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

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Basic information

Entry
Database: PDB / ID: 7zns
TitleInactive D62N mutant of BT1760 Endo-acting levanase from Bacteroides thetaiotaomicron VPI-5482
ComponentsGlycoside hydrolase family 32
KeywordsHYDROLASE / Levan / fructan / endo levanase / gut microbiota
Function / homology
Function and homology information


beta-fructofuranosidase / hydrolase activity, hydrolyzing O-glycosyl compounds / carbohydrate metabolic process / metal ion binding
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
beta-fructofuranosidase
Similarity search - Component
Biological speciesBacteroides thetaiotaomicron VPI-5482 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.3 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
Revision 1.1Jun 19, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / struct_ncs_dom_lim
Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id ..._struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glycoside hydrolase family 32
B: Glycoside hydrolase family 32
hetero molecules


Theoretical massNumber of molelcules
Total (without water)122,47127
Polymers118,5982
Non-polymers3,87325
Water3,621201
1
A: Glycoside hydrolase family 32
hetero molecules


Theoretical massNumber of molelcules
Total (without water)61,80316
Polymers59,2991
Non-polymers2,50415
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Glycoside hydrolase family 32
hetero molecules


Theoretical massNumber of molelcules
Total (without water)60,66811
Polymers59,2991
Non-polymers1,36910
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)175.500, 175.500, 214.530
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-743-

HOH

21B-753-

HOH

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

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: THR / Beg label comp-ID: THR / End auth comp-ID: TYR / End label comp-ID: TYR / Auth asym-ID: A / Label asym-ID: A / Auth seq-ID: 17 - 503 / Label seq-ID: 37 - 523

Dom-ID
1
2

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

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Components

#1: Protein Glycoside hydrolase family 32


Mass: 59298.934 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacteroides thetaiotaomicron VPI-5482 (bacteria)
Gene: BT_1760 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8A6W6
#2: Polysaccharide beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose


Type: oligosaccharide / Mass: 504.438 Da / Num. of mol.: 3
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
[][b-D-Fruf]{[(6+2)][b-D-Fruf]{[(6+2)][a-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: Chemical...
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 21
Source method: isolated from a genetically manipulated source
Formula: SO4
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 201 / Source method: isolated from a natural source / Formula: H2O
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: 3.48 Å3/Da / Density % sol: 64.68 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop
Details: 200 mM ammonium sulfate, 100 mM mes pH 6.5 and 30% PEG 5000 MME

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.9763 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: May 10, 2010
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 2.3→73.71 Å / Num. obs: 73764 / % possible obs: 99.6 % / Redundancy: 6.9 % / CC1/2: 0.995 / Rmerge(I) obs: 0.16 / Rpim(I) all: 0.097 / Rrim(I) all: 0.188 / Net I/σ(I): 9.4
Reflection shellResolution: 2.3→2.35 Å / Redundancy: 4.3 % / Num. unique obs: 4314 / CC1/2: 0.549

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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: 2.3→73.708 Å / Cor.coef. Fo:Fc: 0.945 / Cor.coef. Fo:Fc free: 0.923 / WRfactor Rfree: 0.218 / WRfactor Rwork: 0.184 / SU B: 9.419 / SU ML: 0.2 / Average fsc free: 0.9355 / Average fsc work: 0.9487 / Cross valid method: FREE R-VALUE / ESU R: 0.238 / ESU R Free: 0.204
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.2537 3652 4.952 %
Rwork0.2157 70095 -
all0.218 --
obs-73747 99.557 %
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: 42.585 Å2
Baniso -1Baniso -2Baniso -3
1-2.169 Å2-0 Å20 Å2
2--2.169 Å2-0 Å2
3----4.338 Å2
Refinement stepCycle: LAST / Resolution: 2.3→73.708 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7800 0 230 201 8231
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0070.0128228
X-RAY DIFFRACTIONr_bond_other_d0.0010.0166953
X-RAY DIFFRACTIONr_angle_refined_deg1.5071.67411173
X-RAY DIFFRACTIONr_angle_other_deg0.4721.58716229
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.815972
X-RAY DIFFRACTIONr_dihedral_angle_2_deg9.653538
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.85101278
X-RAY DIFFRACTIONr_dihedral_angle_6_deg15.49710410
X-RAY DIFFRACTIONr_chiral_restr0.0770.21174
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.029254
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021746
X-RAY DIFFRACTIONr_nbd_refined0.1940.21365
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1970.26422
X-RAY DIFFRACTIONr_nbtor_refined0.1810.23842
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0890.24151
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.130.2318
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.3040.263
X-RAY DIFFRACTIONr_nbd_other0.1590.2104
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.0450.27
X-RAY DIFFRACTIONr_mcbond_it3.0234.3363894
X-RAY DIFFRACTIONr_mcbond_other3.0224.3363894
X-RAY DIFFRACTIONr_mcangle_it4.4956.494864
X-RAY DIFFRACTIONr_mcangle_other4.4966.494865
X-RAY DIFFRACTIONr_scbond_it3.7484.6684334
X-RAY DIFFRACTIONr_scbond_other3.4074.594251
X-RAY DIFFRACTIONr_scangle_it5.5076.8986309
X-RAY DIFFRACTIONr_scangle_other5.0266.7766184
X-RAY DIFFRACTIONr_lrange_it7.3650.3168838
X-RAY DIFFRACTIONr_lrange_other7.29350.3048821
X-RAY DIFFRACTIONr_ncsr_local_group_10.0820.0516579
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)Weight position
11AX-RAY DIFFRACTIONLocal ncs0.081550.0501
12AX-RAY DIFFRACTIONLocal ncs0.081550.0501
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
2.3-2.360.3752420.35649250.35754170.8550.85495.38490.333
2.36-2.4240.3622540.35349550.35452530.8620.88499.16240.328
2.424-2.4940.352730.33448670.33551450.9120.91299.90280.302
2.494-2.5710.3532510.28947480.29250000.9210.93999.980.255
2.571-2.6550.3292560.27845830.28148420.9260.94699.9380.237
2.655-2.7480.2812570.25744060.25946630.9420.9541000.22
2.748-2.8520.2542400.24143000.24145410.9570.96199.9780.205
2.852-2.9680.332380.25641090.2643470.9150.9541000.222
2.968-3.10.3031850.24640160.24842020.940.95999.97620.212
3.1-3.2510.2682090.22337880.22639980.950.96899.9750.198
3.251-3.4270.2491860.2136440.21238310.9650.97499.97390.191
3.427-3.6340.231610.18734590.18936210.9680.98199.97240.172
3.634-3.8840.2341790.17532370.17834160.9640.9821000.16
3.884-4.1940.1951530.15430330.15631860.9790.9861000.14
4.194-4.5930.1621370.13728010.13829380.9840.9881000.127
4.593-5.1330.1831250.14225660.14426910.9830.9891000.133
5.133-5.9220.2061060.17422540.17623600.9770.9831000.16
5.922-7.2420.314820.21919510.22220350.950.97199.90170.198
7.242-10.1920.206750.19515410.19516180.9720.97799.87640.191
10.192-73.7080.174430.2559120.2519670.9760.96398.7590.269

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