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- PDB-7zc9: Human Pikachurin/EGFLAM C-terminal Laminin-G domain (LG3) -

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Basic information

Entry
Database: PDB / ID: 7zc9
TitleHuman Pikachurin/EGFLAM C-terminal Laminin-G domain (LG3)
ComponentsPikachurin
KeywordsSIGNALING PROTEIN / Bipolar cells / GPR179 ligand / Proteoglycan / Synapse / Signalosome
Function / homology
Function and homology information


interstitial matrix / photoreceptor ribbon synapse / glycosaminoglycan binding / positive regulation of cell-substrate adhesion / presynaptic active zone / basement membrane / synaptic cleft / extracellular matrix organization / cell projection / calcium ion binding
Similarity search - Function
Laminin G domain / Laminin G domain / Laminin G domain profile. / Laminin G domain / Laminin G domain / EGF-like domain / EGF-like calcium-binding domain / Calcium-binding EGF-like domain / Epidermal growth factor-like domain. / EGF-like domain profile. ...Laminin G domain / Laminin G domain / Laminin G domain profile. / Laminin G domain / Laminin G domain / EGF-like domain / EGF-like calcium-binding domain / Calcium-binding EGF-like domain / Epidermal growth factor-like domain. / EGF-like domain profile. / EGF-like domain signature 2. / EGF-like domain signature 1. / Fibronectin type III domain / EGF-like domain / Fibronectin type 3 domain / Fibronectin type-III domain profile. / Fibronectin type III / Fibronectin type III superfamily / Concanavalin A-like lectin/glucanase domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsPantalone, S. / Forneris, F.
Funding support Switzerland, United States, Italy, 3items
OrganizationGrant numberCountry
Velux Stiftung1375 Switzerland
The Giovanni Armenise-Harvard FoundationCDA 2013 United States
Ministero dell Universita e della RicercaDipartimenti di Eccellenza 2018-2022 Italy
CitationJournal: Sci Signal / Year: 2023
Title: Structure of the photoreceptor synaptic assembly of the extracellular matrix protein pikachurin with the orphan receptor GPR179.
Authors: Dipak N Patil / Serena Pantalone / Yan Cao / Thibaut Laboute / Scott J Novick / Shikha Singh / Simone Savino / Silvia Faravelli / Francesca Magnani / Patrick R Griffin / Appu K Singh / ...Authors: Dipak N Patil / Serena Pantalone / Yan Cao / Thibaut Laboute / Scott J Novick / Shikha Singh / Simone Savino / Silvia Faravelli / Francesca Magnani / Patrick R Griffin / Appu K Singh / Federico Forneris / Kirill A Martemyanov /
Abstract: Precise synapse formation is essential for normal functioning of the nervous system. Retinal photoreceptors establish selective contacts with bipolar cells, aligning the neurotransmitter release ...Precise synapse formation is essential for normal functioning of the nervous system. Retinal photoreceptors establish selective contacts with bipolar cells, aligning the neurotransmitter release apparatus with postsynaptic signaling cascades. This involves transsynaptic assembly between the dystroglycan-dystrophin complex on the photoreceptor and the orphan receptor GPR179 on the bipolar cell, which is mediated by the extracellular matrix protein pikachurin (also known as EGFLAM). This complex plays a critical role in the synaptic organization of photoreceptors and signal transmission, and mutations affecting its components cause blinding disorders in humans. Here, we investigated the structural organization and molecular mechanisms by which pikachurin orchestrates transsynaptic assembly and solved structures of the human pikachurin domains by x-ray crystallography and of the GPR179-pikachurin complex by single-particle, cryo-electron microscopy. The structures reveal molecular recognition principles of pikachurin by the Cache domains of GPR179 and show how the interaction is involved in the transsynaptic alignment of the signaling machinery. Together, these data provide a structural basis for understanding the synaptic organization of photoreceptors and ocular pathology.
History
DepositionMar 26, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 19, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Feb 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
B: Pikachurin
A: Pikachurin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)45,97221
Polymers44,0222
Non-polymers1,95019
Water2,486138
1
B: Pikachurin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)23,19312
Polymers22,0111
Non-polymers1,18211
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
A: Pikachurin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)22,7799
Polymers22,0111
Non-polymers7698
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)73.805, 103.818, 133.534
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Space group name HallC2c2
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y,-z+1/2
#4: -x,-y,z+1/2
#5: x+1/2,y+1/2,z
#6: x+1/2,-y+1/2,-z
#7: -x+1/2,y+1/2,-z+1/2
#8: -x+1/2,-y+1/2,z+1/2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and (resid 829 through 840 or resid 842...
d_2ens_1(chain "B" and (resid 829 through 840 or resid 842...

