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- PDB-3cms: ENGINEERING ENZYME SUB-SITE SPECIFICITY: PREPARATION, KINETIC CHA... -

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Basic information

Entry
Database: PDB / ID: 3cms
TitleENGINEERING ENZYME SUB-SITE SPECIFICITY: PREPARATION, KINETIC CHARACTERIZATION AND X-RAY ANALYSIS AT 2.0-ANGSTROMS RESOLUTION OF VAL111PHE SITE-MUTATED CALF CHYMOSIN
ComponentsCHYMOSIN B
KeywordsHYDROLASE / ACID PROTEINASE
Function / homology
Function and homology information


chymosin / digestion / aspartic-type endopeptidase activity / proteolysis
Similarity search - Function
Pepsin catalytic domain / Aspartic peptidase, N-terminal / A1 Propeptide / Eukaryotic aspartyl protease / Aspartic peptidase A1 family / Peptidase family A1 domain / Peptidase family A1 domain profile. / Cathepsin D, subunit A; domain 1 / Acid Proteases / Aspartic peptidase, active site ...Pepsin catalytic domain / Aspartic peptidase, N-terminal / A1 Propeptide / Eukaryotic aspartyl protease / Aspartic peptidase A1 family / Peptidase family A1 domain / Peptidase family A1 domain profile. / Cathepsin D, subunit A; domain 1 / Acid Proteases / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain superfamily / Beta Barrel / Mainly Beta
Similarity search - Domain/homology
Biological speciesBos taurus (cattle)
MethodX-RAY DIFFRACTION / Resolution: 2 Å
AuthorsNewman, M. / Frazao, C. / Shearer, A. / Tickle, I.J. / Blundell, T.L.
Citation
Journal: Biochemistry / Year: 1990
Title: Engineering enzyme subsite specificity: preparation, kinetic characterization, and X-ray analysis at 2.0-A resolution of Val111Phe site-mutated calf chymosin.
Authors: Strop, P. / Sedlacek, J. / Stys, J. / Kaderabkova, Z. / Blaha, I. / Pavlickova, L. / Pohl, J. / Fabry, M. / Kostka, V. / Newman, M.
#1: Journal: J.Mol.Biol. / Year: 1991
Title: X-Ray Analyses of Aspartic Proteinases Iv: Structure and Refinement at 2.2 Angstroms Resolution of Bovine Chymosin
Authors: Newman, M. / Safro, M. / Frazao, C. / Kahn, G. / Zdanov, A. / Tickle, I.J. / Blundell, T.L. / Andreeva, N.
History
DepositionFeb 26, 1990Processing site: BNL
Revision 1.0Oct 15, 1992Provider: repository / Type: Initial release
Revision 1.1Mar 3, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Nov 29, 2017Group: Derived calculations / Other
Category: pdbx_database_status / struct_conf / struct_conf_type
Item: _pdbx_database_status.process_site

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CHYMOSIN B


Theoretical massNumber of molelcules
Total (without water)35,7211
Polymers35,7211
Non-polymers00
Water2,612145
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)80.210, 114.560, 72.430
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number23
Space group name H-MI222
Atom site foot note1: RESIDUE PRO 23 IS A CIS-PROLINE.
2: THESE RESIDUES ARE IN POORLY DEFINED REGIONS IN THE ELECTRON DENSITY MAP.
3: RESIDUES 72 TO 79 HAVE BEEN REFINED IN TWO ALTERNATE CONFORMATIONS. THESE RESIDUES ARE IN POORLY DEFINED REGIONS IN THE ELECTRON DENSITY MAP. CONFORMATION B HAS THE HIGHER RELATIVE OCCUPANCY (O.60) ...3: RESIDUES 72 TO 79 HAVE BEEN REFINED IN TWO ALTERNATE CONFORMATIONS. THESE RESIDUES ARE IN POORLY DEFINED REGIONS IN THE ELECTRON DENSITY MAP. CONFORMATION B HAS THE HIGHER RELATIVE OCCUPANCY (O.60) AND IS ASSOCIATED WITH SUPERIOR ELECTRON DENSITY. THUS IT APPEARS THAT CONFORMATION B IS SIGNIFICANTLY MORE HIGHLY POPULATED THAN CONFORMATION A. CONFORMATION A WITH OCCUPANCY OF 0.40 MUST BE REGARDED AS A TENTATIVE INTERPRETATION.

