Difference: CemEngProjects (23 vs. 24)
Revision 2410 Sep 2013 - Main.EdEng
Contents
ProjectsNYUNed SeemanJune 29, 2012
Steve Hubbard/Todd MillerFebruary 23, 2012
March 9, 2012
April 6, 2012* F20 with Richie
May 4, 2012
June 12, 2012
June 20, 2012
July 10, 2012
August 21, 2012
K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast K10 - 60 sec stain - better, but dark areas too much stain and light areas too light L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain N1 - tmv tracer - 1 min incubation, unblotted - too much stain O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact O4 - PTA 2% in purification buffer - tmv tracer - too much stain September 26, 2012
November 07-08, 2012
June 2013Ed, Here are the .box files for the IR EM images. I went picked particles from a little over 100 images. I had to rename some files so I included a key in the folder but I will explain it here too. Essentially, I have three folders from images. Folders "P3 Round 1" and "P3 Round 2" from 6/12/13 and "P3" from 6/19/13. The files were renamed to the following... P3_A_001 to P3_A_005 = Images from 6/12/13 P3 Round 1 P3_B_001 to P3_B_050 = Images from 6/12/13 P3 Round 2 P3_C_001 to P3_C_150 = Images from 6/19/13 I am attempting to continue with analysis but I have been receiving a error stating "The data area passed to a system call is too small". I'm not completely sure what that error means but my guess is my laptop might not have the processing power necessary to complete the function. I might have to install EMAN2 on our lab's desktop computer and try it on that. I'll keep you updated on my progress. RichieMSKCCDirk RemusAugust 9, 2012Initial meetingAugust 15, 2012First 120kV session with JulienGros Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl?. The complexes were assembled on 1 kb linear DNA.buffer: 1 wash+0.1 M K-glutamate 2 wash+0.1 M K-Ac 3 wash+0.1 M NaCl? UrFormate?: 0.75%, pH 4.5 Grids: Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain 1 - sample 1 2 - sample 2 3 - sample 3 Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain 1B - sample 1 2B - sample 2 3B - sample 3 August 22, 20122nd 120kV session with JulienGros Mcm2-7 - 600 kDaNotes: Purification: Endogenous protein purified from yeast. Enriched with DNA coupled to magnetic beads. ORC (?300 kDa complex) and Cdc6 (60KDa protein) required to lead protein onto DNA. buffer:
Prasad JallepalliJuly 10, 2012Initial meetingJuly 23 2012First 80kV session with JohnMaciejowski Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa buffer: Tris, pH 7.4, 150 mM NaCl? , 1 mM EDTA, 1 mM DTT First conditions: Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stainA1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein A2 - 10 ug/mL - Ska1/2/3 wt A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain B1 - 2.5 ug/mL - Ska1/2/3 wt B2 - 10 ug/mL - Ska1/2/3 wt B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain C1 - 2.5 ug/mL - Ska1/2/3 wt C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain D1 - 2.5 ug/mL - Ska1/2/3 wt D2 - 10 ug/mL - Ska1/2/3 wt D3 - 25 ug/mL - Ska1/2/3 wt D4 - 10 ug/mL - Ska1/2/3 wt A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers B5 - 10 ug/mL - Ska1/2/3 S34D C5 - 10 ug/mL - Ska1/2 D5 - 10 ug/mL - Ska1/2 - monomers and oligomers Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain E1 - 10 ug/mL - Ska1/2/3 wt E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation? E3 - 10 ug/mL - Ska1/2/3 S34D E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation? Image stack
RCT
# rct macro v2
# 05-21-12
# should have opened file for writing
# sould be eucentric on square
# should have set defocu target
# should be in low dose
# roll buffer limit set to M or less
# this one is for plain carbon -- no alignment to hole is done
# no pause if drift fails, continues macro
MyAngle = 50 # set as desired
interval = 5 # set interval between photos for drift measurement
times = 8 # number of pictures
crit = 0.5 # drift must fall below this (nm/sec)
shot = Trial # type of image to take for drift measurement
T
copy A O # save current position
WalkUpTo $MyAngle
T # to minimize image shift
ResetImageShift
T
AlignTo B
Delay $interval
G
G # autofocus twice
$shot
loop $times index
$shot
AlignTo B
ReportAlignShift
ClearAlignment
dx = $reportedValue3
dy = $reportedValue4
dist = sqrt $dx * $dx + $dy * $dy
echo Distance = $dist nm
if $dist < $crit
echo drift is low enough after shot $index
break
endif
if $index < $times
echo warning drift is high in this image
endif
EndLoop
R
S
WalkUpto 0
ResetImageShift
G
G
AlignTo O
$shot
loop $times index
$shot
AlignTo B
ReportAlignShift
ClearAlignment
dx = $reportedValue3
dy = $reportedValue4
dist = sqrt $dx * $dx + $dy * $dy
echo Distance = $dist nm
if $dist < $crit
echo drift is low enough after shot $index
break
endif
if $index < $times
echo warning drift is high in this image
endif
EndLoop
R
S
#
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