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11SERSERPHEPHEAB829 - 84010 - 21
d_12GLYGLYGLYGLYAB84223
d_13SERSERLEULEUAB844 - 84625 - 27
d_14TYRTYRPROPROAB848 - 85129 - 32
d_15ILEILELYSLYSAB853 - 85534 - 36
d_16VALVALGLYGLYAB857 - 85938 - 40
d_17ARGARGARGARGAB861 - 93242 - 113
d_18GLYGLYILEILEAB934 - 939115 - 120
d_19VALVALTHRTHRAB941 - 995122 - 176
d_110TYRTYRLEULEUAB997 - 1001178 - 182
d_111ASPASPALAALAAB1004 - 1005185 - 186
d_112ASPASPASNASNAB1007 - 1010188 - 191
d_113ASNASNLYSLYSAB1012 - 1017193 - 198
d_21SERSERPHEPHEBA829 - 84010 - 21
d_22GLYGLYGLYGLYBA84223
d_23SERSERLEULEUBA844 - 84625 - 27
d_24TYRTYRPROPROBA848 - 85129 - 32
d_25ILEILELYSLYSBA853 - 85534 - 36
d_26VALVALGLYGLYBA857 - 85938 - 40
d_27ARGARGARGARGBA861 - 93242 - 113
d_28GLYGLYILEILEBA934 - 939115 - 120
d_29VALVALTHRTHRBA941 - 995122 - 176
d_210TYRTYRLEULEUBA997 - 1001178 - 182
d_211ASPASPALAALABA1004 - 1005185 - 186
d_212ASPASPASNASNBA1007 - 1010188 - 191
d_213ASNASNLYSLYSBA1012 - 1017193 - 198

NCS oper: (Code: givenMatrix: (-0.989926193842, -0.077796521342, 0.11829552829), (0.0687717957614, -0.994535068142, -0.0785521377409), (0.123760134349, -0.0696254228209, 0.989866521124)Vector: 44. ...NCS oper: (Code: given
Matrix: (-0.989926193842, -0.077796521342, 0.11829552829), (0.0687717957614, -0.994535068142, -0.0785521377409), (0.123760134349, -0.0696254228209, 0.989866521124)
Vector: 44.4180938738, 43.4217849964, 2.95110047811)

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Components

#1: Protein Pikachurin / Agrin-like protein / EGF-like / fibronectin type-III and laminin G-like domain-containing protein


Mass: 22010.971 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: N-terminal GSHHHHHHGSA sequence and C-terminal AAA sequence were introduced by cloning into the expression vector for recombinant expression
Source: (gene. exp.) Homo sapiens (human) / Gene: EGFLAM, AGRINL, AGRNL, PIKA / Plasmid: pUPE.06.03 / Cell line (production host): HEK293-F / Production host: Homo sapiens (human) / References: UniProt: Q63HQ2
#2: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#3: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: SO4
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 138 / Source method: isolated from a natural source / Formula: H2O
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: 2.91 Å3/Da / Density % sol: 57.66 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.6
Details: 1.5-2.0 M ammonium sulfate 100 mM sodium acetate pH 4.6

