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Title:Second 3D model of the MjHSP16.5 mutant R107G at 60 degree Celsius by CryoEM.
Authors:Roy AQ, Yan Z, Andrew L, Ian W, Ehmke P, Fei S
Sample:R107G mutation of small heat shock protein (sHSP) HSP16.5 from Methanocaldococcus jannaschii (MjHSP16.5)
Method:Single particle reconstruction (15 angstroms resolution)
Red flagLatest update:2013-07-24
Sample
Sample name: R107G mutation of small heat shock protein (sHSP) HSP16.5 from Methanocaldococcus jannaschii (MjHSP16.5)
Oligomeric state: 24mer
Experimental molecular weight of the sample: 0.396
Components:
ID Type Name Exp. MW (MDa) Theo. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteinMjHSP16.5 R107G mutation0.01650.016524mertrueMethanocaldococcus jannaschiiQ57733GO:0005737, GO:0006950IPR002068, IPR008978
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.40.8 mg/mL10mM HEPES, 100mM NaCl.GIG holey grids (LifeTrust, China) were treated with a glow discharge machine (Master Plasmer)
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE%93 KFEI VITROBOT MARK IVThe samples were heated to 60 degree Celsius in boiler, then 3.5 microliter samples were added to the grid and blotted for 2 s with blot force 2 at 90% humidity. ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TITAN KRIOS300 kVFLOOD BEAMBRIGHT FIELD2.7 mm2000 nm3000 nm96000FIELD EMISSION GUNGATAN ULTRASCAN 4000 (4k x 4k) mm

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
Liquid nitrogen cooledFEI TITAN KRIOS AUTOGRID HOLDER°° eV85 K K K mrad20 e/Å231-DEC-2011
Processing
Protocol:projection matching
Software:EMAN1
CTF correction:Each image
Number of particles:5010
Number of class averages:193
Imposed symmetry:O
Resolution by author:15 Å
Resolution method:FSC 0.5
Processing details:The final reconstructed density map was further sharpened by application of an amplitude correction algorithm in the program BFACTOR. The particles were selected using an automatic selection program Gautomatch developed in Fei Sun lab (to be published). Octahedron symmetry were imposed during 3D reconstructing.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
1308 μm/pixel32Electron micrograph exposures were made with the automatic collection package Leginon.
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails