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- EMDB-8222: MicroED structure of thermolysin at 2.5 A resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-8222
TitleMicroED structure of thermolysin at 2.5 A resolution
Map dataThermolysin
Sample
  • Organelle or cellular component: Thermolysin
    • Protein or peptide: Thermolysin
  • Ligand: CALCIUM ION
  • Ligand: ZINC ION
  • Ligand: DIMETHYL SULFOXIDE
  • Ligand: ISOPROPYL ALCOHOL
  • Ligand: water
KeywordsHydrolase
Function / homology
Function and homology information


thermolysin / metalloendopeptidase activity / proteolysis / extracellular region / metal ion binding
Similarity search - Function
PepSY domain / Peptidase propeptide and YPEB domain / : / Peptidase M4, C-terminal / FTP domain / Peptidase M4 domain / Peptidase M4 / Thermolysin metallopeptidase, catalytic domain / Thermolysin metallopeptidase, alpha-helical domain / Fungalysin/Thermolysin Propeptide Motif ...PepSY domain / Peptidase propeptide and YPEB domain / : / Peptidase M4, C-terminal / FTP domain / Peptidase M4 domain / Peptidase M4 / Thermolysin metallopeptidase, catalytic domain / Thermolysin metallopeptidase, alpha-helical domain / Fungalysin/Thermolysin Propeptide Motif / Peptidase M4/M1, CTD superfamily / Neutral zinc metallopeptidases, zinc-binding region signature.
Similarity search - Domain/homology
Biological speciesBacillus thermoproteolyticus (bacteria)
Methodelectron crystallography / cryo EM / Resolution: 2.5 Å
Authorsde la Cruz MJ / Hattne J
CitationJournal: Nat Methods / Year: 2017
Title: Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.
Authors: M Jason de la Cruz / Johan Hattne / Dan Shi / Paul Seidler / Jose Rodriguez / Francis E Reyes / Michael R Sawaya / Duilio Cascio / Simon C Weiss / Sun Kyung Kim / Cynthia S Hinck / Andrew P ...Authors: M Jason de la Cruz / Johan Hattne / Dan Shi / Paul Seidler / Jose Rodriguez / Francis E Reyes / Michael R Sawaya / Duilio Cascio / Simon C Weiss / Sun Kyung Kim / Cynthia S Hinck / Andrew P Hinck / Guillermo Calero / David Eisenberg / Tamir Gonen /
Abstract: Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from ...Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.
History
DepositionMay 26, 2016-
Header (metadata) releaseJun 22, 2016-
Map releaseJun 22, 2016-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.25
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8222.png

Downloads & links

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Map

FileDownload / File: emd_8222.map.gz / Format: CCP4 / Size: 2.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThermolysin
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
0.82 Å/pix.
x 71 pix.
= 128.498 Å		0.81 Å/pix.
x 99 pix.
= 92.065 Å		0.81 Å/pix.
x 82 pix.
= 92.065 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 0.80759 Å / Y: 0.80759 Å / Z: 0.82371 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.27 / Movie #1: 0.25
Minimum - Maximum-0.5022738 - 0.9751885
Average (Standard dev.)-0.0005916783 (±0.14839673)
SymmetrySpace group: 178
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-152-37
Dimensions998271
Spacing114114156
CellA: 92.065094 Å / B: 92.065094 Å / C: 128.498 Å
α: 90.0 ° / β: 90.0 ° / γ: 120.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.807587719298250.807587719298250.82370512820513
M x/y/z114114156
origin x/y/z0.0000.0000.000
length x/y/z92.06592.065128.498
α/β/γ90.00090.000120.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S213
start NC/NR/NS2-15-37
NC/NR/NS829971
D min/max/mean-0.5020.975-0.001

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Supplemental data

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Sample components

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Entire : Thermolysin

EntireName: Thermolysin
Components
  • Organelle or cellular component: Thermolysin
    • Protein or peptide: Thermolysin
  • Ligand: CALCIUM ION
  • Ligand: ZINC ION
  • Ligand: DIMETHYL SULFOXIDE
  • Ligand: ISOPROPYL ALCOHOL
  • Ligand: water

