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- PDB-5mlb: Crystal structure of human RAS in complex with darpin K27 -

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Basic information

Entry
Database: PDB / ID: 5mlb
TitleCrystal structure of human RAS in complex with darpin K27
Components
  • DARPin K27
  • GTPase KRas
KeywordsHYDROLASE / RAS / darpin / nucleotide exchange / inhibition
Function / homology
Function and homology information


forebrain astrocyte development / regulation of synaptic transmission, GABAergic / negative regulation of epithelial cell differentiation / epithelial tube branching involved in lung morphogenesis / type I pneumocyte differentiation / Rac protein signal transduction / skeletal muscle cell differentiation / positive regulation of Rac protein signal transduction / Signaling by RAS GAP mutants / Signaling by RAS GTPase mutants ...forebrain astrocyte development / regulation of synaptic transmission, GABAergic / negative regulation of epithelial cell differentiation / epithelial tube branching involved in lung morphogenesis / type I pneumocyte differentiation / Rac protein signal transduction / skeletal muscle cell differentiation / positive regulation of Rac protein signal transduction / Signaling by RAS GAP mutants / Signaling by RAS GTPase mutants / Activation of RAS in B cells / RAS signaling downstream of NF1 loss-of-function variants / RUNX3 regulates p14-ARF / SOS-mediated signalling / Activated NTRK3 signals through RAS / Activated NTRK2 signals through RAS / SHC1 events in ERBB4 signaling / Signalling to RAS / glial cell proliferation / SHC-related events triggered by IGF1R / Activated NTRK2 signals through FRS2 and FRS3 / Estrogen-stimulated signaling through PRKCZ / SHC-mediated cascade:FGFR3 / MET activates RAS signaling / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / Signaling by PDGFRA transmembrane, juxtamembrane and kinase domain mutants / Signaling by PDGFRA extracellular domain mutants / SHC-mediated cascade:FGFR2 / SHC-mediated cascade:FGFR4 / protein-membrane adaptor activity / Signaling by FGFR4 in disease / SHC-mediated cascade:FGFR1 / Erythropoietin activates RAS / homeostasis of number of cells within a tissue / positive regulation of glial cell proliferation / FRS-mediated FGFR3 signaling / Signaling by CSF3 (G-CSF) / Signaling by FLT3 ITD and TKD mutants / FRS-mediated FGFR2 signaling / FRS-mediated FGFR4 signaling / Signaling by FGFR3 in disease / p38MAPK events / FRS-mediated FGFR1 signaling / Tie2 Signaling / Signaling by FGFR2 in disease / GRB2 events in EGFR signaling / striated muscle cell differentiation / SHC1 events in EGFR signaling / EGFR Transactivation by Gastrin / Signaling by FLT3 fusion proteins / Ras activation upon Ca2+ influx through NMDA receptor / FLT3 Signaling / Signaling by FGFR1 in disease / GRB2 events in ERBB2 signaling / NCAM signaling for neurite out-growth / CD209 (DC-SIGN) signaling / SHC1 events in ERBB2 signaling / Downstream signal transduction / Insulin receptor signalling cascade / Constitutive Signaling by Overexpressed ERBB2 / small monomeric GTPase / G protein activity / Signaling by phosphorylated juxtamembrane, extracellular and kinase domain KIT mutants / VEGFR2 mediated cell proliferation / FCERI mediated MAPK activation / regulation of long-term neuronal synaptic plasticity / Signaling by ERBB2 TMD/JMD mutants / RAF activation / Signaling by high-kinase activity BRAF mutants / Constitutive Signaling by EGFRvIII / visual learning / MAP2K and MAPK activation / Signaling by ERBB2 ECD mutants / Signaling by ERBB2 KD Mutants / cytoplasmic side of plasma membrane / Signaling by SCF-KIT / Regulation of RAS by GAPs / Negative regulation of MAPK pathway / RAS processing / GDP binding / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / Signaling by CSF1 (M-CSF) in myeloid cells / MAPK cascade / Signaling by BRAF and RAF1 fusions / Constitutive Signaling by Ligand-Responsive EGFR Cancer Variants / DAP12 signaling / Ca2+ pathway / gene expression / actin cytoskeleton organization / RAF/MAP kinase cascade / neuron apoptotic process / Ras protein signal transduction / negative regulation of neuron apoptotic process / mitochondrial outer membrane / positive regulation of protein phosphorylation / Golgi membrane / focal adhesion
Similarity search - Function
Ankyrin repeat-containing domain / Small GTPase, Ras-type / small GTPase Ras family profile. / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Serine Threonine Protein Phosphatase 5, Tetratricopeptide repeat ...Ankyrin repeat-containing domain / Small GTPase, Ras-type / small GTPase Ras family profile. / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Serine Threonine Protein Phosphatase 5, Tetratricopeptide repeat / Alpha Horseshoe / Small GTP-binding protein domain / P-loop containing nucleotide triphosphate hydrolases / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GTPase KRas
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.22 Å
AuthorsDebreczeni, J.E. / Guillard, S. / Kolasinska-Zwierz, P. / Breed, J. / Zhang, J. / Bery, N. / Marwood, R. / Tart, J. / Overman, R. / Stocki, P. ...Debreczeni, J.E. / Guillard, S. / Kolasinska-Zwierz, P. / Breed, J. / Zhang, J. / Bery, N. / Marwood, R. / Tart, J. / Overman, R. / Stocki, P. / Mistry, B. / Phillips, C. / Rabbitts, T. / Jackson, R. / Minter, R.
CitationJournal: To Be Published
Title: INhibition of RAS nucleotide exchange by a DARPin: structural characterisation and effects on downstream signalling by active RAS
Authors: Guillard, S. / Kolasinska-Zwierz, P. / Debreczeni, J. / Breed, J. / Zhang, J. / Bery, N. / Marwood, R. / Tart, J. / Overman, R. / Stocki, P. / Minstry, B. / Phillips, C. / Rabbitts, T. / Jackson, R. / Minter, R.
History
DepositionDec 6, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 20, 2017Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: GTPase KRas
B: DARPin K27
C: GTPase KRas
D: DARPin K27
E: GTPase KRas
F: DARPin K27
G: GTPase KRas
H: DARPin K27
hetero molecules


