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- PDB-6s6d: Crystal structure of RagA-Q66L-GTP/RagC-S75N-GDP GTPase heterodim... -

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Basic information

Entry
Database: PDB / ID: 6s6d
TitleCrystal structure of RagA-Q66L-GTP/RagC-S75N-GDP GTPase heterodimer complex
Components(Ras-related GTP-binding protein ...) x 2
KeywordsSIGNALING PROTEIN / small GTPases / mTORC1 activator / roadblock domain / GTPase domain
Function / homology
Function and homology information


Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / regulation of TORC1 signaling / protein localization to lysosome / regulation of TOR signaling / MTOR signalling / Amino acids regulate mTORC1 / Energy dependent regulation of mTOR by LKB1-AMPK / protein localization to membrane / enzyme-substrate adaptor activity ...Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / regulation of TORC1 signaling / protein localization to lysosome / regulation of TOR signaling / MTOR signalling / Amino acids regulate mTORC1 / Energy dependent regulation of mTOR by LKB1-AMPK / protein localization to membrane / enzyme-substrate adaptor activity / Macroautophagy / small GTPase-mediated signal transduction / mTORC1-mediated signalling / cellular response to nutrient levels / positive regulation of TOR signaling / response to amino acid / protein-membrane adaptor activity / positive regulation of TORC1 signaling / tumor necrosis factor-mediated signaling pathway / cellular response to amino acid starvation / cellular response to starvation / negative regulation of autophagy / RNA splicing / Regulation of PTEN gene transcription / cellular response to amino acid stimulus / TP53 Regulates Metabolic Genes / phosphoprotein binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / protein localization / GDP binding / glucose homeostasis / E3 ubiquitin ligases ubiquitinate target proteins / GTPase binding / lysosome / molecular adaptor activity / intracellular signal transduction / protein heterodimerization activity / lysosomal membrane / intracellular membrane-bounded organelle / GTPase activity / DNA-templated transcription / apoptotic process / ubiquitin protein ligase binding / GTP binding / magnesium ion binding / protein homodimerization activity / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
Beta-Lactamase - #190 / RagA/B / Gtr1/RagA G protein / RagC/D / Gtr1/RagA G protein conserved region / Beta-Lactamase / P-loop containing nucleotide triphosphate hydrolases / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / 2-Layer Sandwich ...Beta-Lactamase - #190 / RagA/B / Gtr1/RagA G protein / RagC/D / Gtr1/RagA G protein conserved region / Beta-Lactamase / P-loop containing nucleotide triphosphate hydrolases / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
pentane-1,5-diol / GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Ras-related GTP-binding protein A / Ras-related GTP-binding protein C
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.5 Å
AuthorsAnandapadamanaban, M. / Masson, G.R. / Perisic, O. / Kaufman, J. / Williams, R.L.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_U105184308 United Kingdom
Cancer Research UKC14801/A21211 United Kingdom
CitationJournal: Science / Year: 2019
Title: Architecture of human Rag GTPase heterodimers and their complex with mTORC1.
Authors: Madhanagopal Anandapadamanaban / Glenn R Masson / Olga Perisic / Alex Berndt / Jonathan Kaufman / Chris M Johnson / Balaji Santhanam / Kacper B Rogala / David M Sabatini / Roger L Williams /
Abstract: The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag ...The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo-electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagA/RagC nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes.
History
DepositionJul 2, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 16, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2May 15, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_conn_angle / struct_conn
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_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ras-related GTP-binding protein A
C: Ras-related GTP-binding protein C
B: Ras-related GTP-binding protein A
D: Ras-related GTP-binding protein C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)163,98812
Polymers161,7984
Non-polymers2,1908
Water93752
1
A: Ras-related GTP-binding protein A
C: Ras-related GTP-binding protein C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)81,8905
Polymers80,8992
Non-polymers9913
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5330 Å2
ΔGint-42 kcal/mol
Surface area26220 Å2
MethodPISA
2
B: Ras-related GTP-binding protein A
D: Ras-related GTP-binding protein C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)82,0987
Polymers80,8992
Non-polymers1,1995
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4680 Å2
ΔGint-40 kcal/mol
Surface area26540 Å2
MethodPISA
Unit cell
Length a, b, c (Å)71.504, 81.439, 246.033
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

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Ras-related GTP-binding protein ... , 2 types, 4 molecules ABCD

