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- PDB-7zcb: Human Pikachurin/EGFLAM N-terminal Fibronectin-III (1-2) domains -

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Basic information

Entry
Database: PDB / ID: 7zcb
TitleHuman Pikachurin/EGFLAM N-terminal Fibronectin-III (1-2) domains
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. / Fibronectin type III domain / EGF-like domain signature 1. / 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: 2.5 Å
AuthorsPantalone, S. / Savino, S. / Viti, L.V. / 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
C: Pikachurin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,9369
Polymers49,5862
Non-polymers1,3507
Water724
1
B: Pikachurin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,3802
Polymers24,7931
Non-polymers5871
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
C: Pikachurin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,5577
Polymers24,7931
Non-polymers7646
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)70.902, 62.077, 106.863
Angle α, β, γ (deg.)90.000, 104.280, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "B" and (resid 34 through 148 or resid 150...
d_2ens_1(chain "C" and (resid 34 through 148 or resid 150...

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_11LYSLYSPROPROBA34 - 12820 - 114
d_12VALVALALAALABA131 - 239117 - 225
d_13NAGNAGNAGNAGAC1
d_14NAGNAGNAGNAGAC1
d_15BMABMABMABMAAC3
d_21LYSLYSPROPROCB34 - 12820 - 114
d_22VALVALALAALACB131 - 239117 - 225
d_23NAGNAGNAGNAGDD1
d_24NAGNAGNAGNAGDD1
d_25BMABMABMABMADD3

NCS oper: (Code: givenMatrix: (-0.619191274329, 0.77313600824, 0.13734219511), (0.782347308381, 0.622400315037, 0.0234635229708), (-0.0673413310153, 0.12197770536, -0.99024571927)Vector: -33. ...NCS oper: (Code: given
Matrix: (-0.619191274329, 0.77313600824, 0.13734219511), (0.782347308381, 0.622400315037, 0.0234635229708), (-0.0673413310153, 0.12197770536, -0.99024571927)
Vector: -33.8811326027, 13.3056784404, 70.4488679252)

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Components

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


Mass: 24793.049 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: N-terminal GSHHHHHHGS sequence and C-terminal AAA sequence are due to cloning into the expression vector used for recombinant protein 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: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#3: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Cl
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / 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: 2.3 Å3/Da / Density % sol: 46.48 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.5 / Details: 18% PEG 3350, 180 mM Sodium Bromide

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-2 / Wavelength: 0.873 Å
DetectorType: DECTRIS PILATUS 2M / Detector: PIXEL / Date: Sep 28, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.873 Å / Relative weight: 1
ReflectionResolution: 2.5→46.06 Å / Num. obs: 15494 / % possible obs: 98.8 % / Redundancy: 3.7 % / Biso Wilson estimate: 53.91 Å2 / CC1/2: 0.994 / Rmerge(I) obs: 0.13 / Rpim(I) all: 0.118 / Net I/σ(I): 6.1
Reflection shellResolution: 2.5→2.6 Å / Redundancy: 4 % / Rmerge(I) obs: 1.594 / Mean I/σ(I) obs: 0.8 / Num. unique obs: 1726 / CC1/2: 0.519 / Rpim(I) all: 1.423 / % possible all: 98.9

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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: 5FM4

