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- PDB-3n97: RNA polymerase alpha C-terminal domain (E. coli) and sigma region... -

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Basic information

Entry
Database: PDB / ID: 3n97
TitleRNA polymerase alpha C-terminal domain (E. coli) and sigma region 4 (T. aq. mutant) bound to (UP,-35 element) DNA
Components
  • DNA (5'-D(*CP*CP*AP*TP*GP*TP*CP*AP*AP*GP*TP*AP*CP*TP*TP*TP*TP*TP*TP*CP*C)-3')
  • DNA (5'-D(*TP*GP*GP*AP*AP*AP*AP*AP*AP*GP*TP*AP*CP*TP*TP*GP*AP*CP*AP*TP*GP*G)-3')
  • DNA-directed RNA polymerase subunit alpha
  • RNA polymerase sigma factor
KeywordsGENE REGULATION/DNA / protein-protein interactions / protein-DNA interactions / transcription initiation / GENE REGULATION-DNA complex
Function / homology
Function and homology information


submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / regulation of DNA-templated transcription elongation ...submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription initiation / cell motility / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / intracellular iron ion homeostasis / protein dimerization activity / DNA binding / membrane / cytoplasm / cytosol
Similarity search - Function
RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 ...RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / 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 / 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-like DNA-binding domain superfamily / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
ACETATE ION / DNA / DNA (> 10) / DNA-directed RNA polymerase subunit alpha / RNA polymerase sigma factor SigA
Similarity search - Component
Biological speciesThermus aquaticus (bacteria)
Escherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.252 Å
AuthorsLara-Gonzalez, S. / Birktoft, J.J. / Lawson, C.L.
CitationJournal: 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.
History
DepositionMay 28, 2010Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 8, 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 ...citation / citation_author / struct_ref_seq_dif / 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_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.3Dec 23, 2020Group: Database references / Category: citation / citation_author
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
Revision 1.4Sep 6, 2023Group: 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 / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA polymerase sigma factor
D: RNA polymerase sigma factor
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit alpha
M: DNA (5'-D(*TP*GP*GP*AP*AP*AP*AP*AP*AP*GP*TP*AP*CP*TP*TP*GP*AP*CP*AP*TP*GP*G)-3')
N: DNA (5'-D(*CP*CP*AP*TP*GP*TP*CP*AP*AP*GP*TP*AP*CP*TP*TP*TP*TP*TP*TP*CP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,0327
Polymers48,9736
Non-polymers591
Water181
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)80.710, 86.410, 147.110
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21D
12A
22D
13B
23C
14B
24C

NCS domain segments:

Component-ID: 1

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11LYSLYSGLYGLYchain A and (resseq 375:402 )AA375 - 40210 - 37
21LYSLYSGLYGLYchain D and (resseq 375:402 )DB375 - 40210 - 37
12ALAALAARGARGchain A and (resseq 403:424 )AA403 - 42438 - 59
22ALAALAARGARGchain D and (resseq 403:424 )DB403 - 42438 - 59
13PROPROLEULEUchain B and (resseq 251:318 )BC251 - 3186 - 73
23PROPROLEULEUchain C and (resseq 251:318 )CD251 - 3186 - 73
14GLUGLUPROPROchain B and (resseq 319:322 )BC319 - 32274 - 77
24GLUGLUPROPROchain C and (resseq 319:322 )CD319 - 32274 - 77

NCS ensembles :
ID
1
2
3
4

NCS oper:
IDCodeMatrixVector
1given(0.092064, 0.966746, 0.238591), (0.973654, -0.137598, 0.181837), (0.20862, 0.215564, -0.953944)62.997299, -37.8302, -144.093994
2given(0.102772, 0.9561, 0.274428), (0.966024, -0.161708, 0.201615), (0.237141, 0.244383, -0.940234)65.330498, -36.278198, -143.481003
3given(0.310109, 0.706236, -0.636446), (-0.644763, 0.648199, 0.405117), (0.698651, 0.284726, 0.656367)9.68058, 23.8967, -31.6476
4given(0.316748, 0.668369, -0.673018), (-0.707628, 0.639005, 0.301554), (0.631612, 0.38073, 0.67536)8.19682, 21.9958, -30.367399

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Components

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Protein , 2 types, 4 molecules ADBC

#1: Protein RNA polymerase sigma factor


Mass: 8527.862 Da / Num. of mol.: 2 / Fragment: sigma subunit region 4, residues 366-438 / Mutation: L386M and 424-429 KYHESR replaced with RHPSR
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus aquaticus (bacteria) / Gene: rpoS / Plasmid: pET / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 DE3 / References: UniProt: Q9EZJ8
#2: Protein DNA-directed RNA polymerase subunit alpha / 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 MN

#3: DNA chain DNA (5'-D(*TP*GP*GP*AP*AP*AP*AP*AP*AP*GP*TP*AP*CP*TP*TP*GP*AP*CP*AP*TP*GP*G)-3')


Mass: 6848.466 Da / Num. of mol.: 1 / Source method: obtained synthetically
#4: DNA chain DNA (5'-D(*CP*CP*AP*TP*GP*TP*CP*AP*AP*GP*TP*AP*CP*TP*TP*TP*TP*TP*TP*CP*C)-3')