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Supramolecule #1: Thermolysin

SupramoleculeName: Thermolysin / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bacillus thermoproteolyticus (bacteria)
Molecular weightTheoretical: 34.634 KDa

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Macromolecule #1: Thermolysin

MacromoleculeName: Thermolysin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: thermolysin
Source (natural)Organism: Bacillus thermoproteolyticus (bacteria)
Molecular weightTheoretical: 34.360336 KDa
SequenceString: ITGTSTVGVG RGVLGDQKNI NTTYSTYYYL QDNTRGNGIF TYDAKYRTTL PGSLWADADN QFFASYDAPA VDAHYYAGVT YDYYKNVHN RLSYDGNNAA IRSSVHYSQG YNNAFWNGSQ MVYGDGDGQT FIPLSGGIDV VAHELTHAVT DYTAGLIYQN E SGAINEAI ...String:
ITGTSTVGVG RGVLGDQKNI NTTYSTYYYL QDNTRGNGIF TYDAKYRTTL PGSLWADADN QFFASYDAPA VDAHYYAGVT YDYYKNVHN RLSYDGNNAA IRSSVHYSQG YNNAFWNGSQ MVYGDGDGQT FIPLSGGIDV VAHELTHAVT DYTAGLIYQN E SGAINEAI SDIFGTLVEF YANKNPDWEI GEDVYTPGIS GDSLRSMSDP AKYGDPDHYS KRYTGTQDNG GVHINSGIIN KA AYLISQG GTHYGVSVVG IGRDKLGKIF YRALTQYLTP TSNFSQLRAA AVQSATDLYG STSQEVASVK QAFDAVGVK

UniProtKB: Thermolysin

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #4: DIMETHYL SULFOXIDE

MacromoleculeName: DIMETHYL SULFOXIDE / type: ligand / ID: 4 / Number of copies: 1 / Formula: DMS
Molecular weightTheoretical: 78.133 Da
Chemical component information

ChemComp-DMS:
DIMETHYL SULFOXIDE / DMSO, precipitant*YM

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Macromolecule #5: ISOPROPYL ALCOHOL

MacromoleculeName: ISOPROPYL ALCOHOL / type: ligand / ID: 5 / Number of copies: 1 / Formula: IPA
Molecular weightTheoretical: 60.095 Da
Chemical component information

ChemComp-IPA:
ISOPROPYL ALCOHOL

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 21 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron crystallography
Aggregation state3D array

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationNameFormula
45.0 %dimethyl sulfoxide
50.0 mMTris
2.5 Mcesium chlorideCsCl
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Number grids imaged: 3 / Number real images: 721 / Number diffraction images: 721 / Average exposure time: 4.1 sec. / Average electron dose: 0.004 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Camera length: 1750 mm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES
Molecular replacementSoftware - Name: MOLREP (ver. 11.4.05) / Software - details: Starting model PDB ID 2tli
Symmetry determination software listSoftware - Name: POINTLESS (ver. 1.10.21)
Merging software listSoftware - Name: AIMLESS (ver. 0.5.25)
Crystallography statisticsNumber intensities measured: 224846 / Number structure factors: 25029 / Fourier space coverage: 58.5 / R sym: 0.634 / R merge: 0.634 / Overall phase error: 26.44 / Overall phase residual: 44.76 / Phase error rejection criteria: 0 / High resolution: 1.6 Å / Shell - Shell ID: 1 / Shell - High resolution: 2.5 Å / Shell - Low resolution: 2.75 Å / Shell - Number structure factors: 2741 / Shell - Phase residual: 47.2 / Shell - Fourier space coverage: 96.8 / Shell - Multiplicity: 12.2

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Atomic model buiding 1

Initial modelPDB ID:

2tli


Chain - Chain ID: A / Chain - Residue range: 1-316 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: RECIPROCAL / Protocol: OTHER
Output model

PDB-5k7t:
MicroED structure of thermolysin at 2.5 A resolution

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