+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-29588 | |||||||||
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Title | MicroED structure of Proteinase K from xenon milled lamellae | |||||||||
Map data | Proteinase K structure from xenon milled lamellae | |||||||||
Sample |
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Function / homology | Function and homology information peptidase K / serine-type endopeptidase activity / proteolysis / extracellular space / metal ion binding Similarity search - Function | |||||||||
Biological species | Parengyodontium album (fungus) | |||||||||
Method | electron crystallography / cryo EM / Resolution: 1.45 Å | |||||||||
Authors | Martynowycz MW / Shiriaeva A / Clabbers MTB / Nicolas WJ / Weaver SJ / Hattne J / Gonen T | |||||||||
Funding support | United States, 2 items
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Citation | Journal: Nat Commun / Year: 2023 Title: A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals. Authors: Michael W Martynowycz / Anna Shiriaeva / Max T B Clabbers / William J Nicolas / Sara J Weaver / Johan Hattne / Tamir Gonen / Abstract: Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction ...Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_29588.map.gz | 11.6 MB | EMDB map data format | |
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Header (meta data) | emd-29588-v30.xml emd-29588.xml | 14.7 KB 14.7 KB | Display Display | EMDB header |
Images | emd_29588.png | 88.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-29588 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-29588 | HTTPS FTP |
-Validation report
Summary document | emd_29588_validation.pdf.gz | 497.1 KB | Display | EMDB validaton report |
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Full document | emd_29588_full_validation.pdf.gz | 496.6 KB | Display | |
Data in XML | emd_29588_validation.xml.gz | 4.2 KB | Display | |
Data in CIF | emd_29588_validation.cif.gz | 4.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-29588 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-29588 | HTTPS FTP |
-Related structure data
Related structure data | 8fypMC 8fynC 8fyoC 8fyqC 8fyrC 8fysC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_29588.map.gz / Format: CCP4 / Size: 12.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Proteinase K structure from xenon milled lamellae | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||||||
Voxel size | X: 0.34922 Å / Y: 0.34922 Å / Z: 0.35673 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 96 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Sample components
-Entire : Proteinase K
Entire | Name: Proteinase K |
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Components |
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-Supramolecule #1: Proteinase K
Supramolecule | Name: Proteinase K / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Parengyodontium album (fungus) |
Molecular weight | Theoretical: 28.9 KDa |
-Macromolecule #1: Proteinase K
Macromolecule | Name: Proteinase K / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: peptidase K |
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Source (natural) | Organism: Parengyodontium album (fungus) |
Molecular weight | Theoretical: 28.958791 KDa |
Sequence | String: AAQTNAPWGL ARISSTSPGT STYYYDESAG QGSCVYVIDT GIEASHPEFE GRAQMVKTYY YSSRDGNGHG THCAGTVGSR TYGVAKKTQ LFGVKVLDDN GSGQYSTIIA GMDFVASDKN NRNCPKGVVA SLSLGGGYSS SVNSAAARLQ SSGVMVAVAA G NNNADARN ...String: AAQTNAPWGL ARISSTSPGT STYYYDESAG QGSCVYVIDT GIEASHPEFE GRAQMVKTYY YSSRDGNGHG THCAGTVGSR TYGVAKKTQ LFGVKVLDDN GSGQYSTIIA GMDFVASDKN NRNCPKGVVA SLSLGGGYSS SVNSAAARLQ SSGVMVAVAA G NNNADARN YSPASEPSVC TVGASDRYDR RSSFSNYGSV LDIFGPGTDI LSTWIGGSTR SISGTSMATP HVAGLAAYLM TL GKTTAAS ACRYIADTAN KGDLSNIPFG TVNLLAYNNY QA |
-Macromolecule #2: NITRATE ION
Macromolecule | Name: NITRATE ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: NO3 |
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Molecular weight | Theoretical: 62.005 Da |
Chemical component information | ChemComp-NO3: |
-Macromolecule #3: CALCIUM ION
Macromolecule | Name: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: CA |
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Molecular weight | Theoretical: 40.078 Da |
-Macromolecule #4: water
Macromolecule | Name: water / type: ligand / ID: 4 / Number of copies: 294 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | electron crystallography |
Aggregation state | 3D array |
-Sample preparation
Concentration | 5 mg/mL |
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Buffer | pH: 7.5 |
Grid | Model: Quantifoil R2/2 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10.0 nm |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA PLUNGER |
Details | Milled microcrystals |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 77.0 K / Max: 90.0 K |
Image recording | Film or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Number grids imaged: 1 / Number real images: 1 / Number diffraction images: 840 / Average exposure time: 0.5 sec. / Average electron dose: 0.001 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Calibrated defocus min: 0.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Cs: 2.7 mm / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm / Camera length: 1202 mm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN / Tilt angle: -30.0, 30.0 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Details | Binned by 2 |
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Final reconstruction | Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 1.45 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES |
Merging software list | Software - Name: AIMLESS |
Crystallography statistics | Number intensities measured: 569407 / Number structure factors: 64974 / Fourier space coverage: 87.58 / R sym: 0.073 / R merge: 0.236 / Overall phase error: 30 / Overall phase residual: 0 / Phase error rejection criteria: None / High resolution: 0.87 Å / Shell - Shell ID: 1 / Shell - High resolution: 0.87 Å / Shell - Low resolution: 0.9 Å / Shell - Number structure factors: 2783 / Shell - Phase residual: 30 / Shell - Fourier space coverage: 37.64 / Shell - Multiplicity: 2.1 |
-Atomic model buiding 1
Refinement | Space: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 12.65 / Target criteria: Maximum likelihood |
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Output model | PDB-8fyp: |