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- PDB-5oa5: CELLOBIOHYDROLASE I (CEL7A) FROM HYPOCREA JECORINA WITH IMPROVED ... -

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Basic information

Entry
Database: PDB / ID: 5oa5
TitleCELLOBIOHYDROLASE I (CEL7A) FROM HYPOCREA JECORINA WITH IMPROVED THERMAL STABILITY
ComponentsExoglucanase 1Cellulose 1,4-beta-cellobiosidase
KeywordsHYDROLASE / CELLULASE / PROTEIN ENGINEERING
Function / homology
Function and homology information


cellulose 1,4-beta-cellobiosidase (non-reducing end) / cellulose 1,4-beta-cellobiosidase activity / cellulose binding / cellulose catabolic process / extracellular region
Similarity search - Function
1,4-Beta-D-Glucan Cellobiohydrolase I; Chain A / Glycoside hydrolase, family 7, domain / Glycoside hydrolase, family 7 / Glycoside hydrolase family 7, catalytic domain superfamily / Glycosyl hydrolase family 7 / CBM1 (carbohydrate binding type-1) domain signature. / Cellulose-binding domain, fungal / Cellulose-binding domain superfamily / Fungal cellulose binding domain / CBM1 (carbohydrate binding type-1) domain profile. ...1,4-Beta-D-Glucan Cellobiohydrolase I; Chain A / Glycoside hydrolase, family 7, domain / Glycoside hydrolase, family 7 / Glycoside hydrolase family 7, catalytic domain superfamily / Glycosyl hydrolase family 7 / CBM1 (carbohydrate binding type-1) domain signature. / Cellulose-binding domain, fungal / Cellulose-binding domain superfamily / Fungal cellulose binding domain / CBM1 (carbohydrate binding type-1) domain profile. / Fungal-type cellulose-binding domain / Distorted Sandwich / Concanavalin A-like lectin/glucanase domain superfamily / Mainly Beta
Similarity search - Domain/homology
Biological speciesHypocrea jecorina (fungus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsGoedegebuur, F. / Hansson, H. / Karkehabadi, S. / Mikkelsen, N. / Stahlberg, J. / Sandgren, M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Science Foundation1552355 United States
National Science Foundation (NSF, United States)ACI-1053575 (under allocation MCB090159) United States
CitationJournal: J. Biol. Chem. / Year: 2017
Title: Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution.
Authors: Goedegebuur, F. / Dankmeyer, L. / Gualfetti, P. / Karkehabadi, S. / Hansson, H. / Jana, S. / Huynh, V. / Kelemen, B.R. / Kruithof, P. / Larenas, E.A. / Teunissen, P.J.M. / Stahlberg, J. / ...Authors: Goedegebuur, F. / Dankmeyer, L. / Gualfetti, P. / Karkehabadi, S. / Hansson, H. / Jana, S. / Huynh, V. / Kelemen, B.R. / Kruithof, P. / Larenas, E.A. / Teunissen, P.J.M. / Stahlberg, J. / Payne, C.M. / Mitchinson, C. / Sandgren, M.
History
DepositionJun 20, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 6, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2017Group: Database references / Category: citation
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Nov 1, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 2.0Mar 11, 2020Group: Data collection / Polymer sequence
Category: chem_comp / diffrn_radiation_wavelength / entity_poly
Item: _chem_comp.type / _entity_poly.pdbx_seq_one_letter_code_can
Revision 2.1Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _entity.pdbx_description ..._chem_comp.name / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.2Mar 30, 2022Group: Author supporting evidence / Database references / Structure summary
Category: chem_comp / database_2 / pdbx_audit_support
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_audit_support.funding_organization
Revision 2.3Mar 8, 2023Group: Advisory / Category: pdbx_database_PDB_obs_spr
Revision 2.4Feb 7, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Exoglucanase 1
B: Exoglucanase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)93,9449
Polymers92,5242
Non-polymers1,4197
Water9,836546
1
A: Exoglucanase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,9264
Polymers46,2621
Non-polymers6643
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Exoglucanase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)47,0185
Polymers46,2621
Non-polymers7564
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)102.940, 92.100, 102.200
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212

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Components

#1: Protein Exoglucanase 1 / Cellulose 1,4-beta-cellobiosidase / 1 / 4-beta-cellobiohydrolase / Exocellobiohydrolase I / CBHI / Exoglucanase I


Mass: 46262.238 Da / Num. of mol.: 2 / Fragment: CATALYTIC DOMAIN, RESIDUES 18-451
Mutation: S8P, T41I, N49S, A68T, N89D, S92T, S113N, S196T, P227L, D249K, T255P, S278P, E295K, T296P, T332Y, V304D, S411F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hypocrea jecorina (fungus) / Gene: cbh1 / Plasmid: pRAXDES / Production host: Aspergillus awamori (mold) / Variant (production host): AP4
References: UniProt: P62694, cellulose 1,4-beta-cellobiosidase (non-reducing end)
#2: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 546 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsENGINEERED ENZYME WITH 17 INTRODUCED MUTATIONS. NATURALLY OCCURRING PYROGLUTAMATE AT THE N-TERMINUS.

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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: 2.48 Å3/Da / Density % sol: 50.45 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6
Details: 25.5% POLYETHYLENE GLYCOL (PEG) 4000, 0.17 M AMSO4 AND 15% GLYCEROL

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-2 / Wavelength: 0.8726 Å
DetectorType: MARRESEARCH / Detector: CCD / Date: Oct 5, 2012 / Details: PT COATED MIRRORS
RadiationMonochromator: HORIZONTALLY DIFFRACTING SI (111) MONOCHROMATOR
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.8726 Å / Relative weight: 1
ReflectionResolution: 1.99→41.16 Å / Num. obs: 56990 / % possible obs: 99.6 % / Observed criterion σ(I): 2 / Redundancy: 3.71 % / Rmerge(I) obs: 0.13 / Net I/σ(I): 4.9
Reflection shellResolution: 2.1→2.21 Å / Redundancy: 3.73 % / Rmerge(I) obs: 0.41 / Mean I/σ(I) obs: 1.86 / % possible all: 99.8

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Processing

Software
NameVersionClassification
XDSdata reduction
SCALAdata scaling
PHENIXphasing
REFMAC5.7.0032refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 2V3I

2v3i


Resolution: 2.1→41.16 Å / Cor.coef. Fo:Fc: 0.951 / Cor.coef. Fo:Fc free: 0.926 / SU B: 4.952 / SU ML: 0.13 / Cross valid method: THROUGHOUT / ESU R: 0.226 / ESU R Free: 0.19 / Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflectionSelection details
Rfree0.24381 2882 5.1 %RANDOM
Rwork0.19929 ---
obs0.20154 53986 99.11 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 17.9 Å2
Baniso -1Baniso -2Baniso -3
1--0.96 Å20 Å20 Å2
2--2.48 Å20 Å2
3----1.52 Å2
Refinement stepCycle: LAST / Resolution: 2.1→41.16 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6480 0 90 546 7116
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.026777
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.3091.9529261
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.4245872
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.11525.676296
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.23515958
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.9731514
X-RAY DIFFRACTIONr_chiral_restr0.0880.2996
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.0215318
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.9491.6733479
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it1.6662.5044348
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it1.0981.7643298
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined4.35314.5710782
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.1→2.155 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.303 183 -
Rwork0.257 3978 -
obs--99.64 %

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