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- PDB-6u9l: Imidazole-triggered RAS-specific subtilisin SUBT_BACAM -

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Basic information

Entry
Database: PDB / ID: 6u9l
TitleImidazole-triggered RAS-specific subtilisin SUBT_BACAM
ComponentsSUBTILISIN BPN'
KeywordsHYDROLASE / engineered protease
Function / homology: / THIOCYANATE ION
Function and homology information
Biological speciesBacillus amyloliquefaciens (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.7 Å
AuthorsToth, E.A. / Bryan, P.N. / Orban, J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Commun Biol / Year: 2021
Title: Engineering subtilisin proteases that specifically degrade active RAS.
Authors: Chen, Y. / Toth, E.A. / Ruan, B. / Choi, E.J. / Simmerman, R. / Chen, Y. / He, Y. / Wang, R. / Godoy-Ruiz, R. / King, H. / Custer, G. / Travis Gallagher, D. / Rozak, D.A. / Solomon, M. / ...Authors: Chen, Y. / Toth, E.A. / Ruan, B. / Choi, E.J. / Simmerman, R. / Chen, Y. / He, Y. / Wang, R. / Godoy-Ruiz, R. / King, H. / Custer, G. / Travis Gallagher, D. / Rozak, D.A. / Solomon, M. / Muro, S. / Weber, D.J. / Orban, J. / Fuerst, T.R. / Bryan, P.N.
History
DepositionSep 9, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 16, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 31, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Oct 11, 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
S: SUBTILISIN BPN'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,95610
Polymers26,4571
Non-polymers5009
Water3,333185
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)58.650, 58.650, 124.750
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number92
Space group name H-MP41212

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Components

#1: Protein SUBTILISIN BPN'


Mass: 26456.518 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus amyloliquefaciens (bacteria) / Production host: Escherichia coli (E. coli)
#2: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: K
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical
ChemComp-SCN / THIOCYANATE ION


Mass: 58.082 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: CNS
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 185 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 2.03 Å3/Da / Density % sol: 39.33 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 0.1 M Bis-TRIS propane pH 8.5, 0.2 M KSCN, and 20% PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 / Wavelength: 1.5418 Å
DetectorType: RIGAKU RAXIS IV++ / Detector: IMAGE PLATE / Date: Aug 6, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.7→53.08 Å / Num. obs: 24845 / % possible obs: 100 % / Redundancy: 27.2 % / CC1/2: 0.999 / Rmerge(I) obs: 0.097 / Rpim(I) all: 0.019 / Rrim(I) all: 0.099 / Net I/σ(I): 26.7 / Num. measured all: 675806 / Scaling rejects: 825
Reflection shellResolution: 1.7→1.73 Å / Redundancy: 26.6 % / Rmerge(I) obs: 0.416 / Num. measured all: 34830 / Num. unique obs: 1307 / CC1/2: 0.98 / Rpim(I) all: 0.082 / Rrim(I) all: 0.424 / Net I/σ(I) obs: 8.3 / % possible all: 100

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Phasing

PhasingMethod: molecular replacement
Phasing MRR rigid body: 0.466
Highest resolutionLowest resolution
Rotation42.73 Å1.78 Å

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Processing

Software
NameVersionClassificationNB
MOSFLMdata reduction
Aimless0.5.27data scaling
MOLREPphasing
REFMAC5.8.0155refinement
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1spb

1spb


Resolution: 1.7→53.08 Å / Cor.coef. Fo:Fc: 0.967 / Cor.coef. Fo:Fc free: 0.954 / WRfactor Rfree: 0.1697 / WRfactor Rwork: 0.1458 / FOM work R set: 0.9065 / SU B: 1.499 / SU ML: 0.051 / SU R Cruickshank DPI: 0.0933 / SU Rfree: 0.0888 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.093 / ESU R Free: 0.089 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.1735 1217 4.9 %RANDOM
Rwork0.147 ---
obs0.1483 23555 99.98 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 48.54 Å2 / Biso mean: 13.729 Å2 / Biso min: 7.76 Å2
Baniso -1Baniso -2Baniso -3
1--0.1 Å20 Å20 Å2
2---0.1 Å20 Å2
3---0.2 Å2
Refinement stepCycle: final / Resolution: 1.7→53.08 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1846 0 24 185 2055
Biso mean--34.29 23.36 -
Num. residues----266
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0190.0191908
X-RAY DIFFRACTIONr_bond_other_d00.021765
X-RAY DIFFRACTIONr_angle_refined_deg1.821.9472603
X-RAY DIFFRACTIONr_angle_other_deg3.51434072
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.0845269
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.85825.86258
X-RAY DIFFRACTIONr_dihedral_angle_3_deg11.92615261
X-RAY DIFFRACTIONr_dihedral_angle_4_deg10.156152
X-RAY DIFFRACTIONr_chiral_restr0.1170.2309
X-RAY DIFFRACTIONr_gen_planes_refined0.010.0212229
X-RAY DIFFRACTIONr_gen_planes_other0.0190.02391
LS refinement shellResolution: 1.7→1.744 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.237 72 -
Rwork0.186 1729 -
all-1801 -
obs--100 %

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