The

scientific

impact

of

the

Structural

Genomics

Consortium

a

protein

family

and

ligand-centered

approach

to

medically-relevant

human

proteins

As

many

of

the

structural

genomics

centers

have

ended

their

first

phase

of

operation

it

is

a

good

point

to

evaluate

the

scientific

impact

of

this

endeavour

The

Structural

Genomics

Consortium

-LRB-

SGC

-RRB-

operating

from

three

centers

across

the

Atlantic

investigates

human

proteins

involved

in

disease

processes

and

proteins

from

Plasmodium

falciparum

and

related

organisms

We

present

here

some

of

the

scientific

output

of

the

Oxford

node

of

the

SGC

where

the

target

areas

include

protein

kinases

phosphatases

oxidoreductases

and

other

metabolic

enzymes

as

well

as

signal

transduction

proteins

The

SGC

has

aimed

to

achieve

extensive

coverage

of

human

gene

families

with

a

focus

on

protein

ligand

interactions

The

methods

employed

for

effective

protein

expression

crystallization

and

structure

determination

by

X-ray

crystallography

are

summarized

In

addition

to

the

cumulative

impact

of

accelerated

delivery

of

protein

structures

we

demonstrate

how

family

coverage

generic

screening

methodology

and

the

availability

of

abundant

purified

protein

samples

allow

a

level

of

discovery

that

is

difficult

to

achieve

otherwise

The

contribution

of

NMR

to

structure

determination

and

protein

characterization

is

discussed

To

make

this

information

available

to

a

wide

scientific

audience

a

new

tool

for

disseminating

annotated

structural

information

was

created

that

also

represents

an

interactive

platform

allowing

for

a

continuous

update

of

the

annotation

by

the

scientific

community

Introduction

The

long-term

goal

of

structural

genomics

-LRB-

SG

-RRB-

has

been

ambitiously

defined

as

to

make

three-dimensional

atomic

level

structures

of

most

proteins

easily

obtainable

from

knowledge

of

their

corresponding

DNA

sequences

-LRB-

-RRB-

Long

before

this

goal

is

achieved

the

multiple

specialized

SG

projects

are

expected

to

have

a

significant

impact

on

many

aspects

of

the

biological

sciences

The

most

readily

apparent

contribution

of

SG

is

the

rapid

expansion

in

the

number

of

available

protein

structures

derived

at

a

reduced

cost

because

of

the

efficiency

of

specialized

centers

Proper

target

selection

is

critical

to

ensure

that

the

structures

solved

by

SG

centers

are

indeed

valuable

to

the

research

and

industrial

community

either

because

of

the

intrinsic

interest

of

the

proteins

investigated

or

because

of

the

improved

mapping

of

the

protein

structure

universe

providing

homologous

structural

models

A

second

important

contribution

of

SG

projects

for

the

scientific

community

is

the

development

of

methods

for

efficient

protein

production

and

structure

determination

which

could

be

adopted

in

smaller

research

laboratories

to

improve

productivity

Other

scientific

deliverables

of

structural

genomics

derive

from

the

scale

and

nature

of

the

operations

and

include

comparative

studies

on

members

of

protein

families

identifying

determinants

of

specificity

deriving

general

rules

and

improving

the

capability

to

predict

protein

structure

and

function

from

gene

sequences

The

Structural

Genomics

Consortium

-LRB-

SGC

-RRB-

operating

in

the

Universities

of

Oxford

and

Toronto

and

the

Karolinska

Institute

was

initiated

in

to

address

needs

of

industrial

and

academic

pharmaceutical

research

The

SGC

investigates

human

and

apicomplexan

proteins

the

targets

are

selected

based

on

their

potential

as

drug

targets

or

involvement

in

disease

processes

Technologically

the

SGC

focuses

on

interaction

of

proteins

with

small

molecules

-LRB-

ligands

inhibitors

substrates

and

co-factors

-RRB-

and

on

coverage

of

protein

families

This

report

provides

several

examples

of

the

impact

of

research

undertaken

at

the

Oxford

node

of

the

SGC

including

methodology

for

high-throughput

structure

determination

generic

means

for

ligand

screening

selected

examples

of

insight

from

specific

structures

insights

from

family

coverage

and

the

possibilities

resulting

from

the

availability

of

large

numbers

of

purified

protein

samples

The

other

SGC

nodes

share

the

core

technologies

but

investigate

non-overlapping

target

areas

Finally

the

scientific

impact

depends

on

dissemination

of

structural

data

We

describe

a

new

platform

for

distribution

of

annotated

protein

structures

which

aims

at

making

this

data

more

meaningful

to

an

audience

beyond

the

usual

users

of

the

PDB

Methodology

Protein

production

Method

adaptation

and

development

for

structural

genomics

involved

a

change

of

mindset

no

less

than

developments

in

instrumentation

chemistry

and

computer

software

Industrialization

of

protein

production

applied

to

a

huge

variety

of

proteins

with

very

divergent

chemical

properties

is

not

straightforward

Yet

extensive

work

in

several

SG

centres

have

led

to

a

convergence

to

core

procedures

which

are

widely

applicable

and

often

sufficient

to

generate

purified

proteins

crystals

and

structures

-LRB-

Table

-RRB-

Where

the

core

protocol

fails

additional

steps

-LRB-

e.g.

further

purification

crystal

optimization

-RRB-

or

alternative

methods

-LRB-

e.g.

different

cloning

vectors

-RRB-

are

applied

Table

protocols

employed

at

the

Source

of

Sequence-verified

cDNA

clone

collections

Synthetic

DNA

RT-PCR

site-directed

mutagenesis

Genomic

-LRB-

microbial

-RRB-

CloningLigation-independent

cloning.Recombinase-based

cloning

-LRB-

e.g.

