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- PDB-4e6r: Crystal structure of a Cytoplasmic protein NCK2 (NCK2) from Homo ... -

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Basic information

Entry
Database: PDB / ID: 4e6r
TitleCrystal structure of a Cytoplasmic protein NCK2 (NCK2) from Homo sapiens at 2.20 A resolution
ComponentsCytoplasmic protein NCK2
KeywordsSUGAR BINDING PROTEIN / SH3 domain / protein binding / Structural Genomics / Joint Center for Structural Genomics / JCSG / Protein Structure Initiative / PSI-BIOLOGY / Partnership for T-Cell Biology / TCELL
Function / homology
Function and homology information


Regulation of cortical dendrite branching / negative regulation of endoplasmic reticulum stress-induced eIF2 alpha phosphorylation / positive regulation of peptidyl-serine dephosphorylation / positive regulation of translation in response to endoplasmic reticulum stress / positive regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / immunological synapse formation / dendritic spine development / cytoskeletal anchor activity / vesicle membrane / signal complex assembly ...Regulation of cortical dendrite branching / negative regulation of endoplasmic reticulum stress-induced eIF2 alpha phosphorylation / positive regulation of peptidyl-serine dephosphorylation / positive regulation of translation in response to endoplasmic reticulum stress / positive regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / immunological synapse formation / dendritic spine development / cytoskeletal anchor activity / vesicle membrane / signal complex assembly / Activation of RAC1 / Nephrin family interactions / Ephrin signaling / lamellipodium assembly / RHOV GTPase cycle / positive regulation of actin filament polymerization / negative regulation of PERK-mediated unfolded protein response / RHOU GTPase cycle / ephrin receptor signaling pathway / signaling adaptor activity / positive regulation of T cell proliferation / T cell activation / phosphotyrosine residue binding / Downstream signal transduction / actin filament organization / epidermal growth factor receptor signaling pathway / receptor tyrosine kinase binding / VEGFA-VEGFR2 Pathway / signaling receptor complex adaptor activity / cell migration / scaffold protein binding / postsynaptic density / negative regulation of cell population proliferation / protein-containing complex binding / negative regulation of transcription by RNA polymerase II / endoplasmic reticulum / signal transduction / positive regulation of transcription by RNA polymerase II / cytoplasm / cytosol
Similarity search - Function
Nck2, SH3 domain 1 / Nck2, SH3 domain 2 / Nck2, SH3 domain 3 / Nck2, SH2 domain / Cytoplasmic protein NCK / Variant SH3 domain / SH3 Domains / SH3 domain / SH2 domain / Src homology 2 (SH2) domain profile. ...Nck2, SH3 domain 1 / Nck2, SH3 domain 2 / Nck2, SH3 domain 3 / Nck2, SH2 domain / Cytoplasmic protein NCK / Variant SH3 domain / 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 / Roll / Mainly Beta
Similarity search - Domain/homology
IMIDAZOLE / Unknown ligand / Cytoplasmic protein NCK2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.2 Å
AuthorsJoint Center for Structural Genomics (JCSG) / Partnership for T-Cell Biology (TCELL)
CitationJournal: To be published
Title: Crystal structure of a Cytoplasmic protein NCK2 (NCK2) from Homo sapiens at 2.20 A resolution
Authors: Joint Center for Structural Genomics (JCSG) / Partnership for T-Cell Biology (TCELL)
History
DepositionMar 15, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 4, 2012Provider: repository / Type: Initial release
Revision 1.1Oct 21, 2015Group: Database references / Structure summary
Revision 1.2Nov 15, 2017Group: Refinement description / Category: software / Item: _software.classification / _software.name
Revision 1.3Feb 1, 2023Group: Database references / Derived calculations
Category: database_2 / pdbx_struct_conn_angle ...database_2 / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _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_asym_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_ref_seq_dif.details / _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: Cytoplasmic protein NCK2
B: Cytoplasmic protein NCK2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)14,02411
Polymers13,5592
Non-polymers4659
Water59433
1
A: Cytoplasmic protein NCK2
B: Cytoplasmic protein NCK2
hetero molecules

