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- EMDB-5294: 3D reconstruction of frozen hydrated HIV-1 integrase dimer in com... -

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Entry
Database: EMDB / ID: EMD-5294
Title3D reconstruction of frozen hydrated HIV-1 integrase dimer in complex with two Fabs.
Map dataThis is 3D reconstruction of frozen hydrated HIV-1 integrase dimer in complex with 2 Fabs.
Sample
  • Sample: HIV-1 integrase - Fab complex
  • Protein or peptide: integrase
KeywordsHIV-1 integrase dimer / Fab
Biological speciesHuman immunodeficiency virus 1
Methodsingle particle reconstruction / cryo EM / Resolution: 9.6 Å
AuthorsWu S / Avila-Sakar A / Kim J / Booth DS / Greenberg CH / Rossi A / Liao M / Alian A / Griner SL / Juge N ...Wu S / Avila-Sakar A / Kim J / Booth DS / Greenberg CH / Rossi A / Liao M / Alian A / Griner SL / Juge N / Mergel CM / Chaparro-Riggers J / Strop P / Tampe R / Edwards RH / Stroud RM / Craik CS / Cheng Y
CitationJournal: Structure / Year: 2012
Title: Fabs enable single particle cryoEM studies of small proteins.
Authors: Shenping Wu / Agustin Avila-Sakar / JungMin Kim / David S Booth / Charles H Greenberg / Andrea Rossi / Maofu Liao / Xueming Li / Akram Alian / Sarah L Griner / Narinobu Juge / Yadong Yu / ...Authors: Shenping Wu / Agustin Avila-Sakar / JungMin Kim / David S Booth / Charles H Greenberg / Andrea Rossi / Maofu Liao / Xueming Li / Akram Alian / Sarah L Griner / Narinobu Juge / Yadong Yu / Claudia M Mergel / Javier Chaparro-Riggers / Pavel Strop / Robert Tampé / Robert H Edwards / Robert M Stroud / Charles S Craik / Yifan Cheng /
Abstract: In spite of its recent achievements, the technique of single particle electron cryomicroscopy (cryoEM) has not been widely used to study proteins smaller than 100 kDa, although it is a highly ...In spite of its recent achievements, the technique of single particle electron cryomicroscopy (cryoEM) has not been widely used to study proteins smaller than 100 kDa, although it is a highly desirable application of this technique. One fundamental limitation is that images of small proteins embedded in vitreous ice do not contain adequate features for accurate image alignment. We describe a general strategy to overcome this limitation by selecting a fragment antigen binding (Fab) to form a stable and rigid complex with a target protein, thus providing a defined feature for accurate image alignment. Using this approach, we determined a three-dimensional structure of an ∼65 kDa protein by single particle cryoEM. Because Fabs can be readily generated against a wide range of proteins by phage display, this approach is generally applicable to study many small proteins by single particle cryoEM.
History
DepositionMay 22, 2011-
Header (metadata) releaseOct 26, 2011-
Map releaseMay 29, 2012-
UpdateMay 29, 2012-
Current statusMay 29, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5294_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5294.map.gz / Format: CCP4 / Size: 12.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is 3D reconstruction of frozen hydrated HIV-1 integrase dimer in complex with 2 Fabs.
Voxel sizeX=Y=Z: 1.8 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-1.03870511 - 3.12253904
Average (Standard dev.)-0.00818645 (±0.18489794)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-74-54-74
Dimensions148148148
Spacing148148148
CellA=B=C: 266.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.81.81.8
M x/y/z148148148
origin x/y/z0.0000.0000.000
length x/y/z266.400266.400266.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-54-74-74
NC/NR/NS148148148
D min/max/mean-1.0393.123-0.008

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Supplemental data

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Sample components

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Entire : HIV-1 integrase - Fab complex

EntireName: HIV-1 integrase - Fab complex
Components
  • Sample: HIV-1 integrase - Fab complex
  • Protein or peptide: integrase

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Supramolecule #1000: HIV-1 integrase - Fab complex

SupramoleculeName: HIV-1 integrase - Fab complex / type: sample / ID: 1000 / Oligomeric state: 2 Fabs bind to one integrase dimer / Number unique components: 2
Molecular weightExperimental: 160 KDa / Theoretical: 160 KDa

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Macromolecule #1: integrase

MacromoleculeName: integrase / type: protein_or_peptide / ID: 1 / Details: dimer, total molecular weight 65kDa / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus 1 / synonym: HIV-1
Molecular weightExperimental: 32 KDa / Theoretical: 32 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

GridDetails: 200 mesh Quantifoil
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 100 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.1 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 80000
Sample stageSpecimen holder: CT3500 / Specimen holder model: GATAN LIQUID NITROGEN
DateFeb 1, 2011
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F816 (8k x 8k) / Average electron dose: 30 e/Å2
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Frealign / Number images used: 14000

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Atomic model buiding 1

Initial modelPDB ID:

1exq

SoftwareName: Chimera
DetailsProtocol: rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

1m71

SoftwareName: Chimera
DetailsProtocol: rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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