Theoretical massNumber of molelcules
Total (without water)156,82816
Polymers154,9588
Non-polymers1,8708
Water1,06359
1
A: GTPase KRas
B: DARPin K27
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,2074
Polymers38,7402
Non-polymers4682
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2780 Å2
ΔGint-20 kcal/mol
Surface area13520 Å2
MethodPISA
2
C: GTPase KRas
D: DARPin K27
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,2074
Polymers38,7402
Non-polymers4682
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2670 Å2
ΔGint-20 kcal/mol
Surface area13830 Å2
MethodPISA
3
E: GTPase KRas
F: DARPin K27
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,2074
Polymers38,7402
Non-polymers4682
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2770 Å2
ΔGint-20 kcal/mol
Surface area13360 Å2
MethodPISA
4
G: GTPase KRas
H: DARPin K27
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,2074
Polymers38,7402
Non-polymers4682
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2620 Å2
ΔGint-20 kcal/mol
Surface area14030 Å2
MethodPISA
Unit cell
Length a, b, c (Å)197.140, 197.140, 84.610
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number170
Space group name H-MP65
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21C
12A
22E
13A
23G
14B
24D
15B
25F
16B
26H
17C
27E
18C
28G
19D
29F
110D
210H
111E
211G
112F
212H

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1010A1 - 165
2010C1 - 165
1020A1 - 165
2020E1 - 165
1030A1 - 163
2030G1 - 163
1040B22 - 177
2040D22 - 177
1050B22 - 177
2050F22 - 177
1060B22 - 177
2060H22 - 177
1070C1 - 165
2070E1 - 165
1080C1 - 163
2080G1 - 163
1090D22 - 177
2090F22 - 177
10100D22 - 177
20100H22 - 177
10110E1 - 163
20110G1 - 163
10120F22 - 177
20120H22 - 177

NCS ensembles :
ID
1
2
3
4
5
6
7
8
9
10
11
12

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Components

#1: Protein
GTPase KRas / K-Ras 2 / Ki-Ras / c-K-ras / c-Ki-ras


Mass: 19208.643 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KRAS, KRAS2, RASK2 / Production host: Escherichia coli (E. coli) / References: UniProt: P01116
#2: Protein
DARPin K27


Mass: 19530.924 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#3: Chemical
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 59 / Source method: isolated from a natural source / Formula: H2O

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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: 3.3 Å3/Da / Density % sol: 62.73 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 30% PEGMME 2000, 299mM Calcium Acetate, 100mM Sodium Cacodylate pH 6.5

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.927 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Jan 15, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.927 Å / Relative weight: 1
ReflectionResolution: 3.22→98.57 Å / Num. obs: 30646 / % possible obs: 100 % / Redundancy: 10.1 % / CC1/2: 0.988 / Rmerge(I) obs: 0.362 / Net I/σ(I): 7.8
Reflection shellResolution: 3.22→3.3 Å / Redundancy: 9.9 % / Rmerge(I) obs: 0.965 / Mean I/σ(I) obs: 1.8 / CC1/2: 0.562 / % possible all: 100

