PDB-3llu: Crystal structure of the nucleotide-binding domain of Ras-related...

Basic information

• Entry: Database: PDB / ID: 3llu
• Title: Crystal structure of the nucleotide-binding domain of Ras-related GTP-binding protein C
• Components: Ras-related GTP-binding protein C
• Keywords: HYDROLASE / Structural Genomics Consortium / SGC / Cytoplasm / GTP-binding / Nucleotide-binding / Nucleus / Phosphoprotein

Function / homology

Function:

Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / regulation of TORC1 signaling / protein localization to lysosome / regulation of TOR signaling / Amino acids regulate mTORC1 / MTOR signalling / Energy dependent regulation of mTOR by LKB1-AMPK / protein localization to membrane / enzyme-substrate adaptor activity / small GTPase-mediated signal transduction / Macroautophagy / mTORC1-mediated signalling / response to amino acid / cellular response to nutrient levels / protein-membrane adaptor activity / positive regulation of TORC1 signaling / cellular response to amino acid starvation / cellular response to starvation / RNA splicing / negative regulation of autophagy / Regulation of PTEN gene transcription / TP53 Regulates Metabolic Genes / cellular response to amino acid stimulus / GDP binding / protein localization / GTPase binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / molecular adaptor activity / lysosome / protein heterodimerization activity / lysosomal membrane / intracellular membrane-bounded organelle / GTPase activity / DNA-templated transcription / GTP binding / apoptotic process / magnesium ion binding / nucleoplasm / nucleus / cytosol / cytoplasm

Domain/homology:

Gtr1/RagA G protein / RagC/D / Gtr1/RagA G protein conserved region / P-loop containing nucleotide triphosphate hydrolases / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Alpha Beta

Component:

PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / Ras-related GTP-binding protein C

• Biological species: Homo sapiens (human)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.4 Å
• Authors: Nedyalkova, L. / Tempel, W. / Tong, Y. / Crombet, L. / Zhong, N. / Guan, X. / Arrowsmith, C.H. / Edwards, A.M. / Bountra, C. / Weigelt, J. / Bochkarev, A. / Park, H. / Structural Genomics Consortium (SGC)
• Citation: Journal: to be published; Title: Crystal structure of the nucleotide-binding domain of Ras-related GTP-binding protein C; Authors: Nedyalkova, L. / Tempel, W. / Tong, Y. / Crombet, L. / Zhong, N. / Guan, X. / Arrowsmith, C.H. / Edwards, A.M. / Bountra, C. / Weigelt, J. / Bochkarev, A. / Park, H.

History

Deposition	Jan 29, 2010	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Feb 9, 2010	Provider: repository / Type: Initial release
Revision 1.1	Jul 13, 2011	Group: Version format compliance
Revision 1.2	Nov 1, 2017	Group: Refinement description / Category: software
Revision 1.3	Sep 6, 2023	Group: Data collection / Database references / Derived calculations / Refinement description Category: chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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.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.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_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

Assembly

Deposited unit

A: Ras-related GTP-binding protein C

hetero molecules

Summary:

• 23.4 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	23,442	12
Polymers	22,896	1
Non-polymers	547	11
Water	1,531	85

1

A: Ras-related GTP-binding protein C

hetero molecules

A: Ras-related GTP-binding protein C

hetero molecules

A: Ras-related GTP-binding protein C

hetero molecules

A: Ras-related GTP-binding protein C

hetero molecules

Summary:

• defined by software
• 93.8 kDa, 4 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	93,770	48
Polymers	91,584	4
Non-polymers	2,186	44
Water	72	4

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	2_665	-x+1,-y+1,z	1
crystal symmetry operation	3_655	-y+1,x,z	1
crystal symmetry operation	4_565	y,-x+1,z	1

Calculated values:

Buried area	8450 Å2
ΔGint	-91 kcal/mol
Surface area	32420 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	72.220, 72.220, 72.182
Angle α, β, γ (deg.)	90.000, 90.000, 90.000
Int Tables number	79
Space group name H-M	I4

Components

#1: Protein

A Ras-related GTP-binding protein C / Rag C / RagC / GTPase-interacting protein 2 / TIB929

