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- PDB-6mcf: Solution structure of 7SK stem-loop 1 with HIV-1 Tat RNA Binding ... -

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Basic information

Entry
Database: PDB / ID: 6mcf
TitleSolution structure of 7SK stem-loop 1 with HIV-1 Tat RNA Binding Domain
Components
  • 7SK Stem-loop 1 RNA
  • Protein Tat
KeywordsTRANSCRIPTION / RNA Binding Domain / RNA BINDING PROTEIN
Function / homology
Function and homology information


trans-activation response element binding / Interactions of Tat with host cellular proteins / protein serine/threonine phosphatase inhibitor activity / positive regulation of viral transcription / modulation by virus of host chromatin organization / symbiont-mediated suppression of host translation initiation / molecular sequestering activity / evasion of host immune response / host cell nucleolus / actinin binding ...trans-activation response element binding / Interactions of Tat with host cellular proteins / protein serine/threonine phosphatase inhibitor activity / positive regulation of viral transcription / modulation by virus of host chromatin organization / symbiont-mediated suppression of host translation initiation / molecular sequestering activity / evasion of host immune response / host cell nucleolus / actinin binding / negative regulation of peptidyl-threonine phosphorylation / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / RNA-binding transcription regulator activity / Tat-mediated elongation of the HIV-1 transcript / Formation of HIV-1 elongation complex containing HIV-1 Tat / cyclin binding / positive regulation of transcription elongation by RNA polymerase II / PKR-mediated signaling / host cell cytoplasm / protein domain specific binding / DNA-templated transcription / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / host cell nucleus / extracellular region / metal ion binding
Similarity search - Function
Tat domain superfamily / Immunodeficiency virus transactivating regulatory protein (Tat) / Transactivating regulatory protein (Tat)
Similarity search - Domain/homology
RNA / RNA (> 10) / Protein Tat
Similarity search - Component
Biological speciesHomo sapiens (human)
Human immunodeficiency virus type 1 group M subtype B
MethodSOLUTION NMR / simulated annealing
AuthorsPham, V.V. / D'Souza, V.M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103297-01 United States
Howard Hughes Medical Institute (HHMI)55108516 United States
CitationJournal: Nat Commun / Year: 2018
Title: HIV-1 Tat interactions with cellular 7SK and viral TAR RNAs identifies dual structural mimicry.
Authors: Pham, V.V. / Salguero, C. / Khan, S.N. / Meagher, J.L. / Brown, W.C. / Humbert, N. / de Rocquigny, H. / Smith, J.L. / D'Souza, V.M.
History
DepositionAug 31, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 31, 2018Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / pdbx_nmr_software
Item: _pdbx_audit_support.funding_organization / _pdbx_nmr_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: 7SK Stem-loop 1 RNA
B: Protein Tat


Theoretical massNumber of molelcules
Total (without water)20,5212
Polymers20,5212
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: isothermal titration calorimetry, Used to determine the NMR Buffer conditions that led to 1:1 binding of the 7SK RNA:Tat RNA Binding domain complex, SAXS, Used the envelope of the Bound ...Evidence: isothermal titration calorimetry, Used to determine the NMR Buffer conditions that led to 1:1 binding of the 7SK RNA:Tat RNA Binding domain complex, SAXS, Used the envelope of the Bound complex and saw there wasn't much difference between the free and the bound in the SAXS so used this to help model the free 7SK-SL1 structure
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2780 Å2
ΔGint-6 kcal/mol
Surface area10040 Å2
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 20structures with the least restraint violations
RepresentativeModel #1fewest violations

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Components

#1: RNA chain 7SK Stem-loop 1 RNA


Mass: 18358.910 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#2: Protein/peptide Protein Tat / Transactivating regulatory protein


Mass: 2162.533 Da / Num. of mol.: 1 / Fragment: RNA BINDING DOMAIN RESIDUES 44-60 / Source method: obtained synthetically
Source: (synth.) Human immunodeficiency virus type 1 group M subtype B
References: UniProt: P04608

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
116isotropic12D 1H-1H NOESY
157isotropic12D 1H-1H NOESY
121isotropic12D 1H-15N HSQC
134isotropic12D 1H-15N HSQC
143isotropic12D 1H-13C HSQC
165isotropic12D 1H-13C HSQC

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent system
solution10.5 mM 7SK Stem-loop 1 RNA, 0.5 mM [U-13C; U-15N] HIV-1 Tat RNA Binding Domain selectively labeled with the following schema: 1) R49, K50, R57, A58 2) K51, R52, Q54, R55 3) R53, Q54, R56, 90% H2O/10% D2O7SK Stem-loop 1 RNA:Tat RNA Binding Domain-190% H2O/10% D2O
solution40.5 mM [U-13C; U-15N] 7SK Stem-loop 1 RNA, 0.5 mM HIV-1 Tat RNA Binding Domain, 90% H2O/10% D2O7SK Stem-loop 1 RNA:Tat RNA Binding Domain-290% H2O/10% D2O
solution30.5 mM 7SK Stem-loop 1 RNA, 0.5 mM [U-13C; U-15N] HIV-1 Tat RNA Binding Domain selectively labeled with the following schema: 1) R49, K50, R57, A58 2) K51, R52, Q54, R55 3) R53, Q54, R56, 100% D2O7SK Stem-loop 1 RNA:Tat RNA Binding Domain-3100% D2O
solution50.5 mM [U-13C; U-15N] 7SK Stem-loop 1 RNA, 0.5 mM HIV-1 Tat RNA Binding Domain, 100% D2O7SK Stem-loop 1 RNA:Tat RNA Binding Domain-4100% D2O
solution60.5 mM 7SK Stem-loop 1 RNA, 0.5 mM HIV-1 Tat RNA Binding Domain, 90% H2O/10% D2O7SK Stem-loop 1 RNA:Tat RNA Binding Domain-590% H2O/10% D2O
solution70.5 mM 7SK Stem-loop 1 RNA, 0.5 mM HIV-1 Tat RNA Binding Domain, 100% D2O7SK Stem-loop 1 RNA:Tat RNA Binding Domain-6100% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.5 mM7SK Stem-loop 1 RNAnatural abundance1
0.5 mMHIV-1 Tat RNA Binding Domain selectively labeled with the following schema: 1) R49, K50, R57, A58 2) K51, R52, Q54, R55 3) R53, Q54, R56[U-13C; U-15N]1
0.5 mM7SK Stem-loop 1 RNA[U-13C; U-15N]4
0.5 mMHIV-1 Tat RNA Binding Domainnatural abundance4
0.5 mM7SK Stem-loop 1 RNAnatural abundance3
0.5 mMHIV-1 Tat RNA Binding Domain selectively labeled with the following schema: 1) R49, K50, R57, A58 2) K51, R52, Q54, R55 3) R53, Q54, R56[U-13C; U-15N]3
0.5 mM7SK Stem-loop 1 RNA[U-13C; U-15N]5
0.5 mMHIV-1 Tat RNA Binding Domainnatural abundance5
0.5 mM7SK Stem-loop 1 RNAnatural abundance6
0.5 mMHIV-1 Tat RNA Binding Domainnatural abundance6
0.5 mM7SK Stem-loop 1 RNAnatural abundance7
0.5 mMHIV-1 Tat RNA Binding Domainnatural abundance7
Sample conditionsIonic strength: 10mM phosphates, 70mM NaCl mM / Label: 7SK_conditions / pH: 5.4 / Pressure: 100000 Pa / Temperature: 298 K

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NMR measurement

NMR spectrometerType: Bruker DMX / Manufacturer: Bruker / Model: DMX / Field strength: 700 MHz

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Processing

NMR software
NameDeveloperClassification
TopSpinBruker Biospincollection
NMRViewJohnson, One Moon Scientificpeak picking
NMRViewJohnson, One Moon Scientificchemical shift assignment
CYANAGuntert, Mumenthaler and Wuthrichdata analysis
CYANAGuntert, Mumenthaler and Wuthrichprocessing
CYANAGuntert, Mumenthaler and Wuthrichstructure calculation
AmberCase, Darden, Cheatham III, Simmerling, Wang, Duke, Luo, and Kollmanrefinement
RefinementMethod: simulated annealing / Software ordinal: 6
NMR representativeSelection criteria: fewest violations
NMR ensembleConformer selection criteria: structures with the least restraint violations
Conformers calculated total number: 20 / Conformers submitted total number: 10

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