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- PDB-3b7m: Crystal structure of a meso-active thermo-stable cellulase (MT Ce... -

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Basic information

Entry
Database: PDB / ID: 3b7m
TitleCrystal structure of a meso-active thermo-stable cellulase (MT Cel12A) derived by making non-contiguous mutations in the active surface of the Cel12A cellulase of Rhodothermus marinus
ComponentsCELLULASE
KeywordsHYDROLASE / cellulase / endoglucanase / crystal / beta-jelly / beta-sheet
Function / homology
Function and homology information


cellulase / cellulase activity / polysaccharide catabolic process
Similarity search - Function
Glycoside hydrolase family 12 / Glycosyl hydrolase family 12 / Glycoside hydrolase family 11/12, catalytic domain / Glycoside hydrolase family 11/12 / Prokaryotic membrane lipoprotein lipid attachment site profile. / Concanavalin A-like lectin/glucanase domain superfamily / Jelly Rolls / Sandwich / Mainly Beta
Similarity search - Domain/homology
Biological speciesRhodothermus marinus (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsKarthikeyan, S. / Guptasarma, P.
CitationJournal: To be Published
Title: Transplantation of the active surface of a mesophile cellulase onto the structural scaffold of a homologous thermophile cellulase through engineering of a surface beta sheet
Authors: Kapoor, D. / Kumar, V. / Chandrayan, S.K. / Ahmed, S. / Sharma, S. / Datt, M. / Singh, B. / Karthikeyan, S. / Guptasarma, P.
History
DepositionOct 31, 2007Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Nov 27, 2007Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Oct 25, 2017Group: Refinement description / Category: software / Item: _software.name
Revision 1.3Nov 1, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Remark 999SEQUENCE The sequence is based on PDB_ID 1H0B (gi:33356963). THE FOLLOWING MUTATIONS WERE MADE TO ...SEQUENCE The sequence is based on PDB_ID 1H0B (gi:33356963). THE FOLLOWING MUTATIONS WERE MADE TO TRANSFORM THE RHODOTHERMUS MARINUS CEL12A (PDB_ID 1H0B) TO MESO -ACTIVE THERMO-STABLE CEL12A, NAMED MT CEL12A (PDB _ID 3B7M). UNLESS OTHERWISE MENTIONED, THE MUTATIONS REPLACE RESIDUES CONSTITUTING THE ACTIVE SURFACE (CELLULOSE-BINDING AND CATALYTIC SITE RESIDUES)OF 1H0B (RHODOTHERMUS MARINUS CEL12A) BY RESIDUES OCCURRING AT STRUCTURALLY EQUIVALENT POSITIONS ON THE ACTIVE SURFACE OF 1OA2 (TRICHODERMA REESEI CEL12A), TO PRODUCE MT CEL12A. 1H0B SEQUENCE 3B7M SEQUENCE A1 MET A1 SER A8 ARG A8 GLN A11 ALA A11 THR A13 ASP A13 THR A20 ARG A20 THR A22 ILE A22 SER A49 ASP A49 GLU (SEQUENCE ACCORDING TO OUR CLONE) A61 ALA A61 ASN A63 TYR A63 GLN A65 GLY A65 ALA LOOP A66 CYS TO A77 LEU LOOP A66 ILE TO A68 GLN A108 TRP A99 PHE A110 SER A101 ALA A111 PRO A102 ALA A112 VAL A103 ASN A113 THR A104 PRO LOOP A115 SER TO A118 GLY LOOP A106 HIS TO A108 THR A122 GLY A112 ASP A131 TRP A121 LYS A134 GLY A124 ASP A138 GLY A128 ILE A159 TRP A149 ASN A160 ASP A150 GLY BETWEEN A160 ASP AND A161 TRP A151 ALA IS INSERTED A161 TRP A152 MET A163 TYR A154 VAL A165 ALA A156 SER A167 ARG A158 VAL A176 SER A167 THR (ADOPTED FROM APPL MICROBIOL BIOTECHNOL. V55, P578) A200 HIS A191 LEU A201 ALA A192 SER A203 GLU A194 GLN A209 TRP A200 PHE A210 GLU A201 THR