Gateway

InFusion

-RRB-

Expression

vectors

and

promoters

controlled

by

Lac

repressor.Bacterial

vectorsN-terminal

hexahistidine

tag

cleavable

by

specific

proteases

-LRB-

TEV

Thrombin

-RRB-

Host

strains

based

on

-LRB-

-RRB-

often

expressing

rare-codon

tRNAs

or

chaperone

proteins

Eukaryotic

expressionBacoluvirus-infected

insect

cells

Protein

expressionRich

media

grow

at

°C

to

mid-log

then

induce

at

low

temperature

with

IPTG.OR

Similar

protocol

using

minimal

medium

for

Selenomethionine

or

isotopic

labelling

PurificationTwo-step

purification

Affinity

chromatography

Gel

filtration

all

in

high-salt

buffers

-LRB-

M

NaCl

-RRB-

Optional

tag

cleavage

and

Ligand

and

buffer

screeningThermal

denaturation

assays

are

used

to

screen

purified

proteins

against

small

molecules

and

several

buffer

compositions

to

identify

stabilizing

conditions

and

potential

ligands

CrystalliationInitial

coarse

screens

-LRB-

conditions

protein

concentrations

each

-RRB-

Vapour

diffusion

sitting

drops

imaged

by

robots

but

scoring

done

by

humans.Include

ligands

identified

from

screening

or

biochemical

knowledge

to

promote

crystallization.Follow-up

screens

and

crystal

optimization

Data

collection

and

structure

determinationManual

or

robotic

screening

of

crystals

for

diffraction

properties

data

collection

in

rotating

anode

or

synchrotron

sources.Phasing

Molecular

replacement

-LRB-

-RRB-

experimental

phasing

using

SeMet

derivatives

and

MIR

Several

features

of

this

protocol

have

been

optimized

to

capture

a

large

portion

of

target

proteins

Gene

clones

have

been

predominantly

obtained

from

public

and

commercial

cDNA

libraries

However

gene

synthesis

may

become

the

method

of

choice

allowing

to

optimize

codon

frequency

restriction

sites

and

mRNA

structure

and

to

introduce

site-directed

mutations

Ligation-independent

cloning

is

a

generic

high-throughput

process

that

can

be

uniformly

applied

regardless

of

the

target

gene

or

the

cloning

vector

Short

N-terminal

fusion

tags

including

a

hexahistidine

sequence

and

a

specific

protease

cleavage

site

are

almost

universally

used

It

has

been

widely

documented

that

larger

fusion

tags

-LRB-

e.g.

GST

thioredoxin

MBP

-RRB-

can

enhance

solubility

of

proteins

that

are

not

soluble

when

expressed

with

a

short

peptide

tag

However

such

fusion

proteins

have

not

been

widely

used

in

the

SGC

since

removal

of

the

tag

often

leads

to

loss

of

solubility

The

standard

purification

protocol

is

designed

to

be

widely

applicable

and

experience

has

shown

that

it

results

in

effective

purification

of

a

large

fraction

of

proteins

solubly

expressed

in

E.

