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Title:Second 3D model of the MjHSP16.5 mutant R107G at room temperature by CryoEM.
Authors:Roy A Q, 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 (10 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 blotted for 2 s with blot force 2 at 100% 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 nm75000FIELD 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/Å226-OCT-2011
Processing
Protocol:projection matching
Software:EMAN1
CTF correction:Each image
Number of particles:5420
Number of class averages:193
Imposed symmetry:O
Resolution by author:10 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
546 μm/pixel32Electron micrographs were collected manually.
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails