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- PDB-8a67: Branched Lys48- and Lys63-linked tri-ubiquitin (K48-K63-Ub3) in c... -

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Basic information

Entry
Database: PDB / ID: 8a67
TitleBranched Lys48- and Lys63-linked tri-ubiquitin (K48-K63-Ub3) in complex with matured synthetic nanobody NbSL3.3Q (3rd generation)
Components
  • (Polyubiquitin- ...) x 2
  • Synthetic Nanobody NbSL3.3Q
KeywordsSIGNALING PROTEIN / Branched Ubiquitin / Nanobody / complex
Function / homology
Function and homology information


modification-dependent protein catabolic process / protein tag activity / protein ubiquitination / ubiquitin protein ligase binding / nucleus / cytoplasm
Similarity search - Function
Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
ISOPROPYL ALCOHOL / Ubiquitin B
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.86 Å
AuthorsLange, S.M. / Kulathu, Y.
Funding support United Kingdom, European Union, 3items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UU_00018/3 United Kingdom
European Research Council (ERC)Young Investigator ProgrammeEuropean Union
European Research Council (ERC)StressHub 101002428European Union
Citation
Journal: Biorxiv / Year: 2023
Title: Comprehensive approach to study branched ubiquitin chains reveals roles for K48-K63 branches in VCP/p97-related processes
Authors: Lange, S.M. / McFarland, M.R. / Lamoliatte, F. / Kwasna, D. / Shen, L. / Wallace, I. / Cole, I. / Armstrong, L.A. / Knebel, A. / Johnson, C. / De Cesare, V. / Kulathu, Y.
#1: Journal: Acta Crystallogr D Biol Crystallogr / Year: 2010
Title: PHENIX: a comprehensive Python-based system for macromolecular structure solution.
Authors: Paul D Adams / Pavel V Afonine / Gábor Bunkóczi / Vincent B Chen / Ian W Davis / Nathaniel Echols / Jeffrey J Headd / Li-Wei Hung / Gary J Kapral / Ralf W Grosse-Kunstleve / Airlie J McCoy ...Authors: Paul D Adams / Pavel V Afonine / Gábor Bunkóczi / Vincent B Chen / Ian W Davis / Nathaniel Echols / Jeffrey J Headd / Li-Wei Hung / Gary J Kapral / Ralf W Grosse-Kunstleve / Airlie J McCoy / Nigel W Moriarty / Robert Oeffner / Randy J Read / David C Richardson / Jane S Richardson / Thomas C Terwilliger / Peter H Zwart /
Abstract: Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many ...Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
#2: Journal: Acta Crystallogr D Biol Crystallogr / Year: 2011
Title: Data processing and analysis with the autoPROC toolbox.
Authors: Vonrhein, C. / Flensburg, C. / Keller, P. / Sharff, A. / Smart, O. / Paciorek, W. / Womack, T. / Bricogne, G.
History
DepositionJun 16, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 15, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 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: Polyubiquitin-B
B: Polyubiquitin-B
C: Polyubiquitin-B
D: Synthetic Nanobody NbSL3.3Q
E: Polyubiquitin-B
F: Polyubiquitin-B
G: Polyubiquitin-B
H: Synthetic Nanobody NbSL3.3Q
hetero molecules


Theoretical massNumber of molelcules
Total (without water)83,36917
Polymers82,8528
Non-polymers5179
Water9,422523
1
A: Polyubiquitin-B
B: Polyubiquitin-B
C: Polyubiquitin-B
D: Synthetic Nanobody NbSL3.3Q
hetero molecules


Theoretical massNumber of molelcules
Total (without water)41,6447
Polymers41,4264
Non-polymers2183
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6060 Å2
ΔGint-54 kcal/mol
Surface area15470 Å2
MethodPISA
2
E: Polyubiquitin-B
F: Polyubiquitin-B
G: Polyubiquitin-B
H: Synthetic Nanobody NbSL3.3Q
hetero molecules


Theoretical massNumber of molelcules
Total (without water)41,72510
Polymers41,4264
Non-polymers2996
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6320 Å2
ΔGint-78 kcal/mol
Surface area15470 Å2
MethodPISA
Unit cell
Length a, b, c (Å)57.081, 58.243, 61.662
Angle α, β, γ (deg.)78.880, 67.940, 80.160
Int Tables number1
Space group name H-MP1
Space group name HallP1
Symmetry operation#1: x,y,z