coli

A

protein

presented

for

crystallization

must

be

homogeneous

in

composition

post-translational

modification

and

oligomeric

state

the

presence

of

protein

aggregates

may

be

especially

detrimental

to

subsequent

crystallization

Affinity

purification

of

highly-expressed

proteins

eliminates

most

other

proteins

while

gel

filtration

effectively

separates

different

oligomeric

forms

of

the

protein

and

removes

protein

aggregates

which

may

otherwise

promote

irreversible

aggregation

of

the

protein

preparation

The

use

of

high

salt

concentration

-LRB-

typically

M

NaCl

-RRB-

throughout

the

purification

process

seems

to

reduce

protein

aggregation

and

non-specific

binding

of

protein

contaminants

Tag

cleavage

followed

by

another

passage

through

the

affinity

column

provides

a

further

generic

and

highly

effective

purification

step

which

removes

other

proteins

that

bind

adventitiously

to

the

first

affinity

column

The

generic

purification

procedure

has

provided

in

the

majority

of

cases

protein

of

sufficient

purity

to

achieve

crystallization

In

most

other

cases

the

generic

procedure

could

be

followed

by

polishing

and

protein

modification

steps

to

achieve

homogeneous

preparations

The

greatest

barrier

to

production

of

human

proteins

in

bacteria

is

recovery

of

soluble

protein

Less

than

of

protein

targets

yielded

detectable

levels

of

soluble

protein

when

tested

as

full-length

constructs

in

the

SGC

while

more

than

were

expressed

as

insoluble

aggregates

The

key

to

achieving

higher

success

rates

has

been

the

parallel

production

of

large

numbers

of

truncated

constructs

often

containing

a

compact

protein

domain

Construct

design

is

initially

based

on

domain

boundary

analysis

using

a

number

of

bioinformatic

tools

endpoints

are

designated

around

each

of

the

predicted

termini

of

the

domain

resulting

in

constructs

We

have

consistently

found

that

this

approach

results

in

a

increase

in

the

number

of

targets

that

can

be

produced

as

soluble

proteins

a

similar

impact

has

been

seen

on

the

production

of

diffracting

crystals

which

can

be

dramatically

affected

by

minute

changes

in

protein

termini

Although

not

rigorously

tested

it

is

presumed

that

a

protein

construct

that

is

inherently

well-behaved

-LRB-

little

tendency

to

aggregate

or

denature

-RRB-

will

be

less

dependent

on

specialized

conditions

for

expression

and

purification

and

may

crystallize

in

a

wider

range

of

conditions

Crystallization

crystal

screening

and

data

collection

For

successful

crystallization

of

a

given

target

the

SGC

's

phase

I

operation

appears

to

have

confirmed

that

the

most

important

driver

for

success

is

to

explore

protein

diversity

at

the

crystallization

stage

One

major

form

of

variation

was

discussed

above

namely

testing

multiple

constructs

of

the

target

Equally

effective

has

been

setting

up

co-crystallization

with

multiple

ligands

along

with

varying

protein

concentration

in

the

primary

crystallization

screens

At

the

same

time

it

appears

not

to

be

vital

to

explore

chemical

space

extensively

for

any

given

protein

preparation

instead

the

primary

goal

of

the

initial

-LRB-

coarse

-RRB-

screen

can

be

to

identify

which

preparations

are

crystallizable

and

a

limited

set

of

coarse

screen

conditions

-LRB-

-RRB-

generally

seems

sufficient

Practically

this

requires

only

two

crystallization

plates

and

by

setting

up

three

drops

per

condition

at

different

protein-well

ratios

-LRB-

in

Greiner

plates

-RRB-

the

protein

concentration

is

simultaneously

varied

The

conditions

themselves

are

derived

from

those

found

to

be

most

successful

in

other

high-throughput

initiatives

-LSB-

-RSB-

although

according

to

this

crystallizability

philosophy

the

exact

composition

is

probably

not

important

Naturally

coarse

screens

do

not

always

yield

high-quality

crystals

that

can

produce

a

dataset

however

the

SGC

operation

does

not

rely

on

these

crystals

showing

up

in

coarse

screens

and

a

good

optimization

infrastructure

is

in

place

In

practice

this

diversity

exploration

leads

to

large

numbers

of

parallel

crystallization

experiments

presenting

a

logistical

challenge

which

at

this

scale

can

only

be

met

with

an

efficient

robotics

and

IT

infrastructure

For

the

automation

the

SGC

has

been

able

to

exploit

the

devices

developed

on

the

back

of

the

first

wave

of

structural

genomics

initiatives

and

our

investment

has

been

less

in

developing

the

machines

than

in

integrating

them

and

implementing

experimental

best

practices

Particular

examples

by

minimizing

sample

requirements

with

nanolitre

crystallization

the

available

protein

can

be

used

in

more

experiments

The

large

numbers

of

drops

thereby

produced

-LRB-

million/year

-RRB-

would

be

practically

impossible

to

view

by

eye

under

the

microscope

whereas

automatic

drop

imaging

on

a

fixed

schedule

allows

images

to

be

reviewed

at

leisure

at

the

desk

Automation

has

also

played

an

important

role

in

crystal

characterization

An

automatic

sample

changer

has

been

used

for

initial

characterization

of

diffraction

quality

of

a

vast

number

of

crystals

This

allows

to

rank

the