A: Cytoplasmic protein NCK2
B: Cytoplasmic protein NCK2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,04822
Polymers27,1184
Non-polymers93018
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation10_665-y+1,-x+1,-z+1/61
Buried area4500 Å2
ΔGint-270 kcal/mol
Surface area14050 Å2
MethodPISA
Unit cell
Length a, b, c (Å)108.398, 108.398, 68.199
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number179
Space group name H-MP6522
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22B

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1116A0 - 170
2116B0 - 170
1126B201 - 500
2126A206 - 500

NCS ensembles :
ID
1
2
DetailsCRYSTAL PACKING ANALYSIS SUGGESTS THE ASSIGNMENT OF A TETRAMER AS THE SIGNIFICANT OLIGOMERIZATION STATE.

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Components

#1: Protein Cytoplasmic protein NCK2 / Growth factor receptor-bound protein 4 / NCK adaptor protein 2 / Nck-2 / SH2/SH3 adaptor protein NCK-beta


Mass: 6779.484 Da / Num. of mol.: 2 / Fragment: SH3 2 domain residues 114-170
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BC007195, GRB4, NCK2 / Plasmid: SpeedET / Production host: Escherichia Coli (E. coli) / Strain (production host): PB1 / References: UniProt: O43639
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Zn
#3: Chemical ChemComp-IMD / IMIDAZOLE


Mass: 69.085 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H5N2
#4: Chemical ChemComp-UNL / UNKNOWN LIGAND


Num. of mol.: 2 / Source method: obtained synthetically
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 33 / Source method: isolated from a natural source / Formula: H2O
Sequence details1. THIS CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED ...1. THIS CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH TEV PROTEASE LEAVING ONLY A GLYCINE (0) FOLLOWED BY RESIDUES 114-170 OF THE TARGET SEQUENCE. 2. THE PROTEIN WAS REDUCTIVELY METHYLATED PRIOR TO CRYSTALLIZATION.

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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: 4.27 Å3/Da / Density % sol: 71.16 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 8
Details: 40.00% polyethylene glycol 300, 0.20M zinc acetate, 0.1M Imidazole pH 8.0, NANODROP, VAPOR DIFFUSION, SITTING DROP, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL9-2 / Wavelength: 0.91837,0.97949,0.97929
DetectorType: MARMOSAIC 325 mm CCD / Detector: CCD / Date: Jan 12, 2012 / Details: double crystal monochromator
RadiationMonochromator: double crystal / Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.918371
20.979491
30.979291
ReflectionResolution: 2.2→28.862 Å / Num. all: 12480 / Num. obs: 12480 / % possible obs: 100 % / Redundancy: 14 % / Biso Wilson estimate: 40.198 Å2 / Rsym value: 0.2 / Net I/σ(I): 11.4
Reflection shell

Rmerge(I) obs: 0.021 / Diffraction-ID: 1

Resolution (Å)Redundancy (%)Mean I/σ(I) obsNum. measured allNum. unique allRsym value% possible all
2.2-2.2614.40.3128708962.069100
2.26-2.3214.20.4124818761.717100
2.32-2.3914.30.4121598501.822100
2.39-2.4614.40.5119478321.311100
2.46-2.5414.20.6113537991.082100
2.54-2.6314.30.8112987910.887100
2.63-2.7314.31108967640.688100
2.73-2.8414.31.3103787260.532100
2.84-2.9714.21.998356930.373100
2.97-3.1114.22.796736810.267100
3.11-3.2814.2490926420.181100
3.28-3.48145.285876120.137100
3.48-3.72146.379995700.112100
3.72-4.0213.8776275520.097100
4.02-4.413.8868794970.083100
4.4-4.9213.68.562894640.075100
4.92-5.6813.58.755464120.077100
5.68-6.9613946443570.075100
6.96-9.8412.59.436482930.07100
9.84-28.86210.810.818651730.0696.2