5fm4


Resolution: 2.5→46.06 Å / SU ML: 0.4759 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 35.1953
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2771 763 4.94 %
Rwork0.2439 14689 -
obs0.2454 15452 98.24 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 68.02 Å2
Refinement stepCycle: LAST / Resolution: 2.5→46.06 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3052 0 83 4 3139
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0053227
X-RAY DIFFRACTIONf_angle_d0.84454404
X-RAY DIFFRACTIONf_chiral_restr0.0547503
X-RAY DIFFRACTIONf_plane_restr0.0071560
X-RAY DIFFRACTIONf_dihedral_angle_d13.34471214
Refine LS restraints NCSType: Torsion NCS / Rms dev position: 2.43014020407 Å
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.5-2.690.40251610.39312885X-RAY DIFFRACTION98.19
2.69-2.960.36151590.31752928X-RAY DIFFRACTION98.19
2.96-3.390.34391430.30612935X-RAY DIFFRACTION98.62
3.39-4.270.26931590.23142945X-RAY DIFFRACTION98.63
4.27-46.060.21511410.18952996X-RAY DIFFRACTION97.57
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)
14.075207755150.587865022396-0.5834683312215.87842062648-0.9375296426163.622983692930.0453044033911-0.183206049371-0.2787442711540.382186967030.06292837902410.167521433299-0.3650725514520.1420049120060.09894505562230.37600323952-0.05790755647450.04625828472010.4062529043940.0137971508250.45559041794819.13244693154.112255246693.2811310376
20.552567303828-1.362545703-0.7518897017083.482747547232.297998118254.35478802762-0.321715369909-0.831295627130.1485536329870.4527031781830.0967876889430.698640131511-1.11229840213-0.07323627680320.04194783122680.450368515144-0.00334540432669-0.003036257717820.9431847667840.03743869583590.8633341361411.652034835764.338778797293.4521697225
34.75142044983-0.1248732829350.2515804105435.69801367541-2.775365534087.767577044850.0210969019732-0.615384542170.007886633336910.2994943254810.2828270907190.7826177957430.0342021734308-1.19464441165-0.1944238925280.476753322-0.05309439839410.02510062288630.4655019327050.006273952734520.5182097508215.295008067258.095979739592.5707235118
43.241286042061.196260937545.73145011358-0.03679250445181.478226125118.97433404146-0.361734723317-0.03770328579490.515807600097-0.144950650917-0.530166097396-0.00456272932547-1.78875554941-0.1078731085610.3565992570890.574122546289-0.03259303019840.01047170034790.606416951019-0.03197835563260.54473585901416.197812337764.704633277568.0330639746
5-0.02769735576610.370456116379-0.04226311638452.478382155940.6439723006113.396780704160.144323449379-0.910522057396-0.1811113748460.15599761235-0.05986827340020.04717135262290.254358489535-1.23462676657-0.06055527624270.47359221696-0.02673343585790.04386005536280.7719467410950.03129860684280.4056883408834.2803026064655.762268118749.6357406409
63.87718519621-0.1066584684964.236807246290.6036679280130.8263934265046.600026973910.1627373824510.333923562392-0.2682676196330.7459267781610.2579712729870.2236763102850.7519893362520.250381151442-0.2919686748480.793358361517-0.09612824425470.09349958389470.5384217928230.190328468420.7799841244948.0470921471248.534909817151.844528466
74.465902393360.7097197179651.414867672712.608575054340.9484480642178.90641632893-0.11851435803-0.9245951211980.005235326702220.2039913672190.00996923897080.219917958451-0.220906913887-1.579806045860.0367196493650.3739942806730.02841184278730.05954119997670.664617745330.06158461320510.498858431821.1044513440656.809199750448.3477008391
84.144299500161.72214591989-1.124821975143.593272405781.54792990661.72219683408-0.2104662702570.80360854029-0.108558841025-0.7480086832620.0498447580150.213479493222-0.0815978027105-0.252855409040.2573598300410.914734297268-0.031704596188-0.06716623642460.5611363869020.06796715286570.5779572978956.9114945424764.5700431557-17.1892671761
96.840436604324.27255407622-2.125685717492.84831633937-1.717189926821.20730653637-0.252186370351-0.1637098503620.386309173054-0.2481656641620.271736058683-0.2509178212411.339856232130.0221282121051-0.4338161928431.201477766290.0651401521953-0.04049425270620.5800229389550.0414685388640.96843313504120.108148430762.6267941557-13.1169978833
100.6662305238260.225426482507-0.605173072871.78909764681-3.157063024284.00057776254-0.02804245548830.304900637043-0.11039080214-0.3606279142760.214984086675-0.166392438514-0.0624718274258-0.107681573766-0.1915999370960.790358058354-0.0251721788317-0.01436244832140.589747638364-0.0153962991710.59720055996414.567841534765.1039195432-5.28870622001
112.136919557620.300981234558-0.03170583037734.35968657028-2.301351751735.718281056790.0409152090035-0.0324917906128-0.1254022853310.2362435543640.1049153705650.2141685666360.242080811701-0.0518455036136-0.1726624098720.4565643171480.03010886406610.02737278441570.369958951979-0.0104087313290.40928663772113.414637173851.68427719127.2171856988
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'B' and (resid 34 through 74 )BA34 - 741 - 41
22chain 'B' and (resid 75 through 94 )BA75 - 9442 - 52
33chain 'B' and (resid 95 through 128 )BA95 - 12853 - 86
44chain 'B' and (resid 129 through 148 )BA129 - 14887 - 106
55chain 'B' and (resid 149 through 191 )BA149 - 191107 - 149
66chain 'B' and (resid 192 through 203 )BA192 - 203150 - 161
77chain 'B' and (resid 204 through 239 )BA204 - 239162 - 197
88chain 'C' and (resid 34 through 66 )CE34 - 661 - 33
99chain 'C' and (resid 67 through 94 )CE67 - 9434 - 52
1010chain 'C' and (resid 95 through 148 )CE95 - 14853 - 106
1111chain 'C' and (resid 149 through 239 )CE149 - 239107 - 197

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