Details:

Mass: 22895.889 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RRAGC / Plasmid: pET28-MHL / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-V2R-pRARE2 / References: UniProt: Q9HB90

#2: Chemical

A-501 ChemComp-GNP / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER

Details:

Mass: 522.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₁₀H₁₇N₆O₁₃P₃
Comment: GppNHp, GMPPNP, energy-carrying molecule analogue*YM

#3: Chemical

A-502 ChemComp-MG / MAGNESIUM ION

Details:

Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

#4: Chemical

A-1 A-2 A-3 A-4 A-5 A-6 A-7 A-8 A-9
ChemComp-UNX / UNKNOWN ATOM OR ION

Details:

Num. of mol.: 9 / Source method: obtained synthetically

#5: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 85 / Source method: isolated from a natural source / Formula: H₂O

Experimental details

Experiment

• Experiment: Method: X-RAY DIFFRACTION / Number of used crystals: 1

Sample preparation

• Crystal: Density Matthews: 2.06 ų/Da / Density % sol: 40.16 %
• Crystal grow: Temperature: 291 K / Method: vapor diffusion, sitting drop / pH: 7.5 ; Details: 2.0M ammonium sulfate, 0.2M sodium chloride, 0.1M HEPES. 1:100 (w/w) papain and GMPPNP were also added. Please note: mass spectral analysis of TEV protease digest prior to crystallization suggested that removal of the His-tag was incomplete even after 2 days. However, without TEV protease treatment no crystals were obtained using otherwise identical crystallization conditions. Also note the presence of papain in the crystallization drop., pH 7.5, vapor diffusion, sitting drop, temperature 291K

Data collection

• Diffraction: Mean temperature: 100 K
• Diffraction source: Source: SYNCHROTRON / Site: APS / Beamline: 19-ID / Wavelength: 0.97934 Å
• Detector: Type: ADSC Q315 / Detector: CCD / Date: Dec 16, 2009
• Radiation: Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 0.97934 Å / Relative weight: 1
• Reflection: Resolution: 1.4→25 Å / Num. obs: 36466 / % possible obs: 100 % / Redundancy: 7.3 % / R_merge(I) obs: 0.055 / Χ²: 1.519 / Net I/σ(I): 12.5

Reflection shell

Resolution (Å)	Redundancy (%)	Rmerge(I) obs	Num. unique all	Χ2	% possible all
1.4-1.42	7.2	0.816	1835	1.996	100
1.42-1.45	7.2	0.705	1787	2.023	100
1.45-1.48	7.3	0.589	1836	1.788	100
1.48-1.51	7.3	0.502	1781	1.785	100
1.51-1.54	7.3	0.417	1819	1.589	100
1.54-1.58	7.3	0.333	1841	1.515	100
1.58-1.62	7.3	0.275	1804	1.462	100
1.62-1.66	7.3	0.237	1807	1.396	100
1.66-1.71	7.4	0.2	1826	1.383	100
1.71-1.76	7.4	0.16	1825	1.336	100
1.76-1.83	7.4	0.123	1824	1.337	100
1.83-1.9	7.4	0.097	1865	1.302	100
1.9-1.99	7.4	0.076	1760	1.324	100
1.99-2.09	7.4	0.061	1844	1.338	100
2.09-2.22	7.4	0.057	1841	1.536	100
2.22-2.39	7.4	0.054	1811	1.766	100
2.39-2.63	7.3	0.046	1821	1.581	100
2.63-3.01	7	0.033	1842	1.137	100
3.01-3.8	7.4	0.028	1827	1.177	100
3.8-25	7.4	0.032	1870	1.63	99.3

Phasing

• Phasing: Method: molecular replacement

Processing

Software

Name	Version	Classification	NB
DENZO		data reduction
SCALEPACK		data scaling
PHASER		phasing
REFMAC	5.5.0102	refinement
PDB_EXTRACT	3.005	data extraction
HKL-3000		data reduction
HKL-3000		data scaling

Refinement

Method to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 2Q3F

2q3f

Resolution: 1.4→22.84 Å / Cor.coef. F_o:F_c: 0.969 / Cor.coef. F_o:F_c free: 0.963 / WR_factor R_free: 0.182 / WR_factor R_work: 0.155 / SU B: 1.644 / SU ML: 0.03 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.063 / ESU R Free: 0.056 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED. ARP/WARP, COOT and the MOLPROBITY server were also used during refinement.