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Components

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Polyubiquitin- ... , 2 types, 6 molecules AEBCFG

#1: Protein Polyubiquitin-B


Mass: 8192.375 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Ubiquitin with C-terminal truncation / Source: (gene. exp.) Homo sapiens (human) / Gene: UBB / Production host: Escherichia coli (E. coli) / References: UniProt: J3QS39
#2: Protein
Polyubiquitin-B


Mass: 8632.859 Da / Num. of mol.: 4 / Mutation: K48R K63R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBB / Production host: Escherichia coli (E. coli) / References: UniProt: J3QS39

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Antibody , 1 types, 2 molecules DH

#3: Antibody Synthetic Nanobody NbSL3.3Q


Mass: 15967.914 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Matured Nanobody NbSL3.3Q with N-terminal pelB signal sequence for periplasmic expression and C-terminal 6His affinity tag
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Non-polymers , 6 types, 532 molecules

#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-IPA / ISOPROPYL ALCOHOL / 2-PROPANOL


Mass: 60.095 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O / Comment: alkaloid*YM
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#7: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#8: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 523 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 2.24 Å3/Da / Density % sol: 45.01 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: Protein concentrated to 14.5 mg/ml in 20 mM HEPES pH 7.5, 150 mM NaCl. Mixed 200 nl protein with 100 nl mother liquor (0.1 M HEPES pH 7.5, 10% 2-propanol, 20% PEG4000). Crystals harvested ...Details: Protein concentrated to 14.5 mg/ml in 20 mM HEPES pH 7.5, 150 mM NaCl. Mixed 200 nl protein with 100 nl mother liquor (0.1 M HEPES pH 7.5, 10% 2-propanol, 20% PEG4000). Crystals harvested and cryo-protected with Mother liquor supplemented with 30% glycerol.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-2 / Wavelength: 0.87313 Å
DetectorType: DECTRIS PILATUS3 2M / Detector: PIXEL / Date: Jun 9, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.87313 Å / Relative weight: 1
ReflectionResolution: 1.86→56.58 Å / Num. obs: 36500 / % possible obs: 88.7 % / Redundancy: 3.2 % / Biso Wilson estimate: 13.75 Å2 / CC1/2: 0.987 / Net I/σ(I): 4.1
Reflection shellResolution: 1.86→2.09 Å / Num. unique obs: 1826 / CC1/2: 0.735

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
autoPROCdata processing
autoPROCdata reduction
XSCALEdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7NBB

7nbb


Resolution: 1.86→52.59 Å / SU ML: 0.2054 / Cross valid method: FREE R-VALUE / σ(F): 1.96 / Phase error: 28.1262
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2452 1721 4.72 %
Rwork0.1865 34775 -
obs0.1893 36496 60.76 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 17.36 Å2
Refinement stepCycle: LAST / Resolution: 1.86→52.59 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5445 0 25 523 5993
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0025545
X-RAY DIFFRACTIONf_angle_d0.50277489
X-RAY DIFFRACTIONf_chiral_restr0.0432851
X-RAY DIFFRACTIONf_plane_restr0.0044973
X-RAY DIFFRACTIONf_dihedral_angle_d5.2137759
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.86-1.920.267950.230456X-RAY DIFFRACTION1.22
1.92-1.980.3628110.2334234X-RAY DIFFRACTION4.93
1.98-2.050.3013320.2229730X-RAY DIFFRACTION15.22
2.05-2.130.2806780.21521570X-RAY DIFFRACTION32.88
2.13-2.230.25681140.21712332X-RAY DIFFRACTION49.14
2.23-2.350.26391340.22293004X-RAY DIFFRACTION62.77
2.35-2.490.27261920.22953737X-RAY DIFFRACTION78.61
2.49-2.690.27161970.2224482X-RAY DIFFRACTION93.13
2.69-2.960.28182170.20584668X-RAY DIFFRACTION97.84
2.96-3.380.26642590.18194659X-RAY DIFFRACTION97.97
3.38-4.260.19452420.14434641X-RAY DIFFRACTION97.97
4.26-52.590.22152400.16584662X-RAY DIFFRACTION97.63

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