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- PDB-4orz: HIV-1 Nef protein in complex with single domain antibody sdAb19 a... -

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Basic information

Entry
Database: PDB / ID: 4orz
TitleHIV-1 Nef protein in complex with single domain antibody sdAb19 and an engineered Hck SH3 domain
Components
  • Protein Nef
  • Tyrosine-protein kinase HCK
  • single domain antibody sdAb19
KeywordsTRANSFERASE/APOPTOSIS/IMMUNE SYSTEM / SH3 domain / immunglobolin fold / Antibodies / Epitopes / HIV Antibodies / HIV Accessory Proteins / PxxP motif / Complementarity Determining Regions / TRANSFERASE-APOPTOSIS-IMMUNE SYSTEM complex
Function / homology
Function and homology information


leukocyte degranulation / respiratory burst after phagocytosis / leukocyte migration involved in immune response / innate immune response-activating signaling pathway / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class I / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class II / regulation of podosome assembly / suppression by virus of host autophagy / regulation of phagocytosis / FLT3 signaling through SRC family kinases ...leukocyte degranulation / respiratory burst after phagocytosis / leukocyte migration involved in immune response / innate immune response-activating signaling pathway / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class I / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class II / regulation of podosome assembly / suppression by virus of host autophagy / regulation of phagocytosis / FLT3 signaling through SRC family kinases / Nef and signal transduction / regulation of DNA-binding transcription factor activity / activation of transmembrane receptor protein tyrosine kinase activity / Fc-gamma receptor signaling pathway involved in phagocytosis / positive regulation of actin filament polymerization / mesoderm development / host cell Golgi membrane / FCGR activation / type II interferon-mediated signaling pathway / Signaling by CSF3 (G-CSF) / cell surface receptor protein tyrosine kinase signaling pathway / transport vesicle / lipopolysaccharide-mediated signaling pathway / phosphotyrosine residue binding / FCGR3A-mediated IL10 synthesis / cell projection / integrin-mediated signaling pathway / regulation of actin cytoskeleton organization / Regulation of signaling by CBL / negative regulation of inflammatory response to antigenic stimulus / FCGR3A-mediated phagocytosis / non-specific protein-tyrosine kinase / virion component / non-membrane spanning protein tyrosine kinase activity / Inactivation of CSF3 (G-CSF) signaling / caveola / endocytosis involved in viral entry into host cell / cytokine-mediated signaling pathway / SH3 domain binding / peptidyl-tyrosine phosphorylation / cytoplasmic side of plasma membrane / Signaling by CSF1 (M-CSF) in myeloid cells / regulation of cell shape / regulation of inflammatory response / protein tyrosine kinase activity / protein autophosphorylation / cell differentiation / lysosome / cytoskeleton / cell adhesion / intracellular signal transduction / inflammatory response / protein phosphorylation / intracellular membrane-bounded organelle / innate immune response / signaling receptor binding / focal adhesion / virus-mediated perturbation of host defense response / lipid binding / positive regulation of cell population proliferation / negative regulation of apoptotic process / GTP binding / apoptotic process / host cell plasma membrane / Golgi apparatus / extracellular region / ATP binding / membrane / nucleus / plasma membrane / cytosol
Similarity search - Function
Nef Regulatory Factor / Nef Regulatory Factor / Tyrosine-protein kinase HCK, SH2 domain / HIV-1 Nef protein, anchor domain superfamily / HIV negative factor Nef / HIV-1 Nef protein, core domain superfamily / Negative factor, (F-Protein) or Nef / SH3 Domains / : / SH3 domain ...Nef Regulatory Factor / Nef Regulatory Factor / Tyrosine-protein kinase HCK, SH2 domain / HIV-1 Nef protein, anchor domain superfamily / HIV negative factor Nef / HIV-1 Nef protein, core domain superfamily / Negative factor, (F-Protein) or Nef / SH3 Domains / : / SH3 domain / SH2 domain / Src homology 2 (SH2) domain profile. / Src homology 2 domains / SH3 type barrels. / SH2 domain / Src homology 3 domains / SH2 domain superfamily / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / Tyrosine-protein kinase, catalytic domain / Tyrosine kinase, catalytic domain / Tyrosine protein kinases specific active-site signature. / Tyrosine-protein kinase, active site / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein tyrosine and serine/threonine kinase / Roll / Immunoglobulins / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / Immunoglobulin-like / Sandwich / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Protein Nef / Tyrosine-protein kinase HCK
Similarity search - Component
Biological speciesHomo sapiens (human)
HIV-1 M:B_ARV2/SF2 (virus)
Lama glama (llama)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsGeyer, M. / Lulf, S.
CitationJournal: Retrovirology / Year: 2014
Title: Structural basis for the inhibition of HIV-1 Nef by a high-affinity binding single-domain antibody.
Authors: Lulf, S. / Matz, J. / Rouyez, M.C. / Jarviluoma, A. / Saksela, K. / Benichou, S. / Geyer, M.
History
DepositionFeb 12, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 26, 2014Provider: repository / Type: Initial release
Revision 1.1Sep 20, 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 / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Tyrosine-protein kinase HCK
B: Protein Nef
C: single domain antibody sdAb19