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Processing

Software
NameVersionClassification
REFMAC5.8.0135refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.22→98.57 Å / Cor.coef. Fo:Fc: 0.927 / Cor.coef. Fo:Fc free: 0.894 / SU B: 48.049 / SU ML: 0.387 / Cross valid method: THROUGHOUT / ESU R Free: 0.449 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.23537 1471 4.8 %RANDOM
Rwork0.20096 ---
obs0.20264 29155 99.98 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 81.172 Å2
Baniso -1Baniso -2Baniso -3
1-1.83 Å20.92 Å2-0 Å2
2--1.83 Å20 Å2
3----5.95 Å2
Refinement stepCycle: 1 / Resolution: 3.22→98.57 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9707 0 116 59 9882
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.0199994
X-RAY DIFFRACTIONr_bond_other_d0.0070.029349
X-RAY DIFFRACTIONr_angle_refined_deg1.9821.97213597
X-RAY DIFFRACTIONr_angle_other_deg1.491321366
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.24451279
X-RAY DIFFRACTIONr_dihedral_angle_2_deg39.56424.85468
X-RAY DIFFRACTIONr_dihedral_angle_3_deg18.937151590
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.7231558
X-RAY DIFFRACTIONr_chiral_restr0.120.21585
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.0211537
X-RAY DIFFRACTIONr_gen_planes_other0.0060.022239
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.9574.3485134
X-RAY DIFFRACTIONr_mcbond_other1.9564.3485133
X-RAY DIFFRACTIONr_mcangle_it3.1796.5256407
X-RAY DIFFRACTIONr_mcangle_other3.1786.5256408
X-RAY DIFFRACTIONr_scbond_it2.2894.5954860
X-RAY DIFFRACTIONr_scbond_other2.2834.5894852
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other3.7366.8087178
X-RAY DIFFRACTIONr_long_range_B_refined5.72237.89916250
X-RAY DIFFRACTIONr_long_range_B_other5.68737.87316226
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A194420.08
12C194420.08
21A191440.07
22E191440.07
31A178580.08
32G178580.08
41B185120.06
42D185120.06
51B187420.04
52F187420.04
61B180260.06
62H180260.06
71C190380.08
72E190380.08
81C181440.07
82G181440.07
91D186740.06
92F186740.06
101D185260.04
102H185260.04
111E181680.07
112G181680.07
121F182000.05
122H182000.05
LS refinement shellResolution: 3.22→3.304 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.286 130 -
Rwork0.317 2124 -
obs--99.96 %
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)
12.1056-0.4599-0.2914.46060.37123.93670.0451-0.00710.2015-0.06220.00510.1773-0.3709-0.1303-0.05030.109-0.04030.00250.14570.00570.031140.7048-67.8196-6.1517
22.45470.1411-0.34326.15982.0684.3487-0.01820.0644-0.13260.01490.06080.45060.1055-0.3669-0.04260.0784-0.0907-0.04560.29530.1060.086229.2515-84.1610.5429
32.87150.2792-0.03322.69861.0365.94390.056-0.03060.107-0.0637-0.0926-0.0419-0.15540.00590.03660.0774-0.0586-0.00610.12510.01370.00735.769-80.6985-39.8307
43.2386-0.72811.64642.59190.08965.0784-0.0039-0.34640.1340.0899-0.14240.3531-0.1447-0.3430.14630.115-0.09780.03890.3429-0.02260.226311.1189-84.5832-32.1076
53.6525-1.35310.57623.14130.48284.3397-0.10150.12560.2609-0.13010.065-0.5174-0.2580.39480.03650.2277-0.19770.04090.2001-0.02380.142272.4054-60.5358-7.7103
63.1944-0.01231.92843.13760.50965.4577-0.05290.2630.7403-0.4281-0.03310.0541-0.7752-0.01040.0860.581-0.1117-0.02740.18030.02310.368958.3596-38.8016-6.3989
72.4832-0.8383-0.33881.59541.4094.6934-0.0202-1.02280.49340.26790.15160.328-0.1416-0.819-0.13150.6737-0.0315-0.01431.2744-0.20280.7251-3.2519-55.616-43.0646
82.3961-1.13-1.18622.08181.28526.2341-0.0223-0.74770.60730.31730.074-0.0578-0.3216-0.2048-0.05180.6629-0.0758-0.01520.8774-0.29820.694922.1597-50.6629-41.1973
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 165
2X-RAY DIFFRACTION2B22 - 177
3X-RAY DIFFRACTION3C1 - 165
4X-RAY DIFFRACTION4D22 - 177
5X-RAY DIFFRACTION5E1 - 165
6X-RAY DIFFRACTION6F22 - 177
7X-RAY DIFFRACTION7G1 - 164
8X-RAY DIFFRACTION8H22 - 177

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