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- EMDB-20645: MicroED structure of a FIB-milled CypA Crystal -

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Basic information

Entry
Database: EMDB / ID: EMD-20645
TitleMicroED structure of a FIB-milled CypA Crystal
Map data2mFo-DFc at 1.5 sigma
Sample
  • Complex: Peptidyl-prolyl cis-trans isomerase A
    • Protein or peptide: Peptidyl-prolyl cis-trans isomerase A
  • Ligand: water
KeywordsPeptidyl-prolyl / cis-trans / isomerase / cyclophilin
Function / homology
Function and homology information


negative regulation of protein K48-linked ubiquitination / negative regulation of viral life cycle / regulation of apoptotic signaling pathway / cell adhesion molecule production / lipid droplet organization / heparan sulfate binding / regulation of viral genome replication / leukocyte chemotaxis / negative regulation of stress-activated MAPK cascade / endothelial cell activation ...negative regulation of protein K48-linked ubiquitination / negative regulation of viral life cycle / regulation of apoptotic signaling pathway / cell adhesion molecule production / lipid droplet organization / heparan sulfate binding / regulation of viral genome replication / leukocyte chemotaxis / negative regulation of stress-activated MAPK cascade / endothelial cell activation / virion binding / Basigin interactions / cyclosporin A binding / Minus-strand DNA synthesis / Plus-strand DNA synthesis / Uncoating of the HIV Virion / Early Phase of HIV Life Cycle / Integration of provirus / APOBEC3G mediated resistance to HIV-1 infection / Calcineurin activates NFAT / viral release from host cell / Binding and entry of HIV virion / positive regulation of viral genome replication / protein peptidyl-prolyl isomerization / negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway / positive regulation of protein dephosphorylation / Gene and protein expression by JAK-STAT signaling after Interleukin-12 stimulation / activation of protein kinase B activity / neutrophil chemotaxis / negative regulation of protein phosphorylation / peptidylprolyl isomerase / peptidyl-prolyl cis-trans isomerase activity / positive regulation of protein secretion / negative regulation of protein kinase activity / Assembly Of The HIV Virion / Budding and maturation of HIV virion / neuron differentiation / platelet activation / platelet aggregation / SARS-CoV-1 activates/modulates innate immune responses / unfolded protein binding / integrin binding / protein folding / Platelet degranulation / positive regulation of NF-kappaB transcription factor activity / cellular response to oxidative stress / secretory granule lumen / vesicle / ficolin-1-rich granule lumen / positive regulation of MAPK cascade / response to hypoxia / positive regulation of protein phosphorylation / focal adhesion / apoptotic process / Neutrophil degranulation / protein-containing complex / extracellular space / RNA binding / extracellular exosome / extracellular region / membrane / nucleus / cytosol / cytoplasm
Similarity search - Function
Cyclophilin-type peptidyl-prolyl cis-trans isomerase / Cyclophilin-type peptidyl-prolyl cis-trans isomerase, conserved site / Cyclophilin-type peptidyl-prolyl cis-trans isomerase signature. / Cyclophilin-type peptidyl-prolyl cis-trans isomerase domain profile. / Cyclophilin-type peptidyl-prolyl cis-trans isomerase domain / Cyclophilin type peptidyl-prolyl cis-trans isomerase/CLD / Cyclophilin-like domain superfamily
Similarity search - Domain/homology
Peptidyl-prolyl cis-trans isomerase A
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodelectron crystallography / cryo EM / Resolution: 2.5 Å
AuthorsWolff AM / Martynowycz MW / Zhao W / Gonen T / Fraser JS / Thompson MC
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM123159 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM124149 United States
National Science Foundation (NSF, United States)STC-1231306 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM117126 United States
CitationJournal: IUCrJ / Year: 2020
Title: Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals.
Authors: Alexander M Wolff / Iris D Young / Raymond G Sierra / Aaron S Brewster / Michael W Martynowycz / Eriko Nango / Michihiro Sugahara / Takanori Nakane / Kazutaka Ito / Andrew Aquila / Asmit ...Authors: Alexander M Wolff / Iris D Young / Raymond G Sierra / Aaron S Brewster / Michael W Martynowycz / Eriko Nango / Michihiro Sugahara / Takanori Nakane / Kazutaka Ito / Andrew Aquila / Asmit Bhowmick / Justin T Biel / Sergio Carbajo / Aina E Cohen / Saul Cortez / Ana Gonzalez / Tomoya Hino / Dohyun Im / Jake D Koralek / Minoru Kubo / Tomas S Lazarou / Takashi Nomura / Shigeki Owada / Avi J Samelson / Tomoyuki Tanaka / Rie Tanaka / Erin M Thompson / Henry van den Bedem / Rahel A Woldeyes / Fumiaki Yumoto / Wei Zhao / Kensuke Tono / Sebastien Boutet / So Iwata / Tamir Gonen / Nicholas K Sauter / James S Fraser / Michael C Thompson /
Abstract: Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal ...Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.
History
DepositionAug 27, 2019-
Header (metadata) releaseSep 25, 2019-
Map releaseJan 29, 2020-
UpdateOct 11, 2023-
Current statusOct 11, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.46
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.46
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6u5g
  • Surface level: 1.46
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6u5g
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20645.png

