrient acquisition by our colonic microbiota. Here we show that for both the levan and dextran utilization systems of Bacteroides thetaiotaomicron, the additional outer membrane components assemble on the core SusCD transporter, forming stable glycan-utilizing machines that we term utilisomes. Single-particle cryogenic electron microscopy structures in the absence and presence of substrate reveal concerted conformational changes that demonstrate the mechanism of substrate capture, and rationalize the role of each component in the utilisome.
History
DepositionApr 21, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 21, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Sucrose-6-phosphate hydrolase
B: Sucrose-6-phosphate hydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)123,08419
Polymers118,5982
Non-polymers4,48617
Water905
1
A: Sucrose-6-phosphate hydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)61,87510
Polymers59,2991
Non-polymers2,5769
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Sucrose-6-phosphate hydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)61,2089
Polymers59,2991
Non-polymers1,9108
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)174.720, 174.720, 215.590
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number98
Space group name H-MI4122
Components on special symmetry positions
IDModelComponents
11B-701-

HOH

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21A

NCS domain segments:
Dom-IDComponent-IDEns-IDAuth asym-IDAuth seq-ID
111A17 - 503
211A17 - 503

NCS ensembles : (Details: Local NCS retraints between domains: 1 2)

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Sucrose-6-phosphate hydrolase


Mass: 59298.934 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacteroides thetaiotaomicron (bacteria)
Gene: scrB, BSIG_4326, Btheta7330_01254 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0P0F2Q3, beta-fructofuranosidase

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Sugars , 3 types, 5 molecules

#2: Polysaccharide beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D- ...beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose


Type: oligosaccharide / Mass: 828.719 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DFrufb2-6DFrufb2-6DFrufb2-6DFrufb2-6DFrufb2-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,5,4/[ha122h-2b_2-5]/1-1-1-1-1/a6-b2_b6-c2_c6-d2_d6-e2WURCSPDB2Glycan 1.1.0
[][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{}}}}}LINUCSPDB-CARE
#3: Polysaccharide beta-D-fructofuranose-(2-6)-beta-D-fructofuranose


Type: oligosaccharide / Mass: 342.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DFrufb2-6DFrufb2-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[ha122h-2b_2-5]/1-1/a6-b2WURCSPDB2Glycan 1.1.0
[][b-D-Fruf]{[(6+2)][b-D-Fruf]{}}LINUCSPDB-CARE
#4: Polysaccharide beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose


Type: oligosaccharide / Mass: 504.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DFrufb2-6DFrufb2-6DFrufb2-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,3,2/[ha122h-2b_2-5]/1-1-1/a6-b2_b6-c2WURCSPDB2Glycan 1.1.0
[][<C6O4>]{[(1+2)][b-D-Fruf]{[(6+2)][b-D-Fruf]{}}}LINUCSPDB-CARE

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Non-polymers , 2 types, 17 molecules

#5: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: SO4
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.47 Å3/Da / Density % sol: 64.54 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / Details: 1.5 M lithium sulfate, 100 mM Hepes pH 7.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I02 / Wavelength: 0.9763 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Sep 27, 2009
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 2.65→67.87 Å / Num. obs: 48504 / % possible obs: 99.9 % / Redundancy: 16.3 % / CC1/2: 0.997 / Net I/σ(I): 12.1
Reflection shellResolution: 2.65→2.74 Å / Redundancy: 15.2 % / Num. unique obs: 4407 / CC1/2: 0.708 / Rpim(I) all: 0.911

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Processing

Software
NameVersionClassification
REFMAC5.8.0352refinement
REFMAC5.8.0352refinement
xia2data reduction
XDSdata reduction
Aimlessdata scaling
CRANK2phasing
Cootmodel building
MolProbitymodel building
RefinementMethod to determine structure: SAD / Resolution: 2.65→67.87 Å / Cor.coef. Fo:Fc: 0.956 / Cor.coef. Fo:Fc free: 0.928 / WRfactor Rfree: 0.206 / WRfactor Rwork: 0.159 / SU B: 12.454 / SU ML: 0.24 / Average fsc free: 0.9434 / Average fsc work: 0.9607 / Cross valid method: FREE R-VALUE / ESU R: 0.396 / ESU R Free: 0.278
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.246 2381 4.911 %
Rwork0.1914 46103 -
all0.194 --
obs-48484 99.916 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 59.916 Å2
Baniso -1Baniso -2Baniso -3
1-3.516 Å20 Å2-0 Å2
2--3.516 Å20 Å2
3----7.032 Å2
Refinement stepCycle: LAST / Resolution: 2.65→67.87 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7800 0 284 5 8089
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0128299
X-RAY DIFFRACTIONr_bond_other_d0.0010.0167033
X-RAY DIFFRACTIONr_angle_refined_deg1.9911.6911246
X-RAY DIFFRACTIONr_angle_other_deg0.5881.60316425
X-RAY DIFFRACTIONr_dihedral_angle_1_deg8.2025972
X-RAY DIFFRACTIONr_dihedral_angle_2_deg9.792538
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.922101278
X-RAY DIFFRACTIONr_dihedral_angle_6_deg15.41310410
X-RAY DIFFRACTIONr_chiral_restr0.0780.21188
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.029254
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021746
X-RAY DIFFRACTIONr_nbd_refined0.1950.21366
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1980.26571
X-RAY DIFFRACTIONr_nbtor_refined0.1850.23907
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0930.24362
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1710.2181
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.0270.22
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.3490.255
X-RAY DIFFRACTIONr_nbd_other0.1470.288
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.170.212
X-RAY DIFFRACTIONr_mcbond_it5.775.9323894
X-RAY DIFFRACTIONr_mcbond_other5.7695.9323894
X-RAY DIFFRACTIONr_mcangle_it7.8288.884864
X-RAY DIFFRACTIONr_mcangle_other7.8288.884865
X-RAY DIFFRACTIONr_scbond_it7.1236.3924405
X-RAY DIFFRACTIONr_scbond_other6.8556.3464358
X-RAY DIFFRACTIONr_scangle_it9.6649.4066382
X-RAY DIFFRACTIONr_scangle_other9.2399.3386311
X-RAY DIFFRACTIONr_lrange_it10.9269.8888766
X-RAY DIFFRACTIONr_lrange_other10.9269.8948767
X-RAY DIFFRACTIONr_ncsr_local_group_10.0870.0516081
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)Weight position
11AX-RAY DIFFRACTIONLocal ncs0.087390.05009
12AX-RAY DIFFRACTIONLocal ncs0.087390.05009
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc freeFsc work% reflection obs (%)WRfactor Rwork
2.65-2.7190.4021940.33933170.34235270.870.88799.54640.325
2.719-2.7930.3291550.31632910.31634500.9070.92299.88410.29
2.793-2.8740.3321620.27232000.27533670.9210.94399.85150.244
2.874-2.9620.2971450.27230930.27332390.9260.94499.96910.24
2.962-3.0590.321610.25630160.25931780.9320.95399.96850.216
3.059-3.1660.31810.2428740.24430560.9380.95899.96730.203
3.166-3.2860.271550.22528040.22729630.9490.96599.8650.188
3.286-3.4190.3071530.21127140.21628670.9340.9691000.179
3.419-3.5710.2411320.17826140.18127470.9610.97999.96360.152
3.571-3.7450.2471060.17524990.17726050.960.981000.149
3.745-3.9470.2231260.17423720.17624980.9670.9811000.149
3.947-4.1850.2131130.1522610.15323740.9760.9861000.125
4.185-4.4730.211170.13221210.13622380.9770.9891000.114
4.473-4.830.1551090.12119880.12320970.9870.9911000.106
4.83-5.2880.187860.13918520.14119380.980.9881000.122
5.288-5.9080.22800.16416660.16617460.9780.9841000.141
5.908-6.8130.245740.17314930.17615670.9620.9811000.152
6.813-8.3230.244510.212970.20213480.9640.9731000.178
8.323-11.6830.21510.18210160.18410670.9720.981000.178
11.683-67.870.298300.2776150.2776480.9450.95399.5370.279

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