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- EMDB-5295: 3D reconstruction of negatively stained PCSK9 in complex with a Fab -

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Basic information

Entry
Database: EMDB / ID: EMD-5295
Title3D reconstruction of negatively stained PCSK9 in complex with a Fab
Map data3D reconstruction of negatively stained PCSK9-Fab complex
Sample
  • Sample: human PCSK9 in complex with a Fab
  • Protein or peptide: PCSK9
KeywordsPCSK9-Fab complex
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 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 23, 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: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5295_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5295.map.gz / Format: CCP4 / Size: 825.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of negatively stained PCSK9-Fab complex
Voxel sizeX=Y=Z: 4.26 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-0.82391357 - 3.13429093
Average (Standard dev.)0.00072357 (±0.1562008)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions606060
Spacing606060
CellA=B=C: 255.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.264.264.26
M x/y/z606060
origin x/y/z0.0000.0000.000
length x/y/z255.600255.600255.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS606060
D min/max/mean-0.8243.1340.001

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Supplemental data

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Sample components

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Entire : human PCSK9 in complex with a Fab

EntireName: human PCSK9 in complex with a Fab
Components
  • Sample: human PCSK9 in complex with a Fab
  • Protein or peptide: PCSK9

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Supramolecule #1000: human PCSK9 in complex with a Fab

SupramoleculeName: human PCSK9 in complex with a Fab / type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 2
Molecular weightExperimental: 120 KDa / Theoretical: 120 KDa

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Macromolecule #1: PCSK9

MacromoleculeName: PCSK9 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human
Molecular weightExperimental: 120 KDa / Theoretical: 120 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

StainingType: NEGATIVE / Details: uranyl formate
GridDetails: 200 mesh copper grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 0.0015 µm / Nominal defocus min: 0.0015 µm / Nominal magnification: 50000
Sample stageSpecimen holder: single tilt / Specimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 60
Alignment procedureLegacy - Astigmatism: each particle
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 30 e/Å2
Tilt angle min0

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Image processing

CTF correctionDetails: each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Frealign / Number images used: 3876

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Atomic model buiding 1

Initial modelPDB ID:

2pmw

RefinementSpace: REAL

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