Theoretical massNumber of molelcules
Total (without water)37,4403
Polymers37,4403
Non-polymers00
Water2,252125
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)73.000, 73.000, 71.000
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number76
Space group name H-MP41

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Components

#1: Protein Tyrosine-protein kinase HCK / Hematopoietic cell kinase / Hemopoietic cell kinase / p59-HCK/p60-HCK / p59Hck / p61Hck


Mass: 7614.431 Da / Num. of mol.: 1 / Fragment: SH3 domain, UNP residues 77-138 / Mutation: E90Y, A91S, I92P, H93F, H94S, E95W
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HCK / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: P08631, non-specific protein-tyrosine kinase
#2: Protein Protein Nef / 3'ORF / Negative factor / F-protein / C-terminal core protein


Mass: 16817.979 Da / Num. of mol.: 1 / Fragment: Nef protein, UNP residues 45-210 / Mutation: I47M, T48A, C59S, C210A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HIV-1 M:B_ARV2/SF2 (virus) / Gene: nef / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P03407
#3: Antibody single domain antibody sdAb19


Mass: 13007.562 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 125 / 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: 2.53 Å3/Da / Density % sol: 51.31 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 9
Details: About 0.1 micro L of protein solution at 10 mg/ml concentration was mixed with 0.1 micro L of reservoir solution from a 70 L reservoir in 96-well Hampton 3553 crystallization plates. Initial ...Details: About 0.1 micro L of protein solution at 10 mg/ml concentration was mixed with 0.1 micro L of reservoir solution from a 70 L reservoir in 96-well Hampton 3553 crystallization plates. Initial crystals of NefSF2 sdAb19 SH3B6 could be obtained in 0.2 M potassium formate and 20% polyethylene glycol (PEG) 3350. Crystal conditions were optimized to 0.2 M potassium formate, 17.5% polyethylene glycol (PEG) 3350 and 0.35 M ammonium chloride grown by hanging-drop vapor diffusion in Linbro crystallization plates. , pH 9.0, VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X10SA / Wavelength: 0.9794 Å
DetectorType: PSI PILATUS 6M / Detector: PIXEL / Date: Feb 10, 2012
RadiationMonochromator: Diamond(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9794 Å / Relative weight: 1
ReflectionResolution: 2→41.75 Å / Num. obs: 47918 / % possible obs: 100 % / Observed criterion σ(F): -3 / Observed criterion σ(I): -3
Reflection shellResolution: 2.1→2.15 Å / % possible all: 100

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Processing

Software
NameVersionClassification
ADSCQuantumdata collection
PHASERphasing
PHENIX(phenix.refine: 1.8.1_1168)refinement
XDSdata reduction
XSCALEdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 3RBB

3rbb


Resolution: 2→41.75 Å / SU ML: 0.21 / σ(F): 2.04 / Phase error: 24.91 / Stereochemistry target values: MLHL
RfactorNum. reflection% reflectionSelection details
Rfree0.2364 1252 5 %random
Rwork0.2018 ---
obs0.2036 25031 98.94 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2→41.75 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2330 0 0 125 2455
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0082399
X-RAY DIFFRACTIONf_angle_d1.2883255
X-RAY DIFFRACTIONf_dihedral_angle_d15.518865
X-RAY DIFFRACTIONf_chiral_restr0.085336
X-RAY DIFFRACTIONf_plane_restr0.006413
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2-2.08010.29151380.252628X-RAY DIFFRACTION98
2.0801-2.17480.27121370.23972592X-RAY DIFFRACTION99
2.1748-2.28940.27151380.23422624X-RAY DIFFRACTION98
2.2894-2.43280.29921390.23872638X-RAY DIFFRACTION99
2.4328-2.62060.26511390.23492645X-RAY DIFFRACTION99
2.6206-2.88430.24121380.21892621X-RAY DIFFRACTION99
2.8843-3.30150.23911400.21622658X-RAY DIFFRACTION99
3.3015-4.1590.21941390.17842656X-RAY DIFFRACTION99
4.159-41.760.2011440.16862717X-RAY DIFFRACTION100

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