crystals

for

more

careful

data

collection

especially

at

the

synchrotron

and

to

direct

further

efforts

at

crystal

optimization

A

significant

saver

of

upstream

efforts

has

been

to

exploit

each

crystal

's

diffraction

as

efficiently

as

possible

even

those

traditionally

considered

to

be

marginal

or

problematic

Marginal

diffractors

would

include

crystals

that

are

very

small

-LRB-

μm

in

longest

dimension

-RRB-

twinned

or

have

streaky

or

anisotropic

diffraction

The

latter

cases

generally

require

the

undivided

attention

of

experienced

crystallographers

Small

crystals

require

an

excellent

X-ray

beam

the

PXII

beamline

of

the

Swiss

Light

Source

synchrotron

provides

a

beam

which

is

reliably

small

but

also

well-aligned

and

very

stable

Most

efficient

use

of

the

beamline

relied

on

pre-screening

all

crystals

at

the

laboratory

source

for

thorough

work

prioritization

real-time

data

processing

during

data

collection

and

close

attention

to

radiation

damage

of

crystals

It

has

been

crucial

to

have

experienced

crystallographers

on

site

Adherence

to

these

good

practices

has

been

highly

productive

of

datasets

collected

on

trips

to

SLS

were

used

for

final

structures

while

of

all

depositions

relied

on

synchrotron

data

The

ability

to

extract

useful

data

from

marginal

crystals

has

been

especially

productive

in

combination

with

the

protein/ligand

diversity

approach

of

the

SGC

as

a

significant

fraction

of

structures

-LRB-

-RRB-

could

be

derived

from

crystals

emerging

from

the

primary

screens

saving

the

need

for

further

optimization

Phasing

and

structure

solution

Due

to

the

family-based

approach

for

most

SGC

targets

a

homologous

structure

is

already

known

and

most

structures

-LRB-

-RRB-

can

be

phased

by

molecular

replacement

-LRB-

MR

-RRB-

While

this

saves

significant

experimental

efforts

upstream

compared

to

experimental

phasing

by

eliminating

the

need

for

selenomethionine-derived

protein

or

heavy

atom

soaks

we

find

this

does

not

actually

save

time

overall

because

starting

phases

from

MR

are

heavily

phase

biased

Removing

the

bias

has

required

many

iterations

of

careful

and

incremental

model

building

and

refinement

by

experienced

crystallographers

who

can

see

the

danger

signs

of

a

poorly-refined

model

and

know

how

to

deal

with

it

-LSB-

-RSB-

The

final

step

namely

finalizing

and

depositing

the

model

is

in

fact

a

frequent

stalling

point

not

only

in

high-throughput

contexts

The

reason

is

that

the

final

model

is

not

merely

a

result

that

can

be

trivially

read

off

a

few

measurements

but

instead

is

an

interpretation

of

often

rather

noisy

data

with

a

lot

of

detail

that

is

easy

to

miss

where

individual

errors

influence

the

clarity

in

all

areas

Moreover

poor

model

definition

affects

biologically

interesting

parts

of

a

structure

and

interpreting

it

becomes

a

matter

of

judgment

and

using

in

orthogonal

information

Indeed

the

final

model

is

as

much

scientific

hypothesis

as

result

and

depositing

the

model

means

signing

off

on

the

hypothesis

which

is

why

it

has

traditionally

been

a

bottleneck

in

structural

genomics

efforts

The

SGC

has

used

a

peer

proofreading

system

combined

with

strict

timelines

to

counteract

the

problem

before

deposition

the

structure

is

reviewed

by

another

crystallographer

for

errors

or

alternative

interpretations

and

comments

passed

back

to

the

original

refiner

The

intention

is

threefold

First

to

introduce

quality

control

on

the

final

output

Second

the

refiner

does

not

feel

compelled

to

spend

excessive

time

on

the

model

to

flush

out

the

final

errors

since

she

knows

it

will

be

checked

Third

by

mixing

up

refiners

and

proofreaders

over

time

this

should

lead

to

common

interpretations

of

marginal

modeling

decisions

The

timelines

depend

on

situation

and

difficulty

but

typically

allow

two

weeks

for

refinement

a

day

for

proofreading

and

two

further

days

for

deposition

This

approach

has

made

it

possible

to

deposit

novel

structures

at

a

considerable

rate

-LRB-

each

month

from

a

team

of

dedicated

and

occasional

crystallographers

-RRB-

without

compromising

quality

Information

infrastructure

An

efficient

laboratory

information

management

system

-LRB-

LIMS

-RRB-

has

been

vital

to

manage

not

only

target

tracking

but

also

capturing

and

integrating

where

possible

information

generated

from

robotics

as

well

as

capturing

human

assessments

of

experimental

outcomes

where

these

could

be

entered

via

a

client

-LRB-

e.g.

scoring

of

crystallization

images

-RRB-

Fortuitously

the

solution

we

settled

on

BeeHive

from

Molsoft

-LRB-

-RRB-

is

in

essence

an

extremely

intuitive

database

query

tool

that

enables

even

inexperienced

users

to

extract

information

relevant

to

their

current

work

including

the

simplification

of

data

entry

This

is

a

weak

point

of

many

LIMS

solutions

whose

focus

often

evolves

around

data

entry

but

have

very

inflexible

retrieval

mechanisms

This

has

proved

to

be

a

powerful

means

of

communication

between

all

persons

involved

in

a

project

allowing

immediate

and

error-free

retrieval

of

hard

information

-LRB-

e.g.