Mass: 6339.106 Da / Num. of mol.: 1 / Source method: obtained synthetically

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H3O2
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence details424-429 KYHESR SEQUENCE IN T. AQUATICUS RNA POLYMERASE IS REPLACED WITH RHPSR SEQUENCE FROM E. COLI ...424-429 KYHESR SEQUENCE IN T. AQUATICUS RNA POLYMERASE IS REPLACED WITH RHPSR SEQUENCE FROM E. COLI RNA POLYMERASE

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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: 2.62 Å3/Da / Density % sol: 53.03 %
Crystal growTemperature: 289.15 K / Method: vapor diffusion, hanging drop / pH: 5.6
Details: 28% (w/v) PEG4000, 0.2 M ammonium acetate, 0.01 M sarcosine, 0.1 M sodium citrate, pH 5.6, VAPOR DIFFUSION, HANGING DROP, temperature 289.15K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X25 / Wavelength: 0.92 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Oct 23, 2008
RadiationMonochromator: Double silicon(111) crystal monochromator with cryogenically-cooled first crystal and sagittally-bent second crystal horizontally-focusing at 3.3:1 demagnification
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.92 Å / Relative weight: 1
ReflectionResolution: 3.25→25.43 Å / Num. all: 7461 / Num. obs: 7461 / % possible obs: 90.1 % / Observed criterion σ(F): -1 / Observed criterion σ(I): 2.5 / Redundancy: 3.3 % / Biso Wilson estimate: 98.8 Å2 / Rmerge(I) obs: 0.081 / Rsym value: 0.081 / Net I/σ(I): 11.1
Reflection shellResolution: 3.25→3.42 Å / Redundancy: 3.3 % / Rmerge(I) obs: 0.816 / Mean I/σ(I) obs: 1.6 / Num. unique all: 1048 / Rsym value: 0.816 / % possible all: 91.8

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Processing

Software
NameVersionClassificationNB
MOSFLMdata reduction
SCALA3.3.16data scaling
PHASERphasing
PHENIXrefinement
PDB_EXTRACT3.1data extraction
CBASSdata collection
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entries 3n4m, 1lb2 and 1ku7

3n4m

1lb2

1ku7


Resolution: 3.252→25.43 Å / Occupancy max: 1 / Occupancy min: 1 / FOM work R set: 0.714 / SU ML: 0.54
Isotropic thermal model: group ADP (297 groups, 1 residue per group) plus TLS (all atoms in 1 group)
Cross valid method: THROUGHOUT / σ(F): 1.13 / Stereochemistry target values: ML
Details: additional pseudo-bond restraints applied to DNA base-pairs
RfactorNum. reflection% reflectionSelection details
Rfree0.295 480 6.47 %random
Rwork0.254 ---
all0.257 8450 --
obs0.257 7422 88.65 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 66.437 Å2 / ksol: 0.279 e/Å3
Displacement parametersBiso max: 250.51 Å2 / Biso mean: 138.929 Å2 / Biso min: 85.36 Å2
Baniso -1Baniso -2Baniso -3
1-39.777 Å2-0 Å2-0 Å2
2---5.676 Å20 Å2
3----34.1 Å2
Refinement stepCycle: LAST / Resolution: 3.252→25.43 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2006 875 4 1 2886
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0063111
X-RAY DIFFRACTIONf_angle_d1.2124237
X-RAY DIFFRACTIONf_chiral_restr0.057488
X-RAY DIFFRACTIONf_plane_restr0.008391
X-RAY DIFFRACTIONf_dihedral_angle_d23.6141232
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDTypeRms dev position (Å)
11A219X-RAY DIFFRACTIONPOSITIONAL0.152
12D219X-RAY DIFFRACTIONPOSITIONAL0.152
21A191X-RAY DIFFRACTIONPOSITIONAL0.203
22D191X-RAY DIFFRACTIONPOSITIONAL0.203
31B526X-RAY DIFFRACTIONPOSITIONAL0.152
32C526X-RAY DIFFRACTIONPOSITIONAL0.152
41B38X-RAY DIFFRACTIONPOSITIONAL0.308
42C38X-RAY DIFFRACTIONPOSITIONAL0.308
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 3

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.252-3.7210.3741500.3762335248590
3.721-4.6840.2951690.2682298246790
4.684-25.4530.2691610.2112309247086
Refinement TLS params.Method: refined / Origin x: 21.4667 Å / Origin y: -15.5546 Å / Origin z: -49.2867 Å
111213212223313233
T0.5358 Å2-0.0575 Å2-0.0907 Å2-0.5278 Å20.0749 Å2--0.665 Å2
L1.6588 °2-0.6447 °2-0.5868 °2-1.6605 °20.5178 °2--6.4742 °2
S0.0696 Å °-0.118 Å °-0.2897 Å °-0.0551 Å °0.1144 Å °0.0057 Å °0.6826 Å °0.0664 Å °-0.0016 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA374 - 426
2X-RAY DIFFRACTION1allD375 - 424
3X-RAY DIFFRACTION1allB250 - 324
4X-RAY DIFFRACTION1allC250 - 323
5X-RAY DIFFRACTION1allM1 - 22
6X-RAY DIFFRACTION1allN1 - 21
7X-RAY DIFFRACTION1allC1 - 2

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