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 0.976 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Apr 20, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976 Å / Relative weight: 1
ReflectionResolution: 2.1→48.38 Å / Num. obs: 30331 / % possible obs: 100 % / Redundancy: 7.3 % / Biso Wilson estimate: 39.94 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.098 / Rpim(I) all: 0.058 / Net I/σ(I): 11.9
Reflection shellResolution: 2.1→2.16 Å / Redundancy: 7.6 % / Rmerge(I) obs: 1.276 / Mean I/σ(I) obs: 1.7 / Num. unique obs: 2451 / CC1/2: 0.689 / Rpim(I) all: 0.742 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.20_4459refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
ARP/wARPmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3SH4

3sh4


Resolution: 2.1→48.38 Å / SU ML: 0.2984 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 27.3669
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2459 1492 4.93 %
Rwork0.2066 28791 -
obs0.2085 30283 99.88 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 50.45 Å2
Refinement stepCycle: LAST / Resolution: 2.1→48.38 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2910 0 104 138 3152
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00313135
X-RAY DIFFRACTIONf_angle_d0.64414258
X-RAY DIFFRACTIONf_chiral_restr0.0501460
X-RAY DIFFRACTIONf_plane_restr0.0041540
X-RAY DIFFRACTIONf_dihedral_angle_d12.55591156
Refine LS restraints NCSType: Torsion NCS / Rms dev position: 0.951435972577 Å
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.1-2.170.35661310.30452585X-RAY DIFFRACTION100
2.17-2.250.27221340.27262582X-RAY DIFFRACTION99.93
2.25-2.340.29551360.26392577X-RAY DIFFRACTION100
2.34-2.440.33771340.2672594X-RAY DIFFRACTION99.93
2.44-2.570.27571390.24412596X-RAY DIFFRACTION99.96
2.57-2.730.31541270.24012610X-RAY DIFFRACTION100
2.73-2.940.26691310.23042604X-RAY DIFFRACTION99.96
2.94-3.240.26591580.21562599X-RAY DIFFRACTION99.89
3.24-3.710.19871240.18142652X-RAY DIFFRACTION99.89
3.71-4.670.19561330.15762637X-RAY DIFFRACTION99.6
4.67-48.380.241450.20212755X-RAY DIFFRACTION99.59
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)
1-0.00119982025341-0.0341319237442-0.1780925837010.326832293980.2259409527110.555768473008-0.07562805431090.2111886624440.007465603427570.120668035462-0.105287503672-0.02734372839280.0997620454037-0.0966134011710.0001061182636170.350512106807-0.0397766746349-0.04532704739580.508062437703-0.001894623639760.31120785849327.7433993359-0.417790392996-24.7339432443
20.665899044956-0.6945003597970.5048163190182.21290494681.102851716081.8960407631-0.1347330255750.005244170145520.1183484903930.2269332954510.03763982009750.0451910696235-0.193449238957-0.04735845598877.59900639516E-50.375595080002-0.00928927143353-0.04657910779780.3260574803510.03219432187270.38081107480527.803550298313.1656748911-12.3187635544
30.397982218069-0.14027605605-0.5619057537210.7922009338660.869342158664.10236248279-0.464497985997-0.1213807404330.4241318784920.5484598245690.152159318773-0.5686074634720.5410718115020.855544543359-0.2847423591580.3758719266230.0035391448896-0.1632526965580.390396229303-0.01361174535790.47150041575336.662961169715.478249125-9.10681846417
40.398560051357-0.824022238318-0.3291401203992.169644558831.175477874390.730527223992-0.281535947813-0.1313676907440.3059065497820.455674994816-0.0355280086350.251254779904-0.120760463192-0.102075823624-0.0004721893703360.489627244526-0.00504540632805-0.09223166592120.3941016185560.02928585510130.3626667253725.960658502213.2630149871-7.90951104278
