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- PDB-7ozt: Nanobodies restore stability to cancer-associated mutants of tumo... -

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Basic information

Entry
Database: PDB / ID: 7ozt
TitleNanobodies restore stability to cancer-associated mutants of tumor suppressor protein p16INK4a
Components
  • Camelid nanobody NB09
  • Cyclin-dependent kinase inhibitor 2A
KeywordsCELL CYCLE / nanobody-p16INK4A complex / tumour-suppressor / Checkpoint / pharmacological chaperone
Function / homology
Function and homology information


senescence-associated heterochromatin focus / positive regulation of macrophage apoptotic process / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 / Evasion of Oxidative Stress Induced Senescence Due to Defective p16INK4A binding to CDK4 / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 and CDK6 / Evasion of Oxidative Stress Induced Senescence Due to Defective p16INK4A binding to CDK4 and CDK6 / positive regulation of smooth muscle cell apoptotic process / negative regulation of phosphorylation / negative regulation of cyclin-dependent protein serine/threonine kinase activity / cyclin-dependent protein serine/threonine kinase inhibitor activity ...senescence-associated heterochromatin focus / positive regulation of macrophage apoptotic process / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 / Evasion of Oxidative Stress Induced Senescence Due to Defective p16INK4A binding to CDK4 / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 and CDK6 / Evasion of Oxidative Stress Induced Senescence Due to Defective p16INK4A binding to CDK4 and CDK6 / positive regulation of smooth muscle cell apoptotic process / negative regulation of phosphorylation / negative regulation of cyclin-dependent protein serine/threonine kinase activity / cyclin-dependent protein serine/threonine kinase inhibitor activity / negative regulation of cell-matrix adhesion / negative regulation of NF-kappaB transcription factor activity / regulation of G1/S transition of mitotic cell cycle / Transcriptional Regulation by VENTX / NF-kappaB binding / replicative senescence / Oncogene Induced Senescence / negative regulation of cell growth / Cyclin D associated events in G1 / cellular senescence / Senescence-Associated Secretory Phenotype (SASP) / Oxidative Stress Induced Senescence / Ras protein signal transduction / regulation of cell cycle / cell cycle / negative regulation of cell population proliferation / negative regulation of DNA-templated transcription / protein kinase binding / RNA binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Ankyrin repeat region circular profile. / Ankyrin repeat-containing domain superfamily
Similarity search - Domain/homology
Cyclin-dependent kinase inhibitor 2A
Similarity search - Component
Biological speciesLama glama (llama)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.74 Å
AuthorsPastok, M.W. / Burbidge, O. / Itzhaki, L. / Endicott, J.A. / Noble, M.E.M.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/N009738/1 United Kingdom
CitationJournal: To Be Published
Title: Nanobodies restore stability to cancer-associated mutants of tumor suppressor protein p16INK4a
Authors: Burbidge, O. / Pastok, M.W. / Hodder, S.L. / Zenkeviciute, G. / Noble, M.E.M. / Endicott, J.A. / Itzhaki, L.
History
DepositionJun 28, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 13, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 31, 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
AAA: Camelid nanobody NB09
BBB: Cyclin-dependent kinase inhibitor 2A


Theoretical massNumber of molelcules
Total (without water)32,9102
Polymers32,9102
Non-polymers00
Water1,62190
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1270 Å2
ΔGint-3 kcal/mol
Surface area11620 Å2
MethodPISA
Unit cell
Length a, b, c (Å)42.119, 182.931, 65.225
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Components on special symmetry positions
IDModelComponents
11AAA-236-

HOH

21BBB-245-

HOH

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Components

#1: Antibody Camelid nanobody NB09


Mass: 14208.778 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Plasmid: pMESy4 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Rosetta2
#2: Protein Cyclin-dependent kinase inhibitor 2A / Cyclin-dependent kinase 4 inhibitor A / CDK4I / Multiple tumor suppressor 1 / MTS-1 / p16-INK4a / ...Cyclin-dependent kinase 4 inhibitor A / CDK4I / Multiple tumor suppressor 1 / MTS-1 / p16-INK4a / p16-INK4 / p16INK4A


Mass: 18701.037 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDKN2A, CDKN2, MTS1 / Plasmid: pOPINF / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): LEMO21 / References: UniProt: P42771
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 90 / 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: 1.91 Å3/Da / Density % sol: 35.56 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / Details: Molecular Dimensions Morpheus screen condition G4

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.97622 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Feb 5, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97622 Å / Relative weight: 1
ReflectionResolution: 1.74→91.47 Å / Num. obs: 26446 / % possible obs: 100 % / Redundancy: 6.8 % / CC1/2: 0.998 / Rmerge(I) obs: 0.101 / Rpim(I) all: 0.061 / Rrim(I) all: 0.119 / Net I/σ(I): 9.2
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) all
9.04-91.475.60.0462390.9990.0290.055
1.74-1.776.20.87814120.3610.5821.058

