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- PDB-4bpd: Structure determination of an integral membrane kinase -

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Basic information

Entry
Database: PDB / ID: 4bpd
TitleStructure determination of an integral membrane kinase
ComponentsDIACYLGLYCEROL KINASE
KeywordsTRANSFERASE / CELL-FREE / DGKA / IN MESO CRYSTALLIZATION / IN VITRO EXPRESSION / LIPID METABOLISM / LIPIDIC MESOPHASE / LIPIDIC CUBIC PHASE / LCP / MEMBRANE PROTEIN / MICROCRYSTAL / 7.8 MAG / THERMOSTABLE MUTANT
Function / homology
Function and homology information


diacylglycerol kinase (ATP) / ATP-dependent diacylglycerol kinase activity / phosphatidic acid biosynthetic process / response to UV / phosphorylation / ATP binding / membrane / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Helix Hairpins - #3610 / DAGK family / Diacylglycerol kinase, prokaryotic / Diacylglycerol kinase (DAGK) superfamily / Prokaryotic diacylglycerol kinase / Prokaryotic diacylglycerol kinase signature. / Helix Hairpins / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
(2S)-2,3-DIHYDROXYPROPYL(7Z)-PENTADEC-7-ENOATE / Diacylglycerol kinase
Similarity search - Component
Biological speciesESCHERICHIA COLI (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.3 Å
AuthorsLi, D. / Boland, C. / Caffrey, M.
CitationJournal: Cell.Mol.Life Sci. / Year: 2014
Title: Cell-Free Expression and in Meso Crystallisation of an Integral Membrane Kinase for Structure Determination.
Authors: Boland, C. / Li, D. / Shah, S.T.A. / Haberstock, S. / Dotsch, V. / Bernhard, F. / Caffrey, M.
History
DepositionMay 24, 2013Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 7, 2014Provider: repository / Type: Initial release
Revision 1.1Oct 22, 2014Group: Database references
Revision 1.2Dec 3, 2014Group: Database references
Revision 1.3Mar 6, 2019Group: Data collection / Experimental preparation / Other
Category: exptl_crystal_grow / pdbx_database_proc / pdbx_database_status
Item: _exptl_crystal_grow.method / _pdbx_database_status.recvd_author_approval
Revision 1.4Apr 3, 2019Group: Data collection / Experimental preparation / Category: exptl_crystal_grow / Item: _exptl_crystal_grow.temp
Revision 1.5Dec 20, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DIACYLGLYCEROL KINASE
B: DIACYLGLYCEROL KINASE
C: DIACYLGLYCEROL KINASE
D: DIACYLGLYCEROL KINASE
E: DIACYLGLYCEROL KINASE
F: DIACYLGLYCEROL KINASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)86,55011
Polymers85,2276
Non-polymers1,3235
Water0
1
A: DIACYLGLYCEROL KINASE
B: DIACYLGLYCEROL KINASE
C: DIACYLGLYCEROL KINASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)43,5576
Polymers42,6133
Non-polymers9433
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7540 Å2
ΔGint-81.1 kcal/mol
Surface area15970 Å2
MethodPISA
2
D: DIACYLGLYCEROL KINASE
E: DIACYLGLYCEROL KINASE
F: DIACYLGLYCEROL KINASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)42,9935
Polymers42,6133
Non-polymers3802
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7180 Å2
ΔGint-111.5 kcal/mol
Surface area14640 Å2
MethodPISA
Unit cell
Length a, b, c (Å)74.870, 93.430, 144.020
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein
DIACYLGLYCEROL KINASE / / DIACYLGLYCEROL KINASE - DELTA 7 / DAGK / DIGLYCERIDE KINASE / DGK


Mass: 14204.451 Da / Num. of mol.: 6 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ESCHERICHIA COLI (E. coli) / Strain: K-12 / Description: ESCHERICHIA COLI A19 / Plasmid: PET22B-DGKA-DELTA7 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): A19 / References: UniProt: P0ABN1, diacylglycerol kinase (ATP)
#2: Chemical
ChemComp-78M / (2S)-2,3-DIHYDROXYPROPYL(7Z)-PENTADEC-7-ENOATE / 7.8 MONOACYLGLYCEROL