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Phasing

PhasingMethod: MAD

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Processing

Software
NameVersionClassificationNB
MolProbity3beta29model building
PDB_EXTRACT3.1data extraction
SHELXphasing
SHARPphasing
SCALA3.3.20data scaling
REFMAC5.6.0117refinement
MOSFLMdata reduction
SHELXDphasing
RefinementMethod to determine structure: MAD / Resolution: 2.2→28.862 Å / Cor.coef. Fo:Fc: 0.95 / Cor.coef. Fo:Fc free: 0.94 / Occupancy max: 1 / Occupancy min: 0.4 / SU B: 9.612 / SU ML: 0.125 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.165 / ESU R Free: 0.15
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: 1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. 2. ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS. 3. ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS. 4. WATERS WERE ...Details: 1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. 2. ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS. 3. ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS. 4. WATERS WERE EXCLUDED FROM AUTOMATIC TLS ASSIGNMENT. 5. A MET-INHIBITION PROTOCOL WAS USED FOR SELENOMETHIONINE INCORPORATION DURING PROTEIN EXPRESSION. THE OCCUPANCY OF THE SE ATOMS IN THE MSE RESIDUES WAS REDUCED TO 0.75 FOR THE REDUCED SCATTERING POWER DUE TO PARTIAL S-MET INCORPORATION. 6. X-RAY FLUORESCENCE EXCITATION AND WAVELENGTH SCANS, ANOMALOUS DIFFERENCE FOURIERS AND ITS PRESENCE IN CRYSTALLIZATION SOLUTION SUPPORT THE MODELING OF ZINC (ZN) IONS. 7. LYSINE RESIDUES WERE METHYLATED PRIOR TO CRYSTALLIZATION AND ARE MODELED AS DIMETHYL-LYSINE (MLY). THE N-TERMINAL GLYCINE RESIDUES WERE ALSO METHYLATED IN THIS PROCEDURE AND ARE MODELED AS N,N-DIMETHYLGLYCINE (DMG). 8. IMIDAZOLE (IMD) FROM THE CRYSTALLIZATION CONDITION HAS BEEN MODELED. 9. AN UNKNOWN LIGAND (UNL) HAS BEEN MODELED IN EACH CHAIN.
RfactorNum. reflection% reflectionSelection details
Rfree0.2269 599 4.8 %RANDOM
Rwork0.2012 ---
obs0.2024 12456 99.78 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso max: 152.08 Å2 / Biso mean: 58.2369 Å2 / Biso min: 26.18 Å2
Baniso -1Baniso -2Baniso -3
1-2.58 Å21.29 Å20 Å2
2--2.58 Å20 Å2
3----3.87 Å2
Refinement stepCycle: LAST / Resolution: 2.2→28.862 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms952 0 33 33 1018
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.021007
X-RAY DIFFRACTIONr_bond_other_d0.0010.02686
X-RAY DIFFRACTIONr_angle_refined_deg1.7691.9771367
X-RAY DIFFRACTIONr_angle_other_deg3.91631647
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.1765120
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.5923.67349
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.60215131
X-RAY DIFFRACTIONr_dihedral_angle_4_deg21.204156
X-RAY DIFFRACTIONr_chiral_restr0.0910.2141
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.021124
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02242
Refine LS restraints NCS

Dom-ID: 1 / Auth asym-ID: A / Refine-ID: X-RAY DIFFRACTION

Ens-IDNumberTypeRms dev position (Å)Weight position
1797LOOSE POSITIONAL0.555
1797LOOSE THERMAL5.7910
29LOOSE POSITIONAL0.45
29LOOSE THERMAL4.710
LS refinement shellResolution: 2.2→2.257 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.282 40 -
Rwork0.285 746 -
all-786 -
obs--100 %
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)
12.7965-0.45660.28376.3993-0.63644.921-0.0293-0.14630.20060.86850.05470.1527-0.41380.0572-0.02540.29150.05350.08320.02990.04760.6301-17.21988.728.907
25.91710.18631.24123.478-0.25153.32450.0996-0.6244-0.18710.43960.07620.2243-0.1036-0.3075-0.17580.06250.0046-0.00160.0910.05140.4596-11.45365.1519.591
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A0 - 170
2X-RAY DIFFRACTION2B0 - 170

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