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- PDB-3n4m: E. coli RNA polymerase alpha subunit C-terminal domain in complex... -

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Basic information

Entry
Database: PDB / ID: 3n4m
TitleE. coli RNA polymerase alpha subunit C-terminal domain in complex with CAP and DNA
Components
  • Catabolite gene activator
  • DNA (5'-D(*CP*TP*AP*GP*AP*TP*CP*AP*CP*AP*TP*TP*TP*TP*AP*GP*GP*AP*AP*AP*AP*AP*AP*G)-3')
  • DNA (5'-D(*CP*TP*TP*TP*TP*TP*TP*CP*CP*TP*AP*AP*AP*AP*TP*GP*TP*GP*AP*T)-3')
  • DNA-directed RNA polymerase subunit alphaPolymerase
KeywordsGENE REGULATION/DNA / protein-protein interactions / protein-DNA interactions / GENE REGULATION-DNA complex
Function / homology
Function and homology information


carbon catabolite repression of transcription / DNA binding, bending / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / minor groove of adenine-thymine-rich DNA binding / bacterial-type flagellum-dependent cell motility / nitrate assimilation ...carbon catabolite repression of transcription / DNA binding, bending / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / minor groove of adenine-thymine-rich DNA binding / bacterial-type flagellum-dependent cell motility / nitrate assimilation / cAMP binding / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / cell motility / protein-DNA complex / DNA-templated transcription initiation / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / intracellular iron ion homeostasis / sequence-specific DNA binding / protein dimerization activity / DNA-binding transcription factor activity / negative regulation of DNA-templated transcription / DNA-templated transcription / positive regulation of DNA-templated transcription / DNA binding / membrane / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / helix_turn_helix, cAMP Regulatory protein / Crp-type HTH domain profile. / Crp-like helix-turn-helix domain / Crp-type HTH domain / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain ...Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / helix_turn_helix, cAMP Regulatory protein / Crp-type HTH domain profile. / Crp-like helix-turn-helix domain / Crp-type HTH domain / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / Jelly Rolls / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, alpha subunit / 5' to 3' exonuclease, C-terminal subdomain / RmlC-like jelly roll fold / Winged helix-like DNA-binding domain superfamily/Winged helix DNA-binding domain / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / DNA polymerase; domain 1 / Arc Repressor Mutant, subunit A / Winged helix DNA-binding domain superfamily / Jelly Rolls / Winged helix-like DNA-binding domain superfamily / Sandwich / Orthogonal Bundle / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / DI(HYDROXYETHYL)ETHER / DNA / DNA (> 10) / DNA-directed RNA polymerase subunit alpha / DNA-binding transcriptional dual regulator CRP
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.987 Å
AuthorsLara-Gonzalez, S. / Birktoft, J.J. / Lawson, C.L.
Citation
Journal: Biochemistry / Year: 2020
Title: The RNA Polymerase alpha Subunit Recognizes the DNA Shape of the Upstream Promoter Element.
Authors: Lara-Gonzalez, S. / Dantas Machado, A.C. / Rao, S. / Napoli, A.A. / Birktoft, J. / Di Felice, R. / Rohs, R. / Lawson, C.L.
#1: Journal: Science / Year: 2002
Title: Structural basis of transcription activation: the CAP-alpha CTD-DNA complex
Authors: Benoff, B. / Yang, H. / Lawson, C.L. / Parkinson, G. / Liu, J. / Blatter, E. / Ebright, Y.W. / Berman, H.M. / Ebright, R.H.
History
DepositionMay 21, 2010Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 25, 2011Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Nov 25, 2020Group: Database references / Derived calculations / Category: citation / citation_author / struct_site
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.3Jul 26, 2023Group: Database references / Refinement description
Category: citation / citation_author ...citation / citation_author / database_2 / struct_ncs_dom_lim
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id
Revision 1.4Sep 6, 2023Group: 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
A: Catabolite gene activator
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit alpha
D: DNA (5'-D(*CP*TP*TP*TP*TP*TP*TP*CP*CP*TP*AP*AP*AP*AP*TP*GP*TP*GP*AP*T)-3')
E: DNA (5'-D(*CP*TP*AP*GP*AP*TP*CP*AP*CP*AP*TP*TP*TP*TP*AP*GP*GP*AP*AP*AP*AP*AP*AP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)56,6409
Polymers55,7705
Non-polymers8714
Water63135
1
A: Catabolite gene activator
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit alpha
D: DNA (5'-D(*CP*TP*TP*TP*TP*TP*TP*CP*CP*TP*AP*AP*AP*AP*TP*GP*TP*GP*AP*T)-3')
E: DNA (5'-D(*CP*TP*AP*GP*AP*TP*CP*AP*CP*AP*TP*TP*TP*TP*AP*GP*GP*AP*AP*AP*AP*AP*AP*G)-3')
hetero molecules

