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- EMDB-0409: Single-particle cryo-EM reconstruction of the catalytic subunit o... -

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Basic information

Entry
Database: EMDB / ID: EMD-0409
TitleSingle-particle cryo-EM reconstruction of the catalytic subunit of protein kinase A bound to ATP and IP20 using 200 keV
Map dataSingle-particle cryo-EM reconstruction of the catalytic subunit of protein kinase A bound to ATP and IP20 (unsharpened)
Sample
  • Complex: catalytic subunit of mouse protein kinase A bound to ATP and IP20Catalysis
    • Protein or peptide: catalytic subunit of protein kinase ACatalysis
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.0 Å
AuthorsHerzik Jr MA / Wu M / Lander GC
CitationJournal: Nat Commun / Year: 2019
Title: High-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM.
Authors: Mark A Herzik / Mengyu Wu / Gabriel C Lander /
Abstract: Determining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons (kDa) has been a longstanding goal of the cryo-electron microscopy (cryo-EM) community. While ...Determining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons (kDa) has been a longstanding goal of the cryo-electron microscopy (cryo-EM) community. While the Volta phase plate has enabled visualization of specimens in this size range, this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.7 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity to enable investigation of drug-target interactions. Our ~2.8 Å and ~3.2 Å structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the sub-nanometer cryo-EM structure of a sub-50 kDa protein.
History
DepositionDec 6, 2018-
Header (metadata) releaseFeb 13, 2019-
Map releaseFeb 13, 2019-
UpdateMar 20, 2019-
Current statusMar 20, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.012
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0409.png

Downloads & links

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Map

FileDownload / File: emd_0409.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle-particle cryo-EM reconstruction of the catalytic subunit of protein kinase A bound to ATP and IP20 (unsharpened)
Voxel sizeX=Y=Z: 1.1172 Å
Density
Contour LevelBy AUTHOR: 0.012 / Movie #1: 0.012
Minimum - Maximum-0.054195397 - 0.06922188
Average (Standard dev.)0.00016517921 (±0.0033557545)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 143.0016 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1172031251.1172031251.117203125
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z143.002143.002143.002
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0540.0690.000

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Supplemental data

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Additional map: Single-particle cryo-EM reconstruction of the catalytic subunit of...

Fileemd_0409_additional.map
AnnotationSingle-particle cryo-EM reconstruction of the catalytic subunit of protein kinase A bound to ATP and IP20 (sharpened)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Odd half map

Fileemd_0409_half_map_1.map
AnnotationOdd half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Even half map

Fileemd_0409_half_map_2.map
AnnotationEven half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : catalytic subunit of mouse protein kinase A bound to ATP and IP20

EntireName: catalytic subunit of mouse protein kinase A bound to ATP and IP20Catalysis
Components
  • Complex: catalytic subunit of mouse protein kinase A bound to ATP and IP20Catalysis
    • Protein or peptide: catalytic subunit of protein kinase ACatalysis

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Supramolecule #1: catalytic subunit of mouse protein kinase A bound to ATP and IP20

SupramoleculeName: catalytic subunit of mouse protein kinase A bound to ATP and IP20
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 43 KDa

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Macromolecule #1: catalytic subunit of protein kinase A

MacromoleculeName: catalytic subunit of protein kinase A / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GNAAAAKKGS EQESVKEFLA KAKEDFLKKW ETPSQNTAQL DQFDRIKTLG TGSFGRVMLV KHKESGNHYA MKILDKQKVV KLKQIEHTL NEKRILQAVN FPFLVKLEFS FKDNSNLYMV MEYVAGGEMF SHLRRIGRFS EPHARFYAAQ IVLTFEYLHS L DLIYRDLK ...String:
GNAAAAKKGS EQESVKEFLA KAKEDFLKKW ETPSQNTAQL DQFDRIKTLG TGSFGRVMLV KHKESGNHYA MKILDKQKVV KLKQIEHTL NEKRILQAVN FPFLVKLEFS FKDNSNLYMV MEYVAGGEMF SHLRRIGRFS EPHARFYAAQ IVLTFEYLHS L DLIYRDLK PENLLIDQQG YIQVTDFGFA KRVKGRTWTL CGTPEYLAPE IILSKGYNKA VDWWALGVLI YEMAAGYPPF FA DQPIQIY EKIVSGKVRF PSHFSSDLKD LLRNLLQVDL TKRFGNLKNG VNDIKNHKWF ATTDWIAIYQ RKVEAPFIPK FKG PGDTSN FDDYEEEEIR VSINEKCGKE FTEF

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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

Concentration5 mg/mL
BufferpH: 7
Component:
ConcentrationNameFormula
10.0 mMMOPS
50.0 mMSodium chlorideNaClSodium chloride
3.0 mMManganese chlorideMnCl2
0.2 mMATPAdenosine triphosphate
2.0 mMDTT
10.0 XInhibitor peptide IP20
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
Details: Grids were plasma cleaned using a Solarus plasma cleaner (Gatan, Inc.)
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER
Details: Sample was manually blotted for 4-5 seconds using Whatman No. 1 filter paper immediately prior to plunge-freezing..

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 16.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 73000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #0 - Detector mode: COUNTING / #0 - Number grids imaged: 2 / #0 - Number real images: 3176 / #0 - Average exposure time: 11.0 sec. / #0 - Average electron dose: 69.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #1 - Detector mode: COUNTING / #1 - Digitization - Dimensions - Width: 3710 pixel / #1 - Digitization - Dimensions - Height: 3838 pixel / #1 - Digitization - Sampling interval: 5.0 µm / #1 - Digitization - Frames/image: 1-44 / #1 - Number grids imaged: 2 / #1 - Number real images: 2402 / #1 - Average exposure time: 11.0 sec. / #1 - Average electron dose: 69.0 e/Å2 / #1 - Details: Specimen was tilted at 30 degrees
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 788060 / Details: Template-picked
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: OTHER
Details: An ab initio model was generated from 314,001 template-picked particles using cryoSPARC and low-pass filtered to 20 A for use as an initial model for 3D refinement.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 74413
DetailsCounting mode, 250 ms frames, exposure rate of ~1.95 e- pixel-1 s-1, total exposure of ~69 e- angstrom-2 (1.57 e- angstrom-2 frame-1).
Image recording ID1
FSC plot (resolution estimation)

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