CRYSTALLOGRAPHY AND STORYTELLING WITH DATAUnit cell: smallest repetitive volume which contains the...

CRYSTALLOGRAPHY AND STORYTELLING WITH DATA

President, Association of Women in Science, Bethesda Chapter

STEM Consultant

MY STORY

Passion for Science

BS Biology Major

MS Biotechnology & Project in Bioinformatics fascination with computational biology

PhD in Biochemistry + Protein Crystallography

MBA - Science Management

CRUX OF MY STORY

Passion for Science

Excelling at what I do

What Next?

SKILLS COVERED IN THIS PPT

Biochemistry

Molecular biology

Crystallography

Computer Simulations

Creativity in science

Data tools

Storytelling (Communication)

Problem solving

Collaboration

WHAT IS PROTEIN X- RAY CRYSTALLOGRAPHY?

X-ray crystallography is essentially a form of very high resolution microscopy. It enables us to visualize protein structures at the atomic level and enhances our understanding of protein function. Specifically we can study how proteins interact with other molecules, how they undergo conformational changes, and how they perform catalysis in the case of enzymes. Armed with this information we can design novel drugs that target a particular protein, or rationally engineer an enzyme for a specific industrial process.

Steps :

Protein purification

Crystallization

Data collection

Data analysis

Structure solution

Story telling!

THE METHOD

Protein Crystals X-rays

diffraction Refined structure

FT FT-1

TYPES OF MACROMOLECULES 1. Proteins

2. DNA

3. RNA

4. Complexes: protein-protein, protein-DNA/RNA

Lipids, peptides, sugars, etc are categorized as

non macromolecules

Today’s discussion is more focused on protein

molecules

FOUR LEVELS OF PROTEIN STRUCTURE

Diffraction from a single molecule is weak, hence we need crystals that

have ordered three-dimensional array of molecules.

A small protein crystal contains a billion molecules.

Need to get great quality single crystals: Multiple trials & persistence !

Protein Crystals

CRYSTAL SYSTEMS – SOME DEFINITIONAL INFORMATION

7 crystal systems of varying

symmetry are known

These systems are built by

changing the lattice

parameters:

a, b, and c are the edge

lengths

, , and are interaxial

angles

Unit cell: smallest repetitive volume which contains the complete

lattice pattern of a crystal.

a

b

c

Crystal systems

System Interaxial Axes

Angles

Triclinic ≠ ≠ ≠ 90° a ≠ b ≠ c

Monoclinic = = 90° ≠ a ≠ b ≠ c

Orthorhombic = = = 90° a ≠ b ≠ c

Tetragonal = = = 90° a = b ≠ c

Cubic = = = 90° a = b = c

Hexagonal = = 90°, = 120° a = b ≠ c

Trigonal = = 90°, = 120° a = b ≠ c

PEPTIDE BOND LENGTHS

X-RAY DIFFRACTION

Spacing should be comparable to the wavelength of diffracted radiation.

To resolve interatomic distances should be less that the distances.

Spacing is the distance between parallel planes of atoms.

Diffraction Data

Sequence

Initial Phases

Quality Control

Refinement

Validation

Phase Combination

Model Building

The steps to solve the macromolecular crystal structure

X-RAY DIFFRACTOMETER

CRYSTAL MOUNTING AND CRYO-CRYSTALLOGRAPHY

X-ray sources: synchrotron X-rays, 106 times stronger.

CRYSTAL MOUNTING AND CRYO-CRYSTALLOGRAPHY

Data Collection

DIMENSIONS X-ray detector

Beam width: ~0.20 mm

Crystal thickness: 0.10-1.00 mm

10K+ REFLECTIONS

•Moving the X-rays and the detector gives a new set of

planes.

•Changing the angle of reflection changes the spacing

(resolution).

GENERAL AMINO ACID STRUCTURE

Diffraction image from a RAXIS-IV image plate

Frame Oscillation = 1o

Exposure time = 20 min

Maximum resolution = 2.5 Å

Protein bonds & angles

• Torsion angle (dihedral angle)

– Measures orientation of four linked atoms in a molecule: A, B, C, D

THE RAMACHANDRAN PLOT

DATA ANALYSIS SOFTWARE (TRANSFERRABLE SKILLCCP4 suite

Graphic softwares (Coot, Quanta)

Python

Excel

R

SPSS

DATA COLLECTION STRATEGY AND DATA PROCESSING

• How many images to collect?

• Exposure time

• Expected statistics

• Design the right strategy to collect data

and get good results!

• Data processing : index, integrate,

merge and calculate structure factor

amplitudes.

Electron Density Map

TELLING YOUR STORY

Amino-acid alignment at the active site of dGluR

Active site

residues in

Drosophila

Glutamate

Receptors

Tell a great story

with results !

Bioinformatics

Interdisciplinary science :

Mathematics, Biology, chemistry.

Physics, engineering etc.

Career paths:

• Health informatics

• Data scientists

• Financial consultants

• Fintech careers

MY STORY PART 2: MBA AND LEADERSHIP

Skills:

Project Management

Finance

Team working skills

Received Leadership

Award !!

PR0GRAM MANAGEMENT

Managing multiple projects in one portfolio

Organization management

Planning

Time management

Strategy

Finances

Communication

Collaboration

Team work

On this Sunday from

3-5 pm

Collaboration with Astrazeneca/MedImmune,

Booz Allen Hamilton and BioHealth

Innovations

Advocacy at the Capitol hill for science funding ! We got the funding

STEM

Outreach at

Universities

Area Colleges

Pharma

companies

Collaborations!

Mentoring workshop 2018 – 24 mentors and almost 100 participants!

Dr. Judy Stavley was there too!

STEM EDUCATION & OUTREACH

Our mission is to promote student achievement and career

exploration in math and science while increasing student

exposure to the science, technology, engineering and

mathematics communities.

Experimental Design Program teaches students how to

design and conduct experiments for math and science fairs

utilizing research, laboratory and scientific literacy skills

gained through individual and group mentoring and

learning opportunities

WHY I DO WHAT I DO?

Love for Science

Passion for mentoring

Supporting the creative development in STEM professionals

Serve as a leader

Team player

Science communication

STEM outreach

Poorva Dharkar

poorvadharkar@gmail.com

Crystal growth

Diffraction theory

Symmetry

Experimental methods

Interpretation of data

Software