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- EMDB-8220: MicroED structure of trypsin at 1.7 A resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-8220
TitleMicroED structure of trypsin at 1.7 A resolution
Map dataTrypsin
Sample
  • Organelle or cellular component: Trypsin
    • Protein or peptide: Cationic trypsin
  • Ligand: CALCIUM IONCalcium
  • Ligand: water
Function / homology
Function and homology information


trypsin / serpin family protein binding / serine protease inhibitor complex / digestion / endopeptidase activity / serine-type endopeptidase activity / proteolysis / extracellular space / metal ion binding
Similarity search - Function
Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, histidine active site. / Serine proteases, trypsin domain profile. / Trypsin-like serine protease / Serine proteases, trypsin family, serine active site. / Serine proteases, trypsin domain / Trypsin / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan
Similarity search - Domain/homology
Biological speciesBos taurus (cattle) / Bovine (cattle)
Methodelectron crystallography / cryo EM / Resolution: 1.7 Å
Authorsde la Cruz MJ / Hattne J / Shi D / Seidler P / Rodriguez J / Reyes FE / Sawaya MR / Cascio D / Eisenberg D / Gonen T
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-
UpdateAug 22, 2018-
Current statusAug 22, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5k7r
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5k7r
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8220.png

Downloads & links

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Map

FileDownload / File: emd_8220.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTrypsin
Voxel sizeX: 0.554 Å / Y: 0.56429 Å / Z: 0.57743 Å
Density
Contour LevelBy EMDB: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.054148965 - 0.17173952
Average (Standard dev.)0.00002090898 (±0.018716726)
SymmetrySpace group: 19
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-54-80-31
Dimensions10086100
Spacing96100112
CellA: 53.184002 Å / B: 56.428497 Å / C: 64.6722 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.5540.564280.57742857142857
M x/y/z96100112
origin x/y/z0.0000.0000.000
length x/y/z53.18456.42864.672
α/β/γ90.00090.00090.000
start NX/NY/NZ-54-80-31
NX/NY/NZ10086100
MAP C/R/S213
start NC/NR/NS-80-54-31
NC/NR/NS86100100
D min/max/mean-0.0540.1720.000

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Supplemental data

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Sample components

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Entire : Trypsin

EntireName: Trypsin
Components
  • Organelle or cellular component: Trypsin
    • Protein or peptide: Cationic trypsin
  • Ligand: CALCIUM IONCalcium
  • Ligand: water

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Supramolecule #1: Trypsin

SupramoleculeName: Trypsin / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 23.354 KDa

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Macromolecule #1: Cationic trypsin

MacromoleculeName: Cationic trypsin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: trypsin
Source (natural)Organism: Bovine (cattle)
Molecular weightTheoretical: 23.324287 KDa
SequenceString: IVGGYTCGAN TVPYQVSLNS GYHFCGGSLI NSQWVVSAAH CYKSGIQVRL GEDNINVVEG NEQFISASKS IVHPSYNSNT LNNDIMLIK LKSAASLNSR VASISLPTSC ASAGTQCLIS GWGNTKSSGT SYPDVLKCLK APILSDSSCK SAYPGQITSN M FCAGYLEG ...String:
IVGGYTCGAN TVPYQVSLNS GYHFCGGSLI NSQWVVSAAH CYKSGIQVRL GEDNINVVEG NEQFISASKS IVHPSYNSNT LNNDIMLIK LKSAASLNSR VASISLPTSC ASAGTQCLIS GWGNTKSSGT SYPDVLKCLK APILSDSSCK SAYPGQITSN M FCAGYLEG GKDSCQGDSG GPVVCSGKLQ GIVSWGSGCA QKNKPGVYTK VCNYVSWIKQ TIASN

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Macromolecule #2: CALCIUM ION

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

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 195 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / 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: 6.5
Component:
ConcentrationNameFormula
10.0 mg/mlbenzamidine
3.0 mMcalcium chlorideCaCl
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Camera length: 1500 mm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Digitization - Sampling interval: 0.0311999992 µm / Number grids imaged: 3 / Number real images: 1527 / Number diffraction images: 1527 / Average exposure time: 4.1 sec. / Average electron dose: 0.004 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Crystal parametersUnit cell - A: 53.12 Å / Unit cell - B: 56.08 Å / Unit cell - C: 64.38 Å / Unit cell - γ: 90 ° / Unit cell - α: 90 ° / Unit cell - β: 90 ° / Space group: P 21 21 21
Crystallography statisticsNumber intensities measured: 145833 / Number structure factors: 23542 / Fourier space coverage: 73.8 / R sym: 0.773 / R merge: 0.773 / Overall phase error: 28.86 / Overall phase residual: 40.4 / Phase error rejection criteria: 0 / High resolution: 1.5 Å / Shell - Shell ID: 1 / Shell - High resolution: 1.7 Å / Shell - Low resolution: 1.79 Å / Shell - Number structure factors: 1737 / Shell - Phase residual: 60.7 / Shell - Fourier space coverage: 56.2 / Shell - Multiplicity: 3.1
Molecular replacementSoftware - Name: MOLREP (ver. 11.4.05) / Software - details: Starting model PDB ID 2ptn
Symmetry determination software listSoftware - Name: POINTLESS (ver. 1.10.21)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 1.7 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES
Merging software listSoftware - Name: AIMLESS (ver. 0.5.25)

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Atomic model buiding 1

Initial modelPDB ID:

2ptn


Chain - Chain ID: A / Chain - Residue range: 16-245
RefinementSpace: RECIPROCAL / Protocol: OTHER
Output model

PDB-5k7r:
MicroED structure of trypsin at 1.7 A resolution

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