protein

sequence

ligand

and

buffer

conditions

and

project

history

-RRB-

as

well

as

evaluation

and

prioritization

of

crystals

and

of

concurrent

projects

Protein

characterization

and

ligand

screening

One

of

the

major

challenges

in

structural

genomics

is

identifying

the

function

and

evaluating

the

functional

integrity

of

the

proteins

Examining

the

physical

state

of

a

protein

by

methods

such

as

analytical

ultracentrifugation

chromatography

or

dynamic

light

scattering

is

valuable

in

assessing

the

prospects

for

crystallization

In

contrast

specific

activity

assays

need

to

be

tailored

for

each

protein

class

and

may

be

impractical

or

impossible

when

the

activity

of

the

protein

is

not

known

We

have

implemented

a

generic

screen

based

on

the

increase

in

thermal

stability

of

a

protein

upon

ligand

binding

The

fluorescent

readout

is

based

on

monitoring

of

protein

unfolding

using

a

hydrophobicity-sensing

dye

Differential

Scanning

Fluorimetry

-LRB-

DSF

-RRB-

assays

-LSB-

-RSB-

are

ideal

for

screening

a

large

number

of

compounds

for

binding

to

each

target

protein

Significantly

the

shift

in

Tm

-LRB-

the

unfolding

transition

midpoint

-RRB-

measured

by

this

method

is

comparable

to

measurements

obtained

by

differential

scanning

calorimetry

-LRB-

DSC

-RRB-

the

well-established

standard

method

for

thermal

shift

measurements

In

selected

cases

a

direct

correlation

between

Tm

shift

and

binding

constants

has

been

observed

-LSB-

-RSB-

Several

advantages

have

been

derived

from

this

capability

First

the

identification

of

relatively

strong

interacting

molecules

out

of

several

hundreds

of

candidates

As

detailed

below

the

compounds

discovered

in

this

manner

are

then

included

in

crystallization

experiments

in

many

cases

only

protein

ligand

complexes

yielded

diffracting

crystals

Secondly

the

reactivity

profiles

provide

data

on

binding

selectivity

of

the

protein

active

site

which

is

the

most

crucial

information

for

drug

design

we

have

often

followed

up

the

results

from

ligand

screens

by

analyzing

the

structures

of

several

protein

ligand

complexes

In

parallel

the

properties

of

the

protein

ligand

interactions

are

studied

by

biophysical

methods

and

by

enzyme

inhibition

studies

Third

such

screens

have

allowed

us

to

identify

ligands

or

substrates

of

proteins

with

unknown

function

-LRB-

sometimes

termed

de-orphanizing

-RRB-

Finally

DSF-based

screens

can

be

expanded

to

explore

other

conditions

such

as

buffer

composition

that

enhance

the

stability

of

a

protein

These

conditions

may

then

be

introduced

to

improve

the

outcome

of

protein

purification

and

crystallization

-LSB-

-RSB-

The

limited

scale

of

protein

production

and

other

limitations

on

resources

do

not

allow

a

full-scale

screen

as

done

in

the

pharmaceutical

industry

-LRB-

compounds

-RRB-

Rather

we

have

assembled

smaller

family-specific

compound

libraries

-LRB-

compounds

each

-RRB-

which

can

reasonably

be

tested

against

available

amounts

of

protein

-LRB-

μg

for

assays

-RRB-

The

compound

libraries

are

based

on

the

scientific

and

patent

literature

the

chemical

structure

of

prospective

compounds

is

used

to

search

an

in-house

compilation

of

vendor

databases

to

identify

potential

sources

Acquisition

of

desired

compounds

is

not

trivial

not

all

published

compounds

even

those

appearing

in

vendor

catalogues

are

actually

available

when

required

alternative

vendors

or

collaborative

sources

may

then

be

accessed

With

continuous

updating

based

on

current

literature

and

our

own

experimental

results

these

libraries

have

allowed

to

derive

binding

profiles

and

new

insights

on

ligand

specificity

SGC

target

and

biology

area

selection

relevance

for

the

treatment

of

human

diseases

For

any

structural

genomic

organisation

target

selection

is

an

important

consideration

as

it

can

have

a

major

impact

on

the

procedures

that

are

implemented

during

the

process

of

structure

determination

There

are

a

number

of

approaches

applied

by

different

structural

genomics

projects

to

select

targets

for

structural

analysis

such

as

blanket

coverage

of

an

organism

's

genome

targets

with

potential

novel

folds

percentage

cut

off

based

on

sequence

identity

or

total

coverage

of

selected

protein

families

The

SGC

has

opted

for

the

family-based

approach

with

an

emphasis

on

protein

families

whose

members

are

important

in

human

health

disease

and

are

potentially

druggable

From

our

point

of

view

the

main

advantages

of

this

approach

are

Firstly

the

methods

and

procedures

