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- PDB-5e8j: Crystal structure of mRNA cap guanine-N7 methyltransferase in com... -

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Basic information

Entry
Database: PDB / ID: 5e8j
TitleCrystal structure of mRNA cap guanine-N7 methyltransferase in complex with RAM
Components
  • RNMT-activating mini protein
  • mRNA cap guanine-N7 methyltransferase
KeywordsTRANSLATION / mRNA capping / mRNA processing
Function / homology
Function and homology information


: / RNA 5'-cap (guanine-N7)-methylation / : / mRNA capping enzyme complex / RNA Pol II CTD phosphorylation and interaction with CE during HIV infection / RNA Pol II CTD phosphorylation and interaction with CE / mRNA Capping / 7-methylguanosine mRNA capping / enzyme activator activity / cellular response to leukemia inhibitory factor ...: / RNA 5'-cap (guanine-N7)-methylation / : / mRNA capping enzyme complex / RNA Pol II CTD phosphorylation and interaction with CE during HIV infection / RNA Pol II CTD phosphorylation and interaction with CE / mRNA Capping / 7-methylguanosine mRNA capping / enzyme activator activity / cellular response to leukemia inhibitory factor / fibrillar center / mRNA (guanine-N7)-methyltransferase / methylation / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / receptor complex / RNA binding / nucleoplasm / nucleus
Similarity search - Function
RNA guanine-N7 methyltransferase activating subunit / mRNA cap methylation, RNMT-activating mini protein / mRNA cap guanine-N7 methyltransferase, eukaryotes / mRNA (guanine-N(7))-methyltransferase domain / mRNA cap guanine-N7 methyltransferase / mRNA (guanine-N(7))-methyltransferase domain / mRNA (guanine-N(7)-)-methyltransferase (EC 2.1.1.56) domain profile. / Vaccinia Virus protein VP39 / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Rossmann fold ...RNA guanine-N7 methyltransferase activating subunit / mRNA cap methylation, RNMT-activating mini protein / mRNA cap guanine-N7 methyltransferase, eukaryotes / mRNA (guanine-N(7))-methyltransferase domain / mRNA cap guanine-N7 methyltransferase / mRNA (guanine-N(7))-methyltransferase domain / mRNA (guanine-N(7)-)-methyltransferase (EC 2.1.1.56) domain profile. / Vaccinia Virus protein VP39 / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
S-ADENOSYL-L-HOMOCYSTEINE / mRNA cap guanine-N7 methyltransferase / RNA guanine-N7 methyltransferase activating subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.35 Å
AuthorsPetit, P. / Cowling, V.
CitationJournal: Nucleic Acids Res. / Year: 2016
Title: Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM.
Authors: Varshney, D. / Petit, A.P. / Bueren-Calabuig, J.A. / Jansen, C. / Fletcher, D.A. / Peggie, M. / Weidlich, S. / Scullion, P. / Pisliakov, A.V. / Cowling, V.H.
History
DepositionOct 14, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Jul 13, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 3, 2016Group: Database references
Revision 1.2Dec 14, 2016Group: Database references
Revision 1.3Feb 20, 2019Group: Advisory / Data collection / Derived calculations
Category: diffrn_source / pdbx_data_processing_status ...diffrn_source / pdbx_data_processing_status / pdbx_validate_symm_contact / struct_conn / struct_conn_type
Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.4Jul 10, 2019Group: Data collection / Category: diffrn_source / Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.5Jan 10, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: mRNA cap guanine-N7 methyltransferase
B: mRNA cap guanine-N7 methyltransferase
C: RNMT-activating mini protein
D: RNMT-activating mini protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)84,7777
Polymers83,9164
Non-polymers8613
Water3,945219
1
A: mRNA cap guanine-N7 methyltransferase
C: RNMT-activating mini protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)42,4344
Polymers41,9582
Non-polymers4772
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3580 Å2
ΔGint-25 kcal/mol
Surface area16830 Å2
MethodPISA
2
B: mRNA cap guanine-N7 methyltransferase
D: RNMT-activating mini protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)42,3423
Polymers41,9582
Non-polymers3841
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3440 Å2
ΔGint-23 kcal/mol
Surface area16710 Å2
MethodPISA
Unit cell
Length a, b, c (Å)49.147, 50.487, 84.579
Angle α, β, γ (deg.)90.060, 92.410, 115.410
Int Tables number1
Space group name H-MP1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A and segid
21chain B and segid
12chain C and segid
22chain D and segid

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111chain A and segidA0
211chain B and segidB0
112chain C and segidC0
212chain D and segidD0

NCS ensembles :
ID
1
2

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Components

#1: Protein mRNA cap guanine-N7 methyltransferase / RG7MT1 / mRNA (guanine-N(7)-)-methyltransferase / mRNA cap methyltransferase / hcm1p