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CELLULASE
B: CELLULASE
C: CELLULASE
D: CELLULASE


Theoretical massNumber of molelcules
Total (without water)95,0854
Polymers95,0854
Non-polymers00
Water5,278293
1
A: CELLULASE


Theoretical massNumber of molelcules
Total (without water)23,7711
Polymers23,7711
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: CELLULASE


Theoretical massNumber of molelcules
Total (without water)23,7711
Polymers23,7711
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: CELLULASE


Theoretical massNumber of molelcules
Total (without water)23,7711
Polymers23,7711
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: CELLULASE


Theoretical massNumber of molelcules
Total (without water)23,7711
Polymers23,7711
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
5


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7040 Å2
MethodPISA
Unit cell
Length a, b, c (Å)60.398, 111.874, 133.707
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein
CELLULASE /


Mass: 23771.338 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rhodothermus marinus (bacteria) / Gene: celA / Plasmid: pQE30 / Production host: Escherichia coli (E. coli) / Strain (production host): XL1 Blue / References: UniProt: O33897*PLUS, cellulase
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 293 / Source method: isolated from a natural source / Formula: H2O

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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: 2.38 Å3/Da / Density % sol: 48.22 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.5
Details: 0.2M Sodium dihydrogen phosphate monohydrate, 20% W/V PEG 3350, pH 4.5, VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 298 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU ULTRAX 18 / Wavelength: 1.5418 Å
DetectorType: MAR scanner 345 mm plate / Detector: IMAGE PLATE / Date: Jul 11, 2007 / Details: Mirrors
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.1→50 Å / Num. all: 52603 / Num. obs: 52326 / % possible obs: 97.5 % / Observed criterion σ(F): 3 / Observed criterion σ(I): 3 / Redundancy: 3.53 % / Biso Wilson estimate: 27.9 Å2 / Rsym value: 0.062 / Net I/σ(I): 12.5
Reflection shellResolution: 2.1→2.18 Å / Redundancy: 3.38 % / Mean I/σ(I) obs: 3.1 / Num. unique all: 4888 / Rsym value: 0.2867 / % possible all: 99.2

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Processing

Software
NameVersionClassification
REFMAC5.2.0019refinement
MAR345CONTROLdata collection
AUTOMARdata reduction
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1H0B

1h0b


Resolution: 2.1→41.42 Å / Cor.coef. Fo:Fc: 0.954 / Cor.coef. Fo:Fc free: 0.924 / SU B: 5.122 / SU ML: 0.136 / Isotropic thermal model: Isotropic / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.231 / ESU R Free: 0.194 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.23819 2666 5.1 %RANDOM
Rwork0.18596 ---
all0.205 ---
obs0.18861 49610 97.49 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 27.563 Å2
Baniso -1Baniso -2Baniso -3
1--0.27 Å20 Å20 Å2
2--0.68 Å20 Å2
3----0.41 Å2
Refinement stepCycle: LAST / Resolution: 2.1→41.42 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6769 0 0 293 7062
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.0226950
X-RAY DIFFRACTIONr_angle_refined_deg1.1291.9159551
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.1165884
X-RAY DIFFRACTIONr_dihedral_angle_2_deg38.02323.909330
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.483151004
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.7051548
X-RAY DIFFRACTIONr_chiral_restr0.0770.21086
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.025435
X-RAY DIFFRACTIONr_nbd_refined0.2010.22780
X-RAY DIFFRACTIONr_nbtor_refined0.3020.24783
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1190.2307
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.210.222
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1190.29
X-RAY DIFFRACTIONr_mcbond_it0.71.54423
X-RAY DIFFRACTIONr_mcangle_it1.19127004
X-RAY DIFFRACTIONr_scbond_it1.61332990
X-RAY DIFFRACTIONr_scangle_it2.7244.52535
LS refinement shellResolution: 2.101→2.155 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.296 175 -
Rwork0.244 3717 -
obs--99.13 %

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