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- PDB-9d0z: X-ray crystal structure of H157Q variant Thermothelomyces thermop... -

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Basic information

Entry
Database: PDB / ID: 9d0z
TitleX-ray crystal structure of H157Q variant Thermothelomyces thermophilus polysaccharide monooxygenase 9E
ComponentsGlycoside hydrolase family 61 protein
KeywordsOXIDOREDUCTASE / polysaccharide monooxygenase
Function / homology
Function and homology information


lytic cellulose monooxygenase (C4-dehydrogenating) / cellulose catabolic process / monooxygenase activity / hydrolase activity / extracellular region / metal ion binding
Similarity search - Function
Auxiliary Activity family 9 / : / Auxiliary Activity family 9 (formerly GH61)
Similarity search - Domain/homology
COPPER (II) ION / OXYGEN MOLECULE / lytic cellulose monooxygenase (C4-dehydrogenating)
Similarity search - Component
Biological speciesThermothelomyces thermophilus (fungus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.8 Å
AuthorsThomas, W.C. / Batka, A.E. / Marletta, M.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32-GM149060 United States
CitationJournal: Biochemistry / Year: 2024
Title: Second-Sphere Histidine Catalytic Function in a Fungal Polysaccharide Monooxygenase.
Authors: Batka, A.E. / Thomas, W.C. / Tudorica, D.A. / Sayler, R.I. / Marletta, M.A.
History
DepositionAug 7, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Glycoside hydrolase family 61 protein
B: Glycoside hydrolase family 61 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,01510
Polymers48,5762
Non-polymers4398
Water7,098394
1
A: Glycoside hydrolase family 61 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,5075
Polymers24,2881
Non-polymers2204
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Glycoside hydrolase family 61 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,5075
Polymers24,2881
Non-polymers2204
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)102.410, 122.473, 91.478
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Space group name HallC2c2
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y,-z+1/2
#4: -x,-y,z+1/2
#5: x+1/2,y+1/2,z
#6: x+1/2,-y+1/2,-z
#7: -x+1/2,y+1/2,-z+1/2
#8: -x+1/2,-y+1/2,z+1/2

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Components

#1: Protein Glycoside hydrolase family 61 protein / Polysaccharide monooxygenase


Mass: 24287.814 Da / Num. of mol.: 2 / Mutation: H157Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermothelomyces thermophilus (fungus) / Gene: MYCTH_79765 / Plasmid: pPICZa / Production host: Komagataella pastoris (fungus) / Strain (production host): SMD 1163 / References: UniProt: G2Q7A5
#2: Chemical ChemComp-CU / COPPER (II) ION


Mass: 63.546 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cu
#3: Chemical ChemComp-OXY / OXYGEN MOLECULE


Mass: 31.999 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: O2
#4: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H6O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 394 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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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: 2.96 Å3/Da / Density % sol: 58.42 % / Description: 100-200 um, plate-like
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: The crystal was grown by hanging drop in conditions optimized from Hampton Index HT G8 (0.2 M Ammonium acetate, 0.1 M HEPES pH 7.5, 25 % w/v polyethylene glycol 3,350). H157Q Mt PMO9E was ...Details: The crystal was grown by hanging drop in conditions optimized from Hampton Index HT G8 (0.2 M Ammonium acetate, 0.1 M HEPES pH 7.5, 25 % w/v polyethylene glycol 3,350). H157Q Mt PMO9E was first reconstituted with excess copper and buffer exchanged to buffer with 50 mM MOPS and 50 mM HEPES pH 6. 1 ul 30 mg/mL H157Q Mt PMO9E was then mixed with 1 uL with 0.2 M Ammonium acetate, 0.1 M HEPES pH 7.5, and 26 % PEG 3,350 in a hanging drop. Crystals grew within 4 days. The crystal was looped with a 0.1-0.2 mm loop, then cryoprotected for 10 seconds in well solution mixed with cryoprotectants to a final concentration of 8% w/v sucrose, 2% w/v glucose, 8% v/v glycerol, and 8% ethylene glycol
PH range: 7-7.5 / Temp details: Near room temperature

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 1.000046 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Dec 15, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.000046 Å / Relative weight: 1
ReflectionResolution: 1.8→45.74 Å / Num. obs: 53154 / % possible obs: 99.5 % / Redundancy: 3.6 % / Biso Wilson estimate: 18.67 Å2 / CC1/2: 0.985 / Rmerge(I) obs: 0.135 / Rpim(I) all: 0.08 / Rrim(I) all: 0.157 / Χ2: 1.06 / Net I/σ(I): 6
Reflection shellResolution: 1.8→1.84 Å / Redundancy: 3.8 % / Rmerge(I) obs: 0.537 / Mean I/σ(I) obs: 2 / Num. unique obs: 3123 / CC1/2: 0.811 / Rpim(I) all: 0.377 / Rrim(I) all: 0.627 / Χ2: 0.88 / % possible all: 99.7

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Processing

Software
NameVersionClassification
PHENIX1.21.1_5286refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.8→44.68 Å / SU ML: 0.1611 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 23.5048
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2363 2016 3.88 %
Rwork0.2115 49985 -
obs0.2125 52001 97.21 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 23.17 Å2
Refinement stepCycle: LAST / Resolution: 1.8→44.68 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3420 0 22 394 3836
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00653549
X-RAY DIFFRACTIONf_angle_d0.84844845
X-RAY DIFFRACTIONf_chiral_restr0.0554516
X-RAY DIFFRACTIONf_plane_restr0.0066655
X-RAY DIFFRACTIONf_dihedral_angle_d13.50021251
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.850.28341460.2533594X-RAY DIFFRACTION99.57
1.85-1.890.43171130.41042913X-RAY DIFFRACTION79.76
1.89-1.950.37571450.35973618X-RAY DIFFRACTION99.63
1.95-2.010.32071460.29463560X-RAY DIFFRACTION98.15
2.01-2.090.25431440.20713596X-RAY DIFFRACTION99.36
2.09-2.170.22281490.21723666X-RAY DIFFRACTION99.76
2.17-2.270.42481420.37363521X-RAY DIFFRACTION96.65
2.27-2.390.30721420.26133499X-RAY DIFFRACTION95.79
2.39-2.540.23151470.1973649X-RAY DIFFRACTION99.79
2.54-2.730.20621490.18253655X-RAY DIFFRACTION99.53
2.73-3.010.19951470.19453624X-RAY DIFFRACTION98.59
3.01-3.440.21391490.18313700X-RAY DIFFRACTION99.72
3.44-4.340.19211460.16943641X-RAY DIFFRACTION97.53
4.34-44.680.15821510.14083749X-RAY DIFFRACTION97.06

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