Mass: 314.460 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C18H34O4
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
Sequence detailsTHE CONSTRUCT CONTAINS AN N-TERMIANL HIS TAG 'GHHHHHHEL'. COMPARED TO THE WILDTYPE FORM, THE ...THE CONSTRUCT CONTAINS AN N-TERMIANL HIS TAG 'GHHHHHHEL'. COMPARED TO THE WILDTYPE FORM, THE PROTEIN HAS SEVEN MUTATIONS. THEY ARE A41C, C46A, I53V, I70L, M96L, V107D AND C113A.

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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: 3.91 Å3/Da / Density % sol: 68.56 % / Description: NONE
Crystal growTemperature: 277 K / Method: lipidic cubic phase / pH: 5.6
Details: 3-5 %(V/V) 2-METHYL-2, 4-PENTANEDIOL (MPD), 0.1 M SODIUM CHLORIDE, 0.06 M MAGNESIUM ACETATE, 0.1 M SODIUM CITRATE/HCL PH 5.6. CRYSTALLIZED USING THE IN MESO (LIPIDIC CUBIC PHASE, LCP) METHOD ...Details: 3-5 %(V/V) 2-METHYL-2, 4-PENTANEDIOL (MPD), 0.1 M SODIUM CHLORIDE, 0.06 M MAGNESIUM ACETATE, 0.1 M SODIUM CITRATE/HCL PH 5.6. CRYSTALLIZED USING THE IN MESO (LIPIDIC CUBIC PHASE, LCP) METHOD AT 4 DEGREES CELCIUS WITH THE 7.8 MONOACYLGLYCEROL (7.8 MAG) AS THE HOSTING LIPID.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X10SA / Wavelength: 1.03319
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Sep 13, 2012 / Details: MIRRORS
RadiationMonochromator: DOUBLE CRYSTAL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.03319 Å / Relative weight: 1
ReflectionResolution: 3.3→78.38 Å / Num. obs: 15336 / % possible obs: 97.1 % / Observed criterion σ(I): -3 / Redundancy: 4.2 % / Biso Wilson estimate: 95.95 Å2 / Rmerge(I) obs: 0.09 / Net I/σ(I): 10.6
Reflection shellResolution: 3.3→3.39 Å / Redundancy: 4.4 % / Rmerge(I) obs: 0.91 / Mean I/σ(I) obs: 2 / % possible all: 98

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Processing

Software
NameVersionClassification
PHENIX(PHENIX.REFINE)refinement
XDSdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 3ZE3

3ze3


Resolution: 3.3→57.035 Å / SU ML: 0.49 / σ(F): 1.34 / Phase error: 33.75 / Stereochemistry target values: ML
Details: THERE ARE SIX NCS-RELATED MOLECULES IN THE ASYMMETRIC UNIT BUT NCS RESTRAINTS WERE NOT USED IN THE REFINEMENT.
RfactorNum. reflection% reflection
Rfree0.2821 752 5 %
Rwork0.241 --
obs0.2431 15102 95.74 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 75.01 Å2
Refinement stepCycle: LAST / Resolution: 3.3→57.035 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4551 0 89 0 4640
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0014711
X-RAY DIFFRACTIONf_angle_d0.3576410
X-RAY DIFFRACTIONf_dihedral_angle_d9.5561620
X-RAY DIFFRACTIONf_chiral_restr0.025814
X-RAY DIFFRACTIONf_plane_restr0.001765
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.3005-3.55530.33851470.28892914X-RAY DIFFRACTION99
3.5553-3.9130.36961420.32942459X-RAY DIFFRACTION84
3.913-4.4790.24041590.19822890X-RAY DIFFRACTION98
4.479-5.64230.28891420.23022991X-RAY DIFFRACTION99
5.6423-57.04380.25691620.23293096X-RAY DIFFRACTION98

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