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- PDB-8h2w: Cellodextrin phosphorylase from Clostridium thermocellum mutant -... -

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Basic information

Entry
Database: PDB / ID: 8h2w
TitleCellodextrin phosphorylase from Clostridium thermocellum mutant - all cysteine residues were substituted with serines
ComponentsCellodextrin phosphorylase
KeywordsCARBOHYDRATE / Cellulose / Cellodextrin / Synthesis / Phosphorolysis
Function / homology
Function and homology information


transferase activity / carbohydrate binding / carbohydrate metabolic process
Similarity search - Function
Glycosyl hydrolase 94 / Glycosyltransferase family 36 / Glycosyl hydrolase 36, catalytic domain / Glycosyl hydrolase 36 superfamily, catalytic domain / Glycoside hydrolase family 65, N-terminal domain superfamily / Galactose mutarotase-like domain superfamily / Six-hairpin glycosidase-like superfamily / Six-hairpin glycosidase superfamily
Similarity search - Domain/homology
beta-cellotriose / ACETATE ION / DI(HYDROXYETHYL)ETHER / Cellodextrin phosphorylase
Similarity search - Component
Biological speciesAcetivibrio thermocellus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.21 Å
AuthorsKuga, T. / Sunagawa, N. / Igarashi, K.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)22J12566 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)18H05494 Japan
CitationJournal: To Be Published
Title: 11 cysteine-to-serine mutations improve stability of cellodextrin phosphorylase from Clostridium thermocellum
Authors: Kuga, T. / Sunagawa, N. / Igarashi, K.
History
DepositionOct 7, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 11, 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: Cellodextrin phosphorylase
B: Cellodextrin phosphorylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)230,33850
Polymers225,4722
Non-polymers4,86648
Water37,8132099
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area23270 Å2
ΔGint-72 kcal/mol
Surface area66970 Å2
MethodPISA
Unit cell
Length a, b, c (Å)83.208, 88.778, 88.761
Angle α, β, γ (deg.)98.580, 110.550, 110.560
Int Tables number1
Space group name H-MP1
Space group name HallP1
Symmetry operation#1: x,y,z

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Components

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Protein / Sugars , 2 types, 4 molecules AB

#1: Protein Cellodextrin phosphorylase /


Mass: 112736.172 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acetivibrio thermocellus (bacteria) / Gene: cdp-ym4 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q93HT8
#2: Polysaccharide beta-D-glucopyranose-(1-4)-beta-D-glucopyranose-(1-4)-beta-D-glucopyranose


Type: oligosaccharide, Oligosaccharide / Class: Metabolism / Mass: 504.438 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: oligosaccharide / References: beta-cellotriose
DescriptorTypeProgram
DGlcpb1-4DGlcpb1-4DGlcpb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,3,2/[a2122h-1b_1-5]/1-1-1/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][b-D-Glcp]{[(4+1)][b-D-Glcp]{[(4+1)][b-D-Glcp]{}}}LINUCSPDB-CARE

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Non-polymers , 7 types, 2145 molecules

#3: Chemical
ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C2H3O2
#4: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER / Tris


Mass: 122.143 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#5: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-PEG / DI(HYDROXYETHYL)ETHER / Diethylene glycol


Mass: 106.120 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C4H10O3
#7: Chemical...
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 23 / Source method: obtained synthetically / Formula: C3H8O3
#8: Chemical
ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Formula: Cl
#9: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 2099 / 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: 2.45 Å3/Da / Density % sol: 49.78 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 5
Details: 10 mg/ml protein solution was mixed 1:1 with an optimized solution consisting of 100 mM pH 5.0 sodium acetate, 12% PEG4000, and 5% glycerol with streak seeding