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Components

#1: Protein CHYMOSIN B


Mass: 35720.738 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / References: UniProt: P00794, chymosin
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 145 / Source method: isolated from a natural source / Formula: H2O
Compound detailsTHE SPECIFIC GENE MUTATION V111F IS SITUATED BETWEEN THE PRIMARY SPECIFICITY BINDING POCKET S1 AND ...THE SPECIFIC GENE MUTATION V111F IS SITUATED BETWEEN THE PRIMARY SPECIFICITY BINDING POCKET S1 AND THE SECONDARY SPECIFICITY POCKET S3, EFFECTING THE BINDING OF LARGE HYDROPHOBIC RESIDUES IN BOTH THESE POCKETS. THIS MUTATION IS RESPONSIBLE FOR THE REARRANGEMENT OF RESIDUES 72 TO 79 KNOWN AS THE ACTIVE SITE FLAP, A FLEXIBLE BETA-HAIRPIN TURN ABOVE THE ACTIVE SITE.

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION

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Sample preparation

CrystalDensity Matthews: 2.33 Å3/Da / Density % sol: 47.16 %
Crystal grow
*PLUS
pH: 6.5 / Method: microdialysis
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formula
110 mg/mlprotein1drop
250 mMphosphate1drop
32 M1reservoirNaCl

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Data collection

RadiationScattering type: x-ray
Radiation wavelengthRelative weight: 1
Reflection
*PLUS
Highest resolution: 2 Å / Num. obs: 22098 / % possible obs: 96.4 % / Observed criterion σ(I): 0.072 / Num. measured all: 149939 / Rmerge(I) obs: 3

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Processing

SoftwareName: RESTRAIN / Classification: refinement
RefinementResolution: 2→10 Å
Details: THE QUANTITY PRESENTED IN THE TEMPERATURE FACTOR FIELD IS U. RESIDUES 72 TO 79 HAVE BEEN REFINED IN TWO ALTERNATE CONFORMATIONS. TURN 5 WITH THE A CONFORMATION IS CLASSIFED TYPE II' 2:2 ...Details: THE QUANTITY PRESENTED IN THE TEMPERATURE FACTOR FIELD IS U. RESIDUES 72 TO 79 HAVE BEEN REFINED IN TWO ALTERNATE CONFORMATIONS. TURN 5 WITH THE A CONFORMATION IS CLASSIFED TYPE II' 2:2 DISTORTED. TURN 5 WITH THE B CONFORMATION IS UNCLASSIFIED 2:2. INVARIANT RESIDUE TYR 14 HAS BEEN BUILT INTO A CONFORMATION THAT DIFFERS FROM THE WILD-TYPE CHYMOSIN STRUCTURE. HOWEVER, THIS RESIDUE BELONGS TO THE POORLY DEFINED SURFACE REGION BETWEEN STRANDS AN AND BN, AND THUS THE ASSIGNMENT OF ITS POSITION MUST BE REGARDED AS TENTATIVE.
RfactorNum. reflection
obs0.195 21710
Refinement stepCycle: LAST / Resolution: 2→10 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2490 0 0 145 2635
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONp_bond_d0.022
X-RAY DIFFRACTIONp_angle_d0.05
X-RAY DIFFRACTIONp_angle_deg
X-RAY DIFFRACTIONp_planar_d
X-RAY DIFFRACTIONp_hb_or_metal_coord
X-RAY DIFFRACTIONp_mcbond_it
X-RAY DIFFRACTIONp_mcangle_it
X-RAY DIFFRACTIONp_scbond_it
X-RAY DIFFRACTIONp_scangle_it
X-RAY DIFFRACTIONp_plane_restr
X-RAY DIFFRACTIONp_chiral_restr
X-RAY DIFFRACTIONp_singtor_nbd
X-RAY DIFFRACTIONp_multtor_nbd
X-RAY DIFFRACTIONp_xhyhbond_nbd
X-RAY DIFFRACTIONp_xyhbond_nbd
X-RAY DIFFRACTIONp_planar_tor
X-RAY DIFFRACTIONp_staggered_tor
X-RAY DIFFRACTIONp_orthonormal_tor
X-RAY DIFFRACTIONp_transverse_tor
X-RAY DIFFRACTIONp_special_tor
Refinement
*PLUS
Rfactor obs: 0.195
Solvent computation
*PLUS
Displacement parameters
*PLUS
Biso mean: 30.3 Å2
Refine LS restraints
*PLUS
Type: p_plane_restr / Dev ideal: 0.019

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