Difference: CemEngProjects (22 vs. 23)
Revision 2324 Jun 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
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| > > | 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. Richie | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MSKCCDirk 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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