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Data collection

DiffractionMean temperature: 95 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: BL-5A / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Dec 2, 2021
RadiationMonochromator: M / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.21→43.82 Å / Num. obs: 611147 / % possible obs: 94.35 % / Redundancy: 7 % / Biso Wilson estimate: 14.5 Å2 / CC1/2: 1 / CC star: 1 / Rmerge(I) obs: 0.04475 / Rpim(I) all: 0.01816 / Net I/σ(I): 23.95
Reflection shellResolution: 1.21→1.253 Å / Redundancy: 7.2 % / Rmerge(I) obs: 1.682 / Mean I/σ(I) obs: 1.39 / Num. unique obs: 58904 / CC1/2: 0.736 / CC star: 0.921 / Rpim(I) all: 0.6696 / % possible all: 89.89

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Processing

Software
NameVersionClassification
PHENIX1.20_4459refinement
XDSdata reduction
Aimlessdata scaling
PHENIX1.20_4459phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.21→43.82 Å / SU ML: 0.1627 / Cross valid method: FREE R-VALUE / σ(F): 1.96 / Phase error: 18.925
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.163 30525 5.01 %
Rwork0.1428 578920 -
obs0.1438 609445 94.35 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 21.4 Å2
Refinement stepCycle: LAST / Resolution: 1.21→43.82 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms15746 0 312 2099 18157
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.008217539
X-RAY DIFFRACTIONf_angle_d1.087723842
X-RAY DIFFRACTIONf_chiral_restr0.08422564
X-RAY DIFFRACTIONf_plane_restr0.01053138
X-RAY DIFFRACTIONf_dihedral_angle_d8.40552473
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.21-1.220.430710070.426918020X-RAY DIFFRACTION88.37
1.22-1.240.39459930.38618362X-RAY DIFFRACTION90.04
1.24-1.250.33489850.309318648X-RAY DIFFRACTION91.27
1.25-1.270.298710640.275818616X-RAY DIFFRACTION91.36
1.27-1.290.2989220.26118821X-RAY DIFFRACTION91.53
1.29-1.30.33519970.313418577X-RAY DIFFRACTION90.83
1.3-1.320.27669900.253518922X-RAY DIFFRACTION92.36
1.32-1.340.3310200.317618903X-RAY DIFFRACTION92.74
1.34-1.360.295510330.264119006X-RAY DIFFRACTION92.9
1.36-1.390.24769500.209719116X-RAY DIFFRACTION93.14
1.39-1.410.208110090.167619114X-RAY DIFFRACTION93.35
1.41-1.430.188510080.152519036X-RAY DIFFRACTION93.52
1.43-1.460.171110870.138119091X-RAY DIFFRACTION93.69
1.46-1.490.17129260.133719239X-RAY DIFFRACTION93.76
1.49-1.520.161710340.125919323X-RAY DIFFRACTION94.35
1.52-1.560.15749930.130219392X-RAY DIFFRACTION94.67
1.56-1.60.15599930.13119370X-RAY DIFFRACTION94.83
1.6-1.640.157910290.12719486X-RAY DIFFRACTION95.11
1.64-1.690.14629880.116719524X-RAY DIFFRACTION95.4
1.69-1.750.156810580.122719610X-RAY DIFFRACTION95.64
1.75-1.810.14410450.119419593X-RAY DIFFRACTION95.94
1.81-1.880.151610650.122919683X-RAY DIFFRACTION96.15
1.88-1.970.16229530.142119516X-RAY DIFFRACTION95.32
1.97-2.070.142210280.125719813X-RAY DIFFRACTION96.79
2.07-2.20.135610880.122919767X-RAY DIFFRACTION97.09
2.2-2.370.146810790.130419804X-RAY DIFFRACTION96.76
2.37-2.610.140810730.124119993X-RAY DIFFRACTION97.73
2.61-2.980.14229940.129220153X-RAY DIFFRACTION98.14
2.98-3.760.147110310.128920155X-RAY DIFFRACTION98.45
3.76-43.820.146310830.135220267X-RAY DIFFRACTION99.24

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