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
DIALSdata reduction
PHASERphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1BI7, 3OGO

1bi7

3ogo


Resolution: 1.74→91.465 Å / Cor.coef. Fo:Fc: 0.969 / Cor.coef. Fo:Fc free: 0.938 / SU B: 6.123 / SU ML: 0.089 / Cross valid method: FREE R-VALUE / ESU R: 0.106 / ESU R Free: 0.102
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflectionSelection details
Rfree0.2086 930 3.519 %Random selection
Rwork0.1798 25497 --
all0.181 ---
obs-26427 99.996 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 27.959 Å2
Baniso -1Baniso -2Baniso -3
1-1.175 Å20 Å20 Å2
2---1.038 Å20 Å2
3----0.137 Å2
Refinement stepCycle: LAST / Resolution: 1.74→91.465 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1854 0 0 90 1944
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0160.0131919
X-RAY DIFFRACTIONr_bond_other_d0.0010.0171814
X-RAY DIFFRACTIONr_angle_refined_deg2.0811.6392608
X-RAY DIFFRACTIONr_angle_other_deg1.411.5794150
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.8845253
X-RAY DIFFRACTIONr_dihedral_angle_2_deg31.07919.714105
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.26915303
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.6761522
X-RAY DIFFRACTIONr_chiral_restr0.1020.2242
X-RAY DIFFRACTIONr_gen_planes_refined0.010.022422
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02470
X-RAY DIFFRACTIONr_nbd_refined0.2060.2323
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1930.21699
X-RAY DIFFRACTIONr_nbtor_refined0.160.2896
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0860.21034
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.2020.282
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.1580.210
X-RAY DIFFRACTIONr_nbd_other0.2490.237
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.1940.29
X-RAY DIFFRACTIONr_mcbond_it2.0062.2581006
X-RAY DIFFRACTIONr_mcbond_other1.9792.2541005
X-RAY DIFFRACTIONr_mcangle_it3.1843.371261
X-RAY DIFFRACTIONr_mcangle_other3.1863.3741262
X-RAY DIFFRACTIONr_scbond_it3.3822.758913
X-RAY DIFFRACTIONr_scbond_other3.382.761914
X-RAY DIFFRACTIONr_scangle_it5.4613.9641347
X-RAY DIFFRACTIONr_scangle_other5.4593.9681348
X-RAY DIFFRACTIONr_lrange_it6.94626.7852017
X-RAY DIFFRACTIONr_lrange_other6.92726.7132006
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc work% reflection obs (%)WRfactor RworkFsc free
1.74-1.78500.32719110.32719110.7741000.324
1.785-1.83400.29718850.29718850.7991000.292
1.834-1.88700.28418220.28418220.8191000.277
1.887-1.94500.27517660.27517660.8441000.261
1.945-2.0090.259130.22817270.22817400.8881000.2090.879
2.009-2.080.239830.22115760.22216590.9041000.2020.894
2.08-2.1580.226840.20515140.20615980.9181000.1840.923
2.158-2.2460.185980.19414640.19415620.931000.1710.925
2.246-2.3460.226690.17414360.17715050.9411000.1550.93
2.346-2.460.197550.16913510.1714060.951000.1520.946
2.46-2.5930.205890.16312960.16613850.9541000.1490.941
2.593-2.7510.209590.16712190.16912780.9581000.1590.948
2.751-2.940.224640.17911560.18112200.9561000.170.951
2.94-3.1760.189680.17110730.17211420.96399.91240.1680.965
3.176-3.4780.178570.15410070.15510640.9731000.1580.968
3.478-3.8880.201660.1428910.1469570.9781000.1520.966
3.888-4.4880.225340.128100.1248440.9821000.1340.957
4.488-5.4940.212250.1417190.1437440.9781000.1580.965
5.494-7.7550.293400.1845430.1915830.9621000.2050.925
7.755-91.4650.147260.1653300.1643560.9671000.1940.981
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.16240.0131-0.25451.8680.02230.4043-0.0025-0.04310.01520.00690.03510.10770.00570.0552-0.03260.00150.00620.00510.05270.03830.0476-8.24912.42382.5774
20.7638-0.1098-0.27440.7590.03690.2977-0.0197-0.02740.02990.1160.0424-0.0309-0.02060.0377-0.02260.02170.0004-0.00170.0392-0.01840.01864.37335.3509-2.7296
Refinement TLS group
IDRefine-IDRefine TLS-IDSelectionAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1ALLAAA1 - 122
2X-RAY DIFFRACTION2ALLBBB10 - 134

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