#1: Protein Ras-related GTP-binding protein A / RagA / Adenovirus E3 14.7 kDa-interacting protein 1 / FIP-1


Mass: 36600.195 Da / Num. of mol.: 2 / Mutation: Q66L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RRAGA / Production host: Escherichia coli (E. coli) / References: UniProt: Q7L523
#2: Protein Ras-related GTP-binding protein C / RagC / GTPase-interacting protein 2 / TIB929


Mass: 44298.859 Da / Num. of mol.: 2 / Mutation: S75N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RRAGC / Production host: Escherichia coli (E. coli) / References: UniProt: Q9HB90

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Non-polymers , 5 types, 60 molecules

#3: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C10H16N5O14P3 / Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli) / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, energy-carrying molecule*YM
#6: Chemical ChemComp-9JE / pentane-1,5-diol / Pentane-1,5-diol


Mass: 104.148 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C5H12O2
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 52 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: 2.36 Å3/Da / Density % sol: 47.92 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 100mM MOPSO, Bis-Tris pH 6.5, 40mM polyamines ( mixture of spermine tetrahydrochloride, spermidine trihydrochloride, 1,4. diaminobutane dihydrochloride, 0.1 M D/L-ornithine monohydrochloride) ...Details: 100mM MOPSO, Bis-Tris pH 6.5, 40mM polyamines ( mixture of spermine tetrahydrochloride, spermidine trihydrochloride, 1,4. diaminobutane dihydrochloride, 0.1 M D/L-ornithine monohydrochloride), 10% (w/v) PEG 8000. 20% (v/v) 1,5 - Pentanediol and 24 % (v/v) glycerol.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID30B / Wavelength: 0.9762 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: May 11, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9762 Å / Relative weight: 1
ReflectionResolution: 2.5→49.29 Å / Num. obs: 50589 / % possible obs: 99.8 % / Redundancy: 6.558 % / Biso Wilson estimate: 70.517 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.07 / Rrim(I) all: 0.077 / Χ2: 1.037 / Net I/σ(I): 16.69 / Num. measured all: 331756
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsCC1/2Rrim(I) all% possible all
2.5-2.656.6711.0471.9453487809980180.6541.13699
2.65-2.836.8110.643.3551523756675650.8560.693100
2.83-3.066.5230.3555.7746095706770660.9510.386100
3.06-3.356.7060.17111.3943801653465320.9880.186100
3.35-3.746.6920.08620.3740059598859860.9960.093100
3.74-4.326.4140.04930.8833774526852660.9980.053100
4.32-5.286.5760.03639.1229539449344920.9990.04100
5.28-7.426.1060.03936.9421812357535720.9990.04299.9
7.42-49.295.5760.03345.8811666212020920.9980.03698.7

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
REFMAC5.8.0238refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.5→49.29 Å / Cor.coef. Fo:Fc: 0.954 / Cor.coef. Fo:Fc free: 0.918 / SU B: 12.223 / SU ML: 0.268 / SU R Cruickshank DPI: 0.526 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.526 / ESU R Free: 0.325
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2866 2458 4.9 %RANDOM
Rwork0.209 ---
obs0.2127 48131 99.72 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 189.27 Å2 / Biso mean: 72.597 Å2 / Biso min: 33.2 Å2
Baniso -1Baniso -2Baniso -3
1-0.96 Å20 Å20 Å2
2---0.42 Å20 Å2
3----0.54 Å2
Refinement stepCycle: final / Resolution: 2.5→49.29 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9384 0 136 52 9572
Biso mean--66.41 66.79 -
Num. residues----1156
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.0139711
X-RAY DIFFRACTIONr_bond_other_d0.0010.0179039
X-RAY DIFFRACTIONr_angle_refined_deg1.7041.64413112
X-RAY DIFFRACTIONr_angle_other_deg1.2441.58320945
X-RAY DIFFRACTIONr_dihedral_angle_1_deg8.26451152
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.40422.448531
X-RAY DIFFRACTIONr_dihedral_angle_3_deg19.199151779
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.9921562
X-RAY DIFFRACTIONr_chiral_restr0.0740.21285
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.0210622
X-RAY DIFFRACTIONr_gen_planes_other0.0010.022088
LS refinement shellResolution: 2.5→2.565 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.448 189 -
Rwork0.352 3453 -
all-3642 -
obs--97.82 %

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