A: Catabolite gene activator
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit alpha
D: DNA (5'-D(*CP*TP*TP*TP*TP*TP*TP*CP*CP*TP*AP*AP*AP*AP*TP*GP*TP*GP*AP*T)-3')
E: DNA (5'-D(*CP*TP*AP*GP*AP*TP*CP*AP*CP*AP*TP*TP*TP*TP*AP*GP*GP*AP*AP*AP*AP*AP*AP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)113,28118
Polymers111,53910
Non-polymers1,7418
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation11_657-x+y+1,y,-z+21
Unit cell
Length a, b, c (Å)175.730, 175.730, 160.100
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number180
Space group name H-MP6222
Components on special symmetry positions
IDModelComponents
11E-101-

HOH

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11B
21C

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: ASP / Beg label comp-ID: ASP / End auth comp-ID: PRO / End label comp-ID: PRO / Auth seq-ID: 250 - 322 / Label seq-ID: 5 - 77

Dom-IDSelection detailsAuth asym-IDLabel asym-ID
1chain B and (resseq 250:322 )BB
2chain C and (resseq 250:322 )CC

NCS oper: (Code: given
Matrix: (0.709049, -0.218932, -0.670312), (-0.459989, 0.576888, -0.674989), (0.534472, 0.786937, 0.308335)
Vector: 117.997002, 121.581001, 68.516296)

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Components

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Protein , 2 types, 3 molecules ABC

#1: Protein Catabolite gene activator / cAMP receptor protein / cAMP regulatory protein


Mass: 23541.242 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: b3357, cap, crp, csm, JW5702 / Plasmid: pET / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 DE3 / References: UniProt: P0ACJ8
#2: Protein DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / Transcriptase subunit alpha / RNA polymerase subunit alpha


Mass: 9364.795 Da / Num. of mol.: 2 / Fragment: alpha subunit C-terminal domain, residues 246-329
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: b3295, JW3257, pez, phs, rpoA, sez / Plasmid: pET / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 DE3 / References: UniProt: P0A7Z4, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules DE

#3: DNA chain DNA (5'-D(*CP*TP*TP*TP*TP*TP*TP*CP*CP*TP*AP*AP*AP*AP*TP*GP*TP*GP*AP*T)-3')


Mass: 6088.962 Da / Num. of mol.: 1 / Source method: obtained synthetically
#4: DNA chain DNA (5'-D(*CP*TP*AP*GP*AP*TP*CP*AP*CP*AP*TP*TP*TP*TP*AP*GP*GP*AP*AP*AP*AP*AP*AP*G)-3')


Mass: 7409.844 Da / Num. of mol.: 1 / Source method: obtained synthetically

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Non-polymers , 3 types, 39 molecules

#5: Chemical ChemComp-CMP / ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / CYCLIC AMP / CAMP / Cyclic adenosine monophosphate


Mass: 329.206 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H12N5O6P
#6: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER / Diethylene glycol