identified

for

one

family

member

can

be

applied

to

another

family

member

improving

everything

from

expression

solubility

stability

and

purification

to

crystallisation

and

structure

determination

Secondly

analysis

of

the

structures

from

all

family

members

can

reveal

additional

significant

information

such

as

ligand

binding

site

specificity

conformational

dynamics

understanding

of

aberrant

behaviour

of

specific

family

members

or

the

converse

revealing

common

structural

properties

within

all

family

members

The

availability

of

high

resolution

structures

constitutes

the

foundation

for

structure-guided

drug

discovery

projects

In

recent

years

SG

has

significantly

increased

the

number

of

human

protein

structures

available

for

structure-based

design

projects

-LSB-

-RSB-

In

particular

protein

family

focused

efforts

originating

from

high-throughput

structural

biology

projects

have

contributed

to

the

structural

description

of

a

number

of

members

from

human

protein

families

and

thus

provided

valuable

structural

and

chemical

information

for

the

design

of

bioactive

compounds

In

addition

established

expression

and

crystallization

conditions

have

been

used

to

generate

essential

reagents

methodologies

and

technologies

which

have

facilitated

research

projects

in

academia

and

drug

discovery

programs

in

industry

The

SGC

has

focused

on

providing

protein

structures

to

support

drug

development

and

understanding

of

the

structural

determinants

for

human

disease

Of

unique

targets

deposited

by

the

SGC

-LRB-

in

phase

-RRB-

clear

disease

relevance

has

been

established

for

and

a

further

are

likely

to

be

involved

in

at

least

one

disease

This

pattern

holds

true

for

all

the

human

protein

families

the

SGC

is

working

on

The

following

sections

provide

an

overview

of

the

three

distinct

biological

areas

selected

at

the

Oxford

site

of

the

SGC

Biology

area

I

Structural

Genomics

of

human

metabolic

enzymes

Selection

of

metabolic

enzymes

as

biological

target

area

at

the

SGC

was

based

on

two

distinct

features

they

are

fundamentally

involved

in

a

multitude

of

human

diseases

including

cardiovascular

metabolic

diseases

or

cancer

and

in

addition

several

enzymes

constitute

possible

drug

targets

Emphasis

has

been

given

to

certain

metabolic

enzyme

families

such

as

oxidoreductases

-LRB-

mostly

short-chain

dehydrogenases/reductases

-LRB-

SDR

-RRB-

medium-chain

dehydrogenases/reductases

-LRB-

MDR

-RRB-

long-chain

dehydrogenases/reductases

aldehyde

dehydrogenases

-LRB-

ALDH

-RRB-

aldo

keto

reductases

-LRB-

AKR

-RRB-

and

oxoglutarate

dependent

oxygenases

-LRB-

-RRB-

In

addition

pathways

of

importance

e.g.

in

lipid

or

amino

acid

metabolism

were

selected

with

a

distribution

of

about

between

oxidoreductases

and

other

metabolic

enzymes

The

target

list

comprises

about

metabolic

enzymes

and

after

three

years

of

operation

unique

novel

structures

have

been

solved

Three

points

of

importance

are

highlighted

in

this

review

structural

characterization

of

enzymes

shown

to

be

causative

of

metabolic

inherited

diseases

structure

determination

of

drug

discovery

targets

in

metabolic

diseases

such

as

metabolic

syndrome

or

osteoporosis

and

structure-guided

de-orphanization

of

insufficiently

characterized

human

gene

products

or

even

entire

pathways

Structural

basis

of

inherited

metabolic

diseases

Genetic

defects

in

enzymes

involved

in

metabolic

pathways

such

as

amino

acid

or

lipid

catabolism

are

causative

of

a

whole

spectrum

of

symptoms

including

dysmorphologies

mental

retardation

neuropathies

or

life

threatening

situations

like

fasting

induced

hypoglycemia

-LSB-

-RSB-

Understanding

of

molecular

causes

and

possible

interventions

of

inherited

metabolic

diseases

requires

besides

biochemical

and

clinical

management

a

structural

template

for

explanation

of

mutational

effects

Thus

far

the

focus

has

been

to

a

large

extent

on

oxidoreductases

in

the

area

of

metabolic

diseases

Associated

disorders

comprise

electron

transfer

reactions

for

energy

production

-LRB-

e.g.

mitochondrial

myopathies

-RRB-

oxidative

and

reductive

roles

in

the

metabolism

of

amino

acids

-LRB-

e.g.

hyperprolinemia

or

branched-chain

hydroxyacyl

CoA

dehydrogenase

defects

-RRB-

fatty

acids

-LRB-

e.g.

inborn

errors

in

α

and

β-oxidation

of

short

medium

or

long-chain

fatty

acid

metabolites

-RRB-

cofactors

-LRB-

e.g.

phenylketonuria

type

-RRB-

hormones

-LRB-

e.g.

male

pseudohermaphroditism

or

adrenal

hyperplasia

-RRB-

mediators

-LRB-

e.g.

congestive

heart

failure

-RRB-

and

lipids

-LRB-

e.g.

inborn

errors

in

cholesterol

synthesis

CHILD

syndrome

Smitz-Opitz

Laemmli

syndrome

as

examples

-RRB-

The

impact

of

the

structural

approach

is

illustrated

by

the

successful

structure

determination

of

phytanoyl-CoA

hydroxylase

-LSB-

-RSB-

the

major

molecular

cause

of

Refsum

disease

a

peroxisomal

disorder

with

severe

neurological

symptoms

The

structure

provides

a

framework

to

interpret

the

majority

of

the

disease

causing

polymorphic

alleles

and

we

were

able

to

map

those

to

changes

in

the

active

site

around

the

and

binding

sites

in

this

enzyme

-LSB-

-RSB-

Metabolic

enzymes

as

drug

targets

Oxidoreductions

at

specific

positions

of

lipid

hormones

such

as

steroids

selectively

alter

nuclear

receptor

binding

properties

Therefore

inhibition

of

dehydrogenases/reductases

carrying

out

these

reactions

selectively

influences

cellular

hormone

levels

and

transcriptional

responses

This

concept

has

recently

found

great

attention

with

the

development

of

specific

inhibitors

against

dehydrogenase

type

-LRB-

-RRB-

as

a

novel

drug

target

in

diabetes

and

obesity

-LSB-

-RSB-

Similar

drug

development

efforts

are

underway

regulating

androgen

or

estrogen

levels

through

specific

modulation

of

distinct

hydroxysteroid

dehydrogenases

-LRB-

and

in

cancer

inflammation

osteoporosis

ageing

and

autoimmune

diseases

-RRB-

We

determined

the

structure

of

human

in

complex

with

a

clinically

relevant

inhibitor

carbenoxolone

-LRB-

Wu

et

al.

unpublished

-RRB-

and

have

provided

a

platform

for

drug

development

efforts

Other

hydroxysteroid

dehydrogenase

structures

comprise

such

as

types

and

a

novel

type

-LRB-

see

below

-RRB-

necessary

for

determination

of

off-target

activities

of

compounds

directed

against

type

and

Other

targets

of

pharmaceutical

relevance

successfully

pursued

are

farnesyl

diphosphate

synthase

-LRB-

FDPS

-RRB-

and

geranylgeranyl

diphosphate

synthase

-LRB-

GGPS

-RRB-

which

are

critical

in

synthesis

of

isoprenoids

necessary

for

covalent

modification

of

GTPases

involved

in

cell

signalling

and

survival

Crystal

structures

of

FDPS

complexed

with

nitrogen-containing

bisphosphonates

currently

used

for

osteoporosis

therapy

allowed

a

molecular

mechanism

of

action

to

be

postulated

for

these

drugs

-LSB-

-RSB-

-LRB-

Fig.

-RRB-

Furthermore

several

prokaryotic

and

parasitic

dehydrogenases

have

been

identified

as

novel

targets

for

antibiotic

and

antiparasite

drug

development

and

thus

allow

synchronization

with

the

SGC

Toronto

efforts

where

an

apicomplexan/protozoan

SG

program

has

been

established

Thus

structure

determination

of

related

human

enzymes

will

facilitate

structure

aided

drug

design

and

allow

virtual

and

focused

screening

efforts

in

this

emerging

disease

area

Fig.