50.1349624213040.02941909247220.02408465078730.120576642473-0.0626015309890.06101719840710.2400701116830.2097990708760.348990817013-0.202977598815-0.4895759597880.0961105764087-0.533415780479-1.24864804287-0.0006652157485080.5850259834340.0866652484678-0.02943375331380.594893207834-0.01196596347930.42315778971417.968485846215.7533762639-16.5424547049
60.945714754369-0.88208556419-0.3840421127070.9264696645250.861696163740.8228394099250.06084171090340.209511564307-0.119411925120.03123998220910.0055464928684-0.04491786268650.3274070379240.123115261078-0.0001500907908650.3936297866370.0100013799654-0.03160198385070.385216927666-0.02148146535230.34334015717929.96853818751.28046071638-20.1878316617
70.467230887268-0.8747185617830.1043487943291.243016077620.2023132529960.372345624256-0.01101583224570.355483731610.06056821023650.213003396063-0.142139870006-0.144698420932-0.1641396101230.1681357147660.001630621680110.368323057624-0.004311310125170.009404945844440.4241535013670.04158803331860.42219605572810.222631880745.7690539108-19.7657283682
80.381818473044-0.710754879652-0.1222957616142.311492115090.5737745756520.2023808112090.00208185278276-0.02462537208220.05502003953170.3199150456910.100886778035-0.363863773720.1328782147770.127593826983-0.0001602546249660.4480493963420.0478844259839-0.01436336266950.393545600230.003863266008870.44603149863214.68570583634.4879200562-14.9303413248
90.1279679396960.1356006067060.005555635030250.157239362323-0.1855618593390.2453598400210.04793751680030.2256004420550.160356145789-0.115464358104-0.1437605179930.04670760425830.4884957840310.110601804278-0.0001377598623750.4038696218950.0519273018191-0.01818432633240.398621017756-0.02215626167040.42942254030113.716048937328.1037293414-15.2495077349
101.77359983269-1.08775614-0.4060086326922.09912300108-0.7977706471411.41569046537-0.07235370693790.0838978141714-0.1678038341110.2950880254080.06208025919860.2571162832010.07672716531980.0219053090599-3.70415459288E-50.48219390921-0.00324096036325-0.02137532217760.340738714378-0.003123866088120.4245924567928.2972538610927.9643304202-11.8553554883
110.58944172473-0.299767524196-0.2714645828780.310258487097-0.03847078436460.121561602368-0.29424244449-0.234437282910.2220666139060.03647684383150.00722856851493-0.480145508707-0.1013559593160.2311874008083.8299043307E-50.3697535280470.0626856821142-0.03762525947290.4719433964010.07050189939680.49691995610322.009960480734.9735076154-19.2294584039
120.222587824995-0.2520059921690.4249023177490.941377189925-0.1667110150830.556545190521-0.08581721343290.1210233282360.1388236477040.274614374073-0.05230574812520.0586303157914-0.12285585555-0.156192280336-0.0003487366160530.4103055666090.05262156538180.008187348343350.4137054681540.06587935801760.4381667403348.3569209727644.9608579842-21.1113057298
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'B' and (resid 829 through 850 )BA829 - 8501 - 22
22chain 'B' and (resid 851 through 925 )BA851 - 92523 - 97
33chain 'B' and (resid 926 through 941 )BA926 - 94198 - 113
44chain 'B' and (resid 942 through 975 )BA942 - 975114 - 147
55chain 'B' and (resid 976 through 984 )BA976 - 984148 - 156
66chain 'B' and (resid 985 through 1017 )BA985 - 1017157 - 189
77chain 'A' and (resid 829 through 860 )AC829 - 8601 - 32
88chain 'A' and (resid 861 through 890 )AC861 - 89033 - 62
99chain 'A' and (resid 891 through 906 )AC891 - 90663 - 78
1010chain 'A' and (resid 907 through 963 )AC907 - 96379 - 135
1111chain 'A' and (resid 964 through 984 )AC964 - 984136 - 156
1212chain 'A' and (resid 985 through 1017 )AC985 - 1017157 - 189

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