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Yorodumi- EMDB-5295: 3D reconstruction of negatively stained PCSK9 in complex with a Fab -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5295 | |||||||||
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Title | 3D reconstruction of negatively stained PCSK9 in complex with a Fab | |||||||||
Map data | 3D reconstruction of negatively stained PCSK9-Fab complex | |||||||||
Sample |
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Keywords | PCSK9-Fab complex | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 25.0 Å | |||||||||
Authors | Wu 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 | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5295.map.gz | 799.7 KB | EMDB map data format | |
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Header (meta data) | emd-5295-v30.xml emd-5295.xml | 10.4 KB 10.4 KB | Display Display | EMDB header |
Images | emd_5295_1.jpg | 16.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5295 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5295 | HTTPS FTP |
-Validation report
Summary document | emd_5295_validation.pdf.gz | 78.6 KB | Display | EMDB validaton report |
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Full document | emd_5295_full_validation.pdf.gz | 77.7 KB | Display | |
Data in XML | emd_5295_validation.xml.gz | 492 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5295 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5295 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_5295.map.gz / Format: CCP4 / Size: 825.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 3D reconstruction of negatively stained PCSK9-Fab complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.26 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : human PCSK9 in complex with a Fab
Entire | Name: human PCSK9 in complex with a Fab |
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Components |
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-Supramolecule #1000: human PCSK9 in complex with a Fab
Supramolecule | Name: human PCSK9 in complex with a Fab / type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 2 |
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Molecular weight | Experimental: 120 KDa / Theoretical: 120 KDa |
-Macromolecule #1: PCSK9
Macromolecule | Name: PCSK9 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: human |
Molecular weight | Experimental: 120 KDa / Theoretical: 120 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Staining | Type: NEGATIVE / Details: uranyl formate |
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Grid | Details: 200 mesh copper grid |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | FEI TECNAI 20 |
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Alignment procedure | Legacy - Astigmatism: each particle |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 30 e/Å2 |
Tilt angle min | 0 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Calibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 0.0015 µm / Nominal defocus min: 0.0015 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: single tilt / Specimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 60 |
-Image processing
CTF correction | Details: each particle |
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Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Frealign / Number images used: 3876 |
-Atomic model buiding 1
Initial model | PDB ID: |
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Refinement | Space: REAL |