CHEM M230D Course Syllabus
Structural Molecular Biology Laboratory Winter Quarter, 2019
Duilio Cascio & Michael R. Sawaya

Course Description: A laboratory course covering the practical aspects of determining a protein crystal structure by X-ray crystallography. Specifically, students will determine the structure of proteinase K, a 28 kDa enzyme, by performing all the steps of a typical crystallographic experiment: crystallization, screening for heavy atom derivatives, collecting and reducing data, space group determination, solving for the position of heavy atom sites, calculating MIRAS phases, interpreting electron density, refining and validating structure models, and making illustrative figures. Students will meet in small groups, once a week by appointment for 1.5-2 hours. Five assignments will be given to check understanding of course material and prepare the student to publish a crystallographic journal article. Meeting Locations will vary from week to week as noted below.

M230B LECTURE MATERIALS available at http://people.mbi.ucla.edu/cascio/LECTURES/M230B.

When asked for a login name and password, please refer to the syllabus you received on the first day of class.

DATA PROCESSING SPREADSHEET for 2019
DATA PROCESSING SPREADSHEET for 2018
DATA PROCESSING SPREADSHEET for 2017

M230D LAB MATERIALS: Experimental Procedures and Suggested Reading Materials:

Crystallization and Heavy Atom Screening
January 8-9, 2019
Crystallization Facility, Boyer Hall Room 106

Suggested Reading Materials:
     1) What a crystallographer should know about a protein before beginning crystallization trials by Michael Sawaya.
     2) Heavy Atom Safety, Selection, and Screening by Michael Sawaya (2000).
     3) Screening for Phasing Atoms in Protein Crystallography by Boggon & Shapiro (2000).
Experiments Performed in Lab:
     1) Crystallization of Proteinase K and complex with PMSF
     2) Heavy Atom Derivative Screening by Native Gel Electrophoresis
     3) Preparation of Heavy Atom derivatives 

Heavy Atom Soaking
January 9-10, 2019
X-ray Lab, Boyer Hall Room 124
Cryo-crystallography & Data Collection
January 10-18, 2019 
X-ray Lab, Boyer Hall Room 124

Suggested Reading Materials:
     1) Data Collection Strategies by Zbigniew Dauter (1999).
     2) Solvent Content of Protein Crystals by B.W. Matthews (1968).
Activities In Lab:
     1) Cryo-crystallographic technique video (WMV file 65 Mb, open with Windows Media Player)
     2) Practice removing and storing crystals for synchrotron trip
     3) Initiate data collection

Data Reduction
January 23, 2019
Grad Student Computer Lab, Young Hall Room 4067
Activities In Lab:
    1) Data Reduction
Assignments: "Table 1" and crystal data collection reports due following week
Difference Patterson Maps & Determination of Heavy Atom Sites
January 28, 2019
Grad Student Computer Lab, Young Hall Room 4067

Suggested Reading Materials:
     1) Chapter 6 (page 101-127) from Crystallography Made Crystal Clear by Gale Rhodes.
Activities in Lab:
     1) Familiarization with simple Unix commands.
     2) Calculation of isomorphous and anomalous difference Patterson maps.
Assignments: Patterson reports due following week
 

Phasing & Electron Density Map Calculation
February 6, 2019
Computer Lab, Young Hall Room 4067

Suggested Reading Materials:
     1) Chapter 6 (page 101-127) from Crystallography Made Crystal Clear by Gale Rhodes.
Activities in Lab: 
     1) Phasing with MLPhaRe
     2) Solvent Flattening
     3) Chose correct handedness
     4) Calculate electron density
     5) View density with "COOT"
Assignments:  Phasing reports due following week

Model Building
February 13, 2019
Computer Lab, Young Hall Room 4067

Suggested Reading Materials:
     1) Electron density map intepretation by T. A. Jones and M. Kjeldgaard.
Activities in Lab:
     1) Placing Alpha helices and beta strands in density.
     2) Assigning protein sequence to density
     3) Run & evaluate automatic chain tracing program
Assignments:  Model Building reports due following week
 

Refinement & Structure Validation
February 20, 2019
Computer Lab, Young Hall Room 4067

Suggested Reading Materials:
     1) None yet.
Activities in Lab:
     1) Calculation of difference Fourier electron density maps.
     2) Evaluation of maps
     3) Refinement of Structure
     4) Run Procheck, Verify3D and Errat
Assignments:  Final structure reports due following week
 

Pymol tutorial
 


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