Mass: 106.120 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H10O3
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 35 / 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: 6.4 Å3/Da / Density % sol: 80.77 %
Crystal growTemperature: 293.2 K / Method: vapor diffusion, hanging drop / pH: 4.5
Details: 100 mM sodium acetate (pH 4.5), 625 mM NaCl, VAPOR DIFFUSION, HANGING DROP, temperature 293.2K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X29A / Wavelength: 1.1 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: May 11, 2006 / Details: vertical mirror, horizontal monochromator
RadiationMonochromator: horizontally focusing monochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1 Å / Relative weight: 1
ReflectionResolution: 2.987→38.51 Å / Num. all: 29113 / Num. obs: 29113 / % possible obs: 98.9 % / Observed criterion σ(F): -1 / Observed criterion σ(I): 2.5 / Redundancy: 4.5 % / Biso Wilson estimate: 99.7 Å2 / Rmerge(I) obs: 0.085 / Rsym value: 0.085 / Net I/σ(I): 11.5
Reflection shellResolution: 3.02→3.18 Å / Redundancy: 4.6 % / Rmerge(I) obs: 1.106 / Mean I/σ(I) obs: 1.6 / Num. unique all: 4202 / Rsym value: 1.106 / % possible all: 99.5

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Processing

Software
NameVersionClassificationNB
MOSFLMdata reduction
SCALA3.3.16data scaling
PHENIXrefinement
PDB_EXTRACT3.1data extraction
CBASSdata collection
AMoREphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1lb2, 3k4g

1lb2

3k4g


Resolution: 2.987→38.51 Å / Occupancy max: 1 / Occupancy min: 0.5 / FOM work R set: 0.828 / SU ML: 0.61 / Isotropic thermal model: group adp (by residue) / σ(F): 0.17 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.224 1481 5.1 %random
Rwork0.198 ---
all0.199 30104 --
obs0.199 29042 96.5 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 70.566 Å2 / ksol: 0.298 e/Å3
Displacement parametersBiso max: 351.17 Å2 / Biso mean: 110.384 Å2 / Biso min: 38.96 Å2
Baniso -1Baniso -2Baniso -3
1-10.92 Å2-0 Å2-0 Å2
2--10.92 Å20 Å2
3----21.84 Å2
Refinement stepCycle: LAST / Resolution: 2.987→38.51 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2759 896 47 35 3737
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0043852
X-RAY DIFFRACTIONf_angle_d0.9465391
X-RAY DIFFRACTIONf_chiral_restr0.051622
X-RAY DIFFRACTIONf_plane_restr0.003526
X-RAY DIFFRACTIONf_dihedral_angle_d21.3621517
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDTypeRms dev position (Å)
11B572X-RAY DIFFRACTIONPOSITIONAL0.009
12C572X-RAY DIFFRACTIONPOSITIONAL0.009
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 11

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.987-3.0840.4011080.3562039214780
3.084-3.1940.3481350.3192516265199
3.194-3.3220.2911540.2822513266799
3.322-3.4730.251330.2612518265199
3.473-3.6560.2631530.2322525267899
3.656-3.8850.2311390.2152527266699
3.885-4.1840.2081500.1772529267998
4.184-4.6050.1951330.1662539267298
4.605-5.270.1771290.1542569269898
5.27-6.6340.1741220.172606272897
6.634-38.5170.1941250.1652680280595
Refinement TLS params.Method: refined / Origin x: 112.1779 Å / Origin y: -51.2756 Å / Origin z: 149.0329 Å
111213212223313233
T0.4991 Å2-0.0083 Å2-0.0811 Å2-0.3004 Å2-0.0273 Å2--0.2578 Å2
L1.2914 °2-0.2319 °2-0.2558 °2-0.5189 °2-0.1921 °2--0.3702 °2
S0.0181 Å °-0.02 Å °0.1337 Å °0.4331 Å °0.0013 Å °-0.0819 Å °-0.0045 Å °0.1066 Å °-0.0232 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA7 - 209
2X-RAY DIFFRACTION1allA210
3X-RAY DIFFRACTION1allB249 - 322
4X-RAY DIFFRACTION1allC250 - 322
5X-RAY DIFFRACTION1allD1 - 20
6X-RAY DIFFRACTION1allE21 - 44
7X-RAY DIFFRACTION1allD100
8X-RAY DIFFRACTION1allA501
9X-RAY DIFFRACTION1allA502
10X-RAY DIFFRACTION1allA - D7 - 102

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