Mass: 36716.160 Da / Num. of mol.: 2 / Fragment: UNP residues 167-476
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RNMT, KIAA0398 / Plasmid: pET15 / Production host: Escherichia coli (E. coli)
References: UniProt: O43148, mRNA (guanine-N7)-methyltransferase
#2: Protein/peptide RNMT-activating mini protein / RAM / Protein FAM103A1


Mass: 5241.679 Da / Num. of mol.: 2 / Fragment: UNP residues 3-45
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FAM103A1, C15orf18 / Plasmid: pGEX / Production host: Escherichia coli (E. coli) / References: UniProt: Q9BTL3
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE


Type: L-peptide linking / Mass: 384.411 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C14H20N6O5S
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 219 / 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: 2.27 Å3/Da / Density % sol: 45.9 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 6.5 / Details: 0.1M MES pH 6.5, 30% PEG 4000

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I24 / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Apr 17, 2014
RadiationMonochromator: Si111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.35→84.485 Å / Num. all: 28355 / Num. obs: 28355 / % possible obs: 92.8 % / Redundancy: 1.6 % / Biso Wilson estimate: 24.55 Å2 / Rpim(I) all: 0.079 / Rrim(I) all: 0.111 / Rsym value: 0.079 / Net I/av σ(I): 8.154 / Net I/σ(I): 6.6 / Num. measured all: 45164
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allRpim(I) allRsym valueNet I/σ(I) obs% possible all
2.35-2.481.40.3492.2560738880.3490.349287.1
2.48-2.631.60.272.9626339200.270.272.792.7
2.63-2.811.60.1983.8591637210.1980.1983.593.6
2.81-3.031.60.1235.8559234590.1230.1235.195
3.03-3.321.60.0887.9531532590.0880.0886.895.2
3.32-3.721.60.0669.1464928970.0660.0669.394.2
3.72-4.291.60.0510.8412525440.050.0511.894.3
4.29-5.251.60.04113.8351721600.0410.04113.193.8
5.25-7.431.60.04712.9269316360.0470.04711.692.7
7.43-28.1621.70.03215.914878710.0320.03215.189.9

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
SCALA3.3.21data scaling
MOLREPphasing
PHENIXrefinement
PDB_EXTRACT3.15data extraction
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3BGV

3bgv


Resolution: 2.35→28.162 Å / SU ML: 0.35 / Cross valid method: FREE R-VALUE / σ(F): 1.98 / Phase error: 27.91 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2685 1203 4.25 %Random selection
Rwork0.2105 27133 --
obs0.2129 28336 92.82 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 90.68 Å2 / Biso mean: 25.7436 Å2 / Biso min: 9.44 Å2
Refinement stepCycle: final / Resolution: 2.35→28.162 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5807 0 58 219 6084
Biso mean--23.11 28.94 -
Num. residues----705
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0036002
X-RAY DIFFRACTIONf_angle_d0.798069
X-RAY DIFFRACTIONf_chiral_restr0.03839
X-RAY DIFFRACTIONf_plane_restr0.0031034
X-RAY DIFFRACTIONf_dihedral_angle_d15.0252282
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A2981X-RAY DIFFRACTION8.794TORSIONAL
12B2981X-RAY DIFFRACTION8.794TORSIONAL
21C398X-RAY DIFFRACTION8.794TORSIONAL
22D398X-RAY DIFFRACTION8.794TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 9

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.35-2.44410.32641830.25972706288986
2.4441-2.55520.31191430.24312986312992
2.5552-2.68990.33341120.24253034314693
2.6899-2.85820.31451040.22873091319594
2.8582-3.07870.25471180.21233124324295
3.0787-3.3880.25741150.20533111322695
3.388-3.87720.27141270.19533061318894
3.8772-4.88070.22971660.18043020318694
4.8807-28.16370.25421350.21323000313593
Refinement TLS params.Method: refined / Origin x: -9.1914 Å / Origin y: 13.3564 Å / Origin z: 18.3482 Å
111213212223313233
T0.0976 Å2-0.0053 Å2-0.0217 Å2-0.1242 Å20.0256 Å2--0.1307 Å2
L0.3249 °2-0.1682 °2-0.2865 °2-0.4914 °20.3632 °2--0.7464 °2
S-0.0039 Å °-0.0182 Å °0.0125 Å °0.0064 Å °-0.0006 Å °-0.0053 Å °0.0227 Å °-0.0166 Å °0.0047 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA167 - 476
2X-RAY DIFFRACTION1allB167 - 476
3X-RAY DIFFRACTION1allC3 - 44
4X-RAY DIFFRACTION1allD3 - 45
5X-RAY DIFFRACTION1allE1
6X-RAY DIFFRACTION1allW1 - 306
7X-RAY DIFFRACTION1allL1 - 2

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