	Rfactor	Num. reflection	% reflection	Selection details
Rfree	0.181	1811	4.97 %	RANDOM
Rwork	0.154	-	-	-
obs	0.155	36436	99.956 %	-

• Solvent computation: Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK BULK SOLVENT

Displacement parameters

B_iso mean: 14.63 Ų

	Baniso -1	Baniso -2	Baniso -3
1-	-0.282 Å2	0 Å2	0 Å2
2-	-	-0.282 Å2	0 Å2
3-	-	-	0.563 Å2

Refinement step

Cycle: LAST / Resolution: 1.4→22.84 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	1516	0	42	85	1643

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.015	0.022	1600
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.02	1066
X-RAY DIFFRACTION	r_angle_refined_deg	1.515	1.963	2188
X-RAY DIFFRACTION	r_angle_other_deg	0.941	3	2607
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	5.517	5	201
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	33.24	24.444	81
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	11.149	15	275
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	7.143	15	6
X-RAY DIFFRACTION	r_chiral_restr	0.1	0.2	234
X-RAY DIFFRACTION	r_gen_planes_refined	0.007	0.02	1789
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	340
X-RAY DIFFRACTION	r_mcbond_it	1.263	1.5	931
X-RAY DIFFRACTION	r_mcbond_other	0.407	1.5	373
X-RAY DIFFRACTION	r_mcangle_it	1.918	2	1520
X-RAY DIFFRACTION	r_scbond_it	3.197	3	669
X-RAY DIFFRACTION	r_scangle_it	4.536	4.5	657
X-RAY DIFFRACTION	r_rigid_bond_restr	1.169	3	2666

LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)	Rfactor Rfree	Num. reflection Rfree	Rfactor Rwork	Num. reflection Rwork	Num. reflection all	% reflection obs (%)
1.4-1.436	0.209	122	0.183	2532	2654	100
1.436-1.475	0.204	136	0.154	2470	2607	99.962
1.475-1.518	0.197	126	0.146	2435	2562	99.961
1.518-1.565	0.162	127	0.125	2336	2463	100
1.565-1.616	0.181	105	0.122	2279	2384	100
1.616-1.672	0.155	114	0.122	2209	2323	100
1.672-1.735	0.162	100	0.123	2164	2265	99.956
1.735-1.805	0.169	116	0.128	2008	2124	100
1.805-1.885	0.165	107	0.13	1921	2028	100
1.885-1.976	0.171	106	0.139	1868	1974	100
1.976-2.082	0.173	109	0.136	1812	1921	100
2.082-2.207	0.156	88	0.131	1677	1765	100
2.207-2.358	0.178	88	0.141	1574	1662	100
2.358-2.545	0.19	87	0.155	1506	1593	100
2.545-2.784	0.199	58	0.157	1380	1438	100
2.784-3.107	0.175	68	0.169	1237	1305	100
3.107-3.577	0.161	43	0.171	1099	1142	100
3.577-4.354	0.173	55	0.161	935	991	99.899
4.354-6.049	0.214	37	0.177	747	784	100
6.049-22.84	0.264	19	0.238	436	467	97.43

Refinement TLS params.

Method: refined / Origin x: 26.1668 Å / Origin y: 11.7144 Å / Origin z: 26.7548 Å

	11	12	13	21	22	23	31	32	33
T	0.0155 Å2	-0.0029 Å2	0.0096 Å2	-	0.0214 Å2	0.0021 Å2	-	-	0.0246 Å2
L	1.7053 °2	0.2655 °2	0.0425 °2	-	2.1511 °2	-0.0752 °2	-	-	1.4093 °2
S	0.0234 Å °	-0.0319 Å °	-0.0128 Å °	0.0828 Å °	-0.0207 Å °	0.1116 Å °	0.099 Å °	-0.1176 Å °	-0.0028 Å °