binding

to

human

farnesyl

diphosphate

synthase

Electron

density

is

shown

in

green

around

the

clinically

used

inhibitor

risedronate

Deorphanization

of

metabolic

enzymes

and

pathways

A

significant

proportion

of

the

metabolic

enzymes

targeted

were

at

the

time

of

structure

determination

devoid

of

assigned

activity

or

function

High

throughput

protein

production

structure

determination

and

functional

characterization

allowed

deorphanization

of

unknown

enzymes

We

employed

ligand

screening

enzyme

activity

assays

expression

and

subcellular

localization

data

as

well

as

structure

determination

combined

with

docking

analysis

to

describe

novel

human

enzymes

In

the

absence

of

co-crystal

structures

interpretation

of

results

from

biochemical

assays

and

compound

screening

was

rationalized

by

in

silico

docking

of

potential

ligands

into

the

active

site

of

the

orphan

structures

Analysis

of

the

different

docking

poses

was

correlated

with

experimental

results

allowing

direct

visualization

of

the

putative

protein

ligand

complex

In

this

manner

we

determined

a

novel

-LSB-

-RSB-

possibly

involved

in

cancer

and

a

novel

R-hydroxybutyrate

dehydrogenase

involved

in

ketone

body

utilization

-LSB-

-RSB-

Further

emphasis

was

given

on

novel

pathways

such

as

mitochondrial

fatty

acid

synthesis

This

recently

discovered

pathway

is

important

in

the

synthesis

of

lipoic

acid

essential

for

mitochondrial

function

Thus

far

we

have

determined

three

distinct

enzymes

of

this

metabolic

route

namely

the

malonyl

transferase

-LRB-

-RRB-

ketoacyl

synthase

-LRB-

-RRB-

and

the

enoyl-ACP

reductase

-LRB-

-RRB-

These

structures

represent

the

only

higher

eukaryotic

structures

thus

far

available

for

this

pathway

The

data

will

be

instrumental

to

compare

to

the

multidomain

type

I

fatty

acid

synthase

where

we

recently

solved

the

structure

of

the

malonyl/acyl

transferase

domain

-LRB-

-RRB-

This

cytosolic

enzyme

is

involved

in

production

of

endogenous

fatty

acids

and

lipids

and

is

discussed

as

potential

target

in

metabolic

diseases

and

cancer

Biology

area

II

Structural

Genomics

of

transmembrane

receptor

signalling

pathways

Complete

coverage

of

protein

family

A

human

protein

family

that

the

SGC

has

completed

the

structure

determination

of

all

members

is

the

family

This

family

consists

of

seven

members

-LRB-

β

ε

η

γ

σ

τ

and

ζ

-RRB-

of

which

σ

-LSB-

-RSB-

τ

-LSB-

-RSB-

and

ζ

-LSB-

-RSB-

structures

were

previously

determined

This

protein

family

plays

a

central

role

in

many

fundamental

cellular

roles

such

as

cell

cycle

control

apotosis

protein

trafficking

signal

transduction

and

stress

response

-LSB-

-RSB-

Before

the

structural

completion

of

the

family

most

of

the

structural

studies

utilised

which

provided

details

of

the

conserved

peptide

binding

site

-LSB-

-RSB-

the

primary

peptide

interaction

-LSB-

-RSB-

and

secondary

target

domain

interactions

-LSB-

-RSB-

As

all

of

these

structures

displayed

similar

overall

conformations

structurally

it

was

proposed

that

behaved

as

molecular

anvils

in

that

their

overall

structure

remained

unchanged

whether

in

the

apo-form

or

bound

to

their

target

protein

-LSB-

-RSB-

The

structure

determination

of

the

remaining

members

allowed

for

a

family-wide

comparative

study

that

revealed

another

story

with

a

major

emphasis

on

the

flexibility

of

-LSB-

-RSB-

This

was

most

obviously

with

the

apo-form

of

in

which

one

of

the

subunits

was

in

a

similar

conformation

to

all

other

structure

while

the

opposing

monomer

displayed

a

more

open

conformation

for

the

peptide

binding

groove

-LRB-

Fig.

-RRB-

Fig.

flexibility

of

the

is

illustrated

by

the

superimposition

of

-LRB-

blue

-RRB-

with

-LRB-

orange

-RRB-

The

monomer

conformations

of

both

isoforms

are

essentially

identical

on

the

left

hand

side

However

the

beta

monomer

on

the

right

side

has

a

more

open

peptide

binding

groove

and

flexibility

at

the

dimeric

interface

Additional

flexibility

of

proteins

was

observed

when

all

of

the

family

members

were

superimposed

against

one

subunit

It

became

instantly

clear

that

the

position

of

the

second

subunit

varied

between

the

different

isoforms

-LSB-

-RSB-

This

is

achieved

through

the

N-terminal

helices

that

make

up

the

dimeric

interface

sliding

over

one

another

-LRB-

Fig.

-RRB-

The

significance

of

the

interface

flexibility

is

that

it

allows

for

the

widening

or

shortening

of

the

distance

between

the

two

peptide

binding

grooves

hence

allowing

a

to

accommodate

structures

of

varying

shapes

and

sizes

As

are

known

to

have

bind

hundreds

of

partners

-LSB-

-RSB-

this

interface

flexibility

would

provide

the

necessary

structural

adaptability

to

accommodate

the

wide

structural

range

of

target

proteins

As

all

of

the

human

structures

are

now

known

they

allow

for

a

detailed

bioinformatic

analysis

of

the

family

This

approach

identified

common

protein

protein

interaction

patches

at

the

subunit

interfaces

plus

two

additional

non-specific

protein

interaction

sites

that

would

attract

and

bind

the

globular

structured

regions

of

the

target

protein

thus

providing

a

mechanism

by

which

the

can

initially

attract

and

then

bind

a

wide

range

of

structurally

diverse

target

proteins

-LSB-

-RSB-

Another

more

numerous

protein

protein

interaction

family

that

was

targeted

by

the

SGC

are

the

PDZ

domains

which

have

been

implicated

in

the

regulation

of

drug

transporters

-LSB-

-RSB-

and

involved

in

the

clustering

targeting

and

localisation

of

the

target

proteins

-LSB-

-RSB-

These

domains

bind

mostly

to

C-terminal

peptides

that

fall

into

two

classes

class

I

peptides

are

-LRB-

Ser/Thr

-RRB-

X

Φ

COO

while

class

II

peptides

are

Φ

X

Φ

COO

where

X

represents

any

amino

acid

and

Φ

represents

any

hydrophobic

residue

-LSB-

-RSB-

PDZ

domains

Initial

attempts

at

structure

determination

of

unique

human

PDZ

domains

resulted

in

a

successful

outcome

for

only

of

these

targets

To

improve

our

success

rate

we

took

advantage

of

the

family

based

approach

and

generated

new

expression

clones

of

the

remaining

targets

with

generic

class

I

and

II

PDZ

binding

peptides

attached

to

the

C-terminus

of

each

domain

The

idea

was

for

these

peptides

to

bind

adjacent

PDZ

domains

initiating

protein

protein

interactions

and

thus

crystal

nucleation

As

such

the

linker

between

the

predicted

end

of

the

PDZ

domain

and

the

C-terminal

peptide

was

varied

from

to

amino

acids

allowing

for

flexibility

but

restraining

the

distance

between

adjacent

domains

-LSB-

-RSB-

Using

this

approach

we

have

now

solved

of