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- PDB-6bg9: HYBRID NMR/CRYO-EM STRUCTURE OF THE HIV-1 RNA DIMERIZATION SIGNAL -

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Basic information

Entry
Database: PDB / ID: 6bg9
TitleHYBRID NMR/CRYO-EM STRUCTURE OF THE HIV-1 RNA DIMERIZATION SIGNAL
ComponentsRNA dimerization signal
KeywordsRNA / RNA INERNAL LOOPS / SHEARED GA PAIRS / GU WOBBLE / S-TURN THERMODYNAMICS
Function / homologyRNA / RNA (> 10)
Function and homology information
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / SOLUTION NMR / subtomogram averaging / molecular dynamics / cryo EM / Resolution: 9 Å
AuthorsSummers, M.F.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM R01 42561 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM P01 103297 United States
CitationJournal: Structure / Year: 2018
Title: Structure of the 30 kDa HIV-1 RNA Dimerization Signal by a Hybrid Cryo-EM, NMR, and Molecular Dynamics Approach.
Authors: Kaiming Zhang / Sarah C Keane / Zhaoming Su / Rossitza N Irobalieva / Muyuan Chen / Verna Van / Carly A Sciandra / Jan Marchant / Xiao Heng / Michael F Schmid / David A Case / Steven J ...Authors: Kaiming Zhang / Sarah C Keane / Zhaoming Su / Rossitza N Irobalieva / Muyuan Chen / Verna Van / Carly A Sciandra / Jan Marchant / Xiao Heng / Michael F Schmid / David A Case / Steven J Ludtke / Michael F Summers / Wah Chiu /
Abstract: Cryoelectron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy are routinely used to determine structures of macromolecules with molecular weights over 65 and under 25 kDa, ...Cryoelectron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy are routinely used to determine structures of macromolecules with molecular weights over 65 and under 25 kDa, respectively. We combined these techniques to study a 30 kDa HIV-1 dimer initiation site RNA ([DIS]; 47 nt/strand). A 9 Å cryo-EM map clearly shows major groove features of the double helix and a right-handed superhelical twist. Simulated cryo-EM maps generated from time-averaged molecular dynamics trajectories (10 ns) exhibited levels of detail similar to those in the experimental maps, suggesting internal structural flexibility limits the cryo-EM resolution. Simultaneous inclusion of the cryo-EM map and H-edited NMR-derived distance restraints during structure refinement generates a structure consistent with both datasets and supporting a flipped-out base within a conserved purine-rich bulge. Our findings demonstrate the power of combining global and local structural information from these techniques for structure determination of modest-sized RNAs.
History
DepositionOct 27, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 21, 2018Provider: repository / Type: Initial release
Revision 1.1Mar 21, 2018Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jan 1, 2020Group: Author supporting evidence / Data collection
Category: pdbx_audit_support / pdbx_nmr_software / pdbx_nmr_spectrometer
Item: _pdbx_audit_support.funding_organization / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model
Revision 1.3May 1, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: RNA dimerization signal
B: RNA dimerization signal


Theoretical massNumber of molelcules
Total (without water)30,7182
Polymers30,7182
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4500 Å2
ΔGint-23 kcal/mol
Surface area16630 Å2
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 20all calculated structures submitted
RepresentativeModel #1lowest energy

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Components

#1: RNA chain RNA dimerization signal


Mass: 15359.241 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Production host: in vitro transcription vector pT7-TP(deltai) (others)

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Experimental details

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Experiment

Experiment
Method
ELECTRON MICROSCOPY
SOLUTION NMR
EM experimentAggregation state: PARTICLE / 3D reconstruction method: subtomogram averaging
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic12D NOESY (2H-EDITED)
121isotropic1ABUNDANCE 13C-1H HMQC
NMR detailsText: NMR studies conducted with multiple samples prepared by differential nucleotide-specific 2H labeling.

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Sample preparation

ComponentName: Human immunodeficiency virus 1 / Type: ORGANELLE OR CELLULAR COMPONENT
Details: RNA prepared by in vitro transcription using T7 RNA polymerase
Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: in vitro transcription vector pT7-TP(deltai) (others)
Details of virusIsolate: OTHER / Type: VIRUS-LIKE PARTICLE
Buffer solutionpH: 7.2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %
Details
TypeSolution-IDContentsLabelSolvent system
solution1400 uM Fully protonated, 5 mM NaCl, 140 mM KCl, 1 mM MgCl2, Tris, 100% D2OHIV-1 DIS100% D2O
solution2400 uM protonated A-H2 and A,G-ribose carbons A2rGr; all other sites deuterated A2rGr, 5 mM NaCl, 140 mM KCl, 1 mM MgCl2, 100% D2Osample_2100% D2O
solution3400 uM protonated A-H2,H8 and fully protonated G; all other sites deuterated A28GH, 5 mM NaCl, 140 mM KCl, 1 mM MgCl2, 100% D2Osample_3100% D2O
solution4400 uM protonated G-H8, C-H6,ribose; all other sites deuterated G8C6r, 5 mM NaCl, 140 mM KCl, 1 mM MgCl2, 100% D2Osample_4100% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
400 uMFully protonatednatural abundance1
400 uMA2rGrprotonated A-H2 and A,G-ribose carbons; all other sites deuterated2
400 uMA28GHprotonated A-H2,H8 and fully protonated G; all other sites deuterated3
400 uMG8C6rprotonated G-H8, C-H6,ribose; all other sites deuterated4
5 mMNaClnatural abundance1
5 mMNaClnatural abundance2
5 mMNaClnatural abundance3
5 mMNaClnatural abundance4
140 mMKClnatural abundance1
140 mMKClnatural abundance2
140 mMKClnatural abundance3
140 mMKClnatural abundance4
1 mMMgCl2natural abundance1
1 mMMgCl2natural abundance2
1 mMMgCl2natural abundance3
1 mMMgCl2natural abundance4
Sample conditionsDetails: Samples in D2O, 5 mM NaCl, 140 mM KCl, 1 mM MgCl2, Tris, pH 7.2, T= 308K
Ionic strength: 150 mM / Ionic strength err: 10 / Label: All samples / pH: 7.1 pD / Pressure: 1 atm / Temperature: 308 K

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Data collection

MicroscopyModel: JEOL 2200FS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 25000 X
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingElectron dose: 75 e/Å2 / Film or detector model: DIRECT ELECTRON DE-12 (4k x 3k) / Num. of grids imaged: 4
NMR spectrometerType: Bruker AVANCE / Manufacturer: Bruker / Model: AVANCE / Field strength: 600 MHz

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Processing

EM software
IDNameVersionCategory
1EMAN22.1volume selection
4EMAN22.1CTF correction
13EMAN22.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 20366 / Symmetry type: POINT
EM volume selectionNum. of tomograms: 540 / Num. of volumes extracted: 20366
NMR software
NameDeveloperClassification
NMRFxBruce Johnson, One Moon Scientificprocessing
NMRViewJohnson, One Moon Scientificdata analysis
CYANAGuntert, Mumenthaler and Wuthrichstructure calculation
AmberCase, Darden, Cheatham III, Simmerling, Wang, Duke, Luo, and Kollmanrefinement
NMRViewJohnson, One Moon Scientificchemical shift assignment
NMRViewJohnson, One Moon Scientificpeak picking
RefinementMethod: molecular dynamics / Software ordinal: 6
Details: Used GB for solvent simulation, NOEs and EM data simultaneously as restraints
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: all calculated structures submitted
Conformers calculated total number: 20 / Conformers submitted total number: 20

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