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- EMDB-6439: Helical cryo-EM reconstruction of HIV Rev filament -

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Basic information

Entry
Database: EMDB / ID: EMD-6439
TitleHelical cryo-EM reconstruction of HIV Rev filament
Map data
SampleHIV-1 Rev filament:
Rev protein
KeywordsHIV Rev filament / helical assembly / HIV life cycle / viral RNA / nuclear export
Function / homologyAnti-repression trans-activator protein, REV protein / host cell nucleolus / mRNA transport / host cell cytoplasm / DNA-binding transcription factor activity / viral process / RNA binding / Protein Rev
Function and homology information
Biological speciesHuman immunodeficiency virus 1 (HIV-1)
Methodhelical reconstruction / cryo EM / Resolution: 8.3 Å
AuthorsDiMattia MA / Watts NR / Cheng N / Huang R / Heymann JB / Wingfield PT / Grimes JM / Stuart DI / Steven AC
CitationJournal: Structure / Year: 2016
Title: The Structure of HIV-1 Rev Filaments Suggests a Bilateral Model for Rev-RRE Assembly.
Authors: Michael A DiMattia / Norman R Watts / Naiqian Cheng / Rick Huang / J Bernard Heymann / Jonathan M Grimes / Paul T Wingfield / David I Stuart / Alasdair C Steven /
Abstract: HIV-1 Rev protein mediates the nuclear export of viral RNA genomes. To do so, Rev oligomerizes cooperatively onto an RNA motif, the Rev response element (RRE), forming a complex that engages with the ...HIV-1 Rev protein mediates the nuclear export of viral RNA genomes. To do so, Rev oligomerizes cooperatively onto an RNA motif, the Rev response element (RRE), forming a complex that engages with the host nuclear export machinery. To better understand Rev oligomerization, we determined four crystal structures of Rev N-terminal domain dimers, which show that they can pivot about their dyad axis, giving crossing angles of 90° to 140°. In parallel, we performed cryoelectron microscopy of helical Rev filaments. Filaments vary from 11 to 15 nm in width, reflecting variations in dimer crossing angle. These structures contain additional density, indicating that C-terminal domains become partially ordered in the context of filaments. This conformational variability may be exploited in the assembly of RRE/Rev complexes. Our data also revealed a third interface between Revs, which offers an explanation for how the arrangement of Rev subunits adapts to the "A"-shaped architecture of the RRE in export-active complexes.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionAug 25, 2015-
Header (metadata) releaseSep 23, 2015-
Map releaseJun 22, 2016-
UpdateJul 20, 2016-
Current statusJul 20, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.6
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol

Downloads & links

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Map

FileDownload / File: emd_6439.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.02 Å/pix.
x 300 pix.
= 304.65 Å
1.02 Å/pix.
x 300 pix.
= 304.65 Å
1.02 Å/pix.
x 300 pix.
= 304.65 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0155 Å
Density
Contour LevelBy AUTHOR: 1.6 / Movie #1: 1.6
Minimum - Maximum-5.7130022 - 7.22380447
Average (Standard dev.)0.0 (±0.80485672)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-150-149-150
Dimensions300300300
Spacing300300300
CellA=B=C: 304.65 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.01551.01551.0155
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z304.650304.650304.650
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-149-150-150
NC/NR/NS300300300
D min/max/mean-5.7137.2240.000

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Supplemental data

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Sample components

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Entire HIV-1 Rev filament

EntireName: HIV-1 Rev filament / Number of components: 1 / Oligomeric State: polymerized filament of Rev dimers

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Component #1: protein, Rev protein

ProteinName: Rev protein / Oligomeric Details: helical polymer / Recombinant expression: Yes
SourceSpecies: Human immunodeficiency virus 1 (HIV-1)
Source (engineered)Expression System: Escherichia coli (E. coli)
External referencesUniProt: Protein Rev

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Experimental details

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Sample preparation

SpecimenSpecimen state: Filament / Method: cryo EM
Helical parametersAxial symmetry: C6 (6 fold cyclic) / Hand: LEFT HANDED / Delta z: 21.2 Å / Delta phi: 22.1 %deg;
Sample solutionSpecimen conc.: 2 mg/mL
Support filmC-flat holey carbon grids
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Temperature: 100 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300 / Date: Feb 19, 2015
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 20000 X (nominal) / Imaging mode: BRIGHT FIELD / Defocus: 990 - 3090 nm
Specimen HolderModel: GATAN LIQUID NITROGEN
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 93
Details: Every image is the average of 30 frames recorded by the direct electron detector.

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Image processing

ProcessingMethod: helical reconstruction / Details: The particles were aligned using IHRSR.
3D reconstructionAlgorithm: Iterative helical real space reconstruction (IHRSR)
Software: BSOFT / CTF correction: Each particle / Resolution: 8.3 Å / Resolution method: FSC 0.333, semi-independent

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