protocol.md

Fiji

• Function: inspect movies (*.mrc) quality manually

mrc2smv

• Function: convert .mrc file to SMV imgage file (.img)
  • because data reduction software developed for x-ray crystallography does not understand MRC or TVIPS files
• tvips-tools-jiffies has mrc2smv

Adxv

• Open method: (in macOS) double-click Adxv in /Applications
• Function 1: triage image quality manually
  • Input: *.img, *.mrc, *.mrcs file
  • Output example
    • 
• Function 2: locate ORGX and ORGY manually (however, AutoMicroED will estimate ORGX and ORGY automatically if user specified an argument for this function)
  • Example
    • 

xds

• Function
  • index and integrate SMV image files
  • process single-crystal monochromatic diffraction data recorded by the rotation method
• Input
  • SMV image files
• Output
  • XDS_ASCII.HKL that contains corrected intensities of all reflections recorded in the data images.
  • CORRECT.LP that has COMPLETENESS information.
• Automatic troubleshootings will be tried depending on situations
  • These troubleshootings include
    • sliding SPOT RANGE, applying half SPOT RANGE and full BACKGROUND RANGE, running shorter JOBs, applying shorter DATA RANGE and trying DELPHI=10
  • Additionally, once CORRECT.LP is found, AutoMicroED further iterates xds looping various BEAM DIVERGENCE and REFLECTING RANGE values until ISa no longer improves.

spot2pdb

• Main function: To provide final output for publication purpose (showing what range/wedge of data was collected).
• Other function: spot2pdb can visualize which spots were indexed and not indexed by XDS as well. However, AutoMicroED recommends to use generate_adx for this purpose.
• Input (per mrcs)
  • SPOT.XDS
    • contains a list of strong observed diffraction spots that is generated by the "COLSPOT" step
  • XPARM.XDS
    • generated by IDXREF
    • "contains all diffraction parameters necessary for computing the locations of all reflections occurring in the data images."
• Output (per mrcs)
  • SPOT-indexed.pdb, SPOT-notindexed.pdb
• A user needs to inspect manually.
  • To do
    • Copy merged SPOT.pdb files to user's computer.
      • For example, scp -r kimd999@kriosgpu.emsl.pnl.gov:/gpustorage/DN/microED/acet/protein_False/output/20210726_18_50_22/spot2pdb_result_merged .
    • Open with coot
      • For example, coot SPOT-*.pdb
      • Visualiazation example in coot
        • 

generate_adx

• Function: whole frame verification (checking index)
• This is optional, but recommended.
• A user needs to inspect manually.
• Input: XDS_ASCII.HKL in xds folder per each input mrc(s)
• Output: adx files in img folder per each input mrc(s)
  • Example
    • 
• To do
  • Install (once)
    • copy generate_adx_source_file to user's folder
    • gfortran -O -C generate_adx.f90 -o generate_adx
  • Specify generate_adx folder that has compiled generate_adx binary
    • Refer (AutoMicroED git repository)/templates/args_file/explanation_of_args.txt
  • Analysis method
    • (in macOS) double-click Adxv in /Applications
    • Open both input img (or mrcs) and adx file
      • (If generated adx files lacks extension, then put .adx extension)
        • (Welcome to re-use/modify (AutoMicroED git repository)/util/add_extension.py)

xds-viewer

• Function: allows only per frame verification (checking index)
• This is optional, but recommended.
• A user needs to inspect manually.
• To do
  • Install (once)
    • macOS
      • Download xds-viewer binary package from http://xds-viewer.sourceforge.net
      • Move XDS-Viewer binary to /Applications and double-click there
    • Linux
      • Download https://strucbio.biologie.uni-konstanz.de/pub/linux_bin/XDS-viewer
        • (for example,) wget https://strucbio.biologie.uni-konstanz.de/pub/linux_bin/XDS-viewer
      • chmod +rwx XDS-viewer
    • windowsOS
      • We found no evidence of WindowsOS support
  • Input
    • Download FRAME.cbf (Crystallographic Binary File) to user's computer
      • For example, scp kimd999@kriosgpu.emsl.pnl.gov:/gpustorage/DN/microED/acet/protein_False/output/20210625_19_39_22/131749merged/xds/FRAME.cbf .
    • (for mac), double-click xds-viewer binary in /Applications, then File -> Load Image (FRAME.cbf)
    • (for Linux), we couldn't run XDS-viewer even after logging in -X, -XC, -Y with
      • "However,qt.qpa.screen: QXcbConnection: Could not connect to display Could not connect to any X display."
  • Output example
    • 

xscale (1st run)

• Function: scale .mrc files
  • Retrieve LATT, SYMM records
• Input: XDS_ASCII.HKL
• Output: output.ahkl

xscale (2nd run)

• Function: merge (many) XDS_ASCII.HKL files
• Input: Many XDS_ASCII.HKL files from same SPACE_GROUP_NUMBER and similar UNIT_CELL_CONSTANTS
  • (If absolute difference of a, b, c, alpha, beta, gamma of UNIT_CELL < 1 between datasets, it is considered as "similar" enough to be merged).
• Output: XDS_ASCII_merged.hkl

xdsconv

• Function: convert reflection data files from xds or xscale into various formats for crystal structure determination like CCP4, or SHELX
• Input: XDS_ASCII_merged.hkl
• Output (small molecule): shelxt_input_from_xdsconv.hkl SHELX !CCP4_I+F ! or CCP4_I or CCP4_F or SHELX or CNS
• Output (protein): ready_for_f2mtz.hkl CCP4_I+F

(for small molecule)

• link

(for protein)

• link