Downloads & links

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Map

FileDownload / File: emd_20645.map.gz / Format: CCP4 / Size: 2.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation2mFo-DFc at 1.5 sigma
Voxel sizeX: 0.58889 Å / Y: 0.59333 Å / Z: 0.60944 Å
Density
Contour LevelBy AUTHOR: 1.46 / Movie #1: 1.46
Minimum - Maximum-3.1042123 - 5.7321124
Average (Standard dev.)-0.007928065 (±0.973654)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-19-20-38
Dimensions808285
Spacing858082
CellA: 50.055653 Å / B: 47.4664 Å / C: 49.974083 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.588894117647060.5933250.60943902439024
M x/y/z858082
origin x/y/z0.0000.0000.000
length x/y/z50.05647.46649.974
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S321
start NC/NR/NS-20-19-38
NC/NR/NS828085
D min/max/mean-3.1045.732-0.008

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Supplemental data

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Sample components

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Entire : Peptidyl-prolyl cis-trans isomerase A

EntireName: Peptidyl-prolyl cis-trans isomerase A
Components
  • Complex: Peptidyl-prolyl cis-trans isomerase A
    • Protein or peptide: Peptidyl-prolyl cis-trans isomerase A
  • Ligand: water

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Supramolecule #1: Peptidyl-prolyl cis-trans isomerase A

SupramoleculeName: Peptidyl-prolyl cis-trans isomerase A / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: monomer
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Peptidyl-prolyl cis-trans isomerase A

MacromoleculeName: Peptidyl-prolyl cis-trans isomerase A / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: peptidylprolyl isomerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 18.036504 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MVNPTVFFDI AVDGEPLGRV SFELFADKVP KTAENFRALS TGEKGFGYKG SCFHRIIPGF MCQGGDFTRH NGTGGKSIYG EKFEDENFI LKHTGPGILS MANAGPNTNG SQFFICTAKT EWLDGKHVVF GKVKEGMNIV EAMERFGSRN GKTSKKITIA D CGQLE

UniProtKB: Peptidyl-prolyl cis-trans isomerase A

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 32 / 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: 7.5
GridDetails: unspecified
VitrificationCryogen name: ETHANE
Crystal formationTemperature: 296.0 K
Details: 600 uL of protein at 60 mg/mL was combined with 400 uL of 50 percent PEG 3350 in a glass vial and stirred with an Octagon stir bar at 500 RPM

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: DIFFRACTION / Camera length: 2055 mm
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number grids imaged: 1 / Average electron dose: 0.06 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Crystallography statisticsNumber intensities measured: 22370 / Number structure factors: 6236 / Fourier space coverage: 86 / R sym: 0.217 / R merge: 0.217 / Overall phase error: 23.06 / Overall phase residual: 23.06 / Phase error rejection criteria: 0 / High resolution: 2.5 Å
Shell:
Shell IDHigh resolutionLow resolutionNumber structure factorsPhase residualFourier space coverageMultiplicity
13.6045 Å30.4934 Å358114.44999999999999984.03.41
22.8617 Å3.6045 Å371922.05000000000000187.03.64
32.5002 Å2.8617 Å374632.29999999999999787.03.72
Molecular replacementSoftware - Name: PHENIX (ver. dev2880) / Software - details: PHASER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: PHENIX (ver. dev2880)

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Atomic model buiding 1

Initial modelPDB ID:

4yum


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: RECIPROCAL
Output model

PDB-6u5g:
MicroED structure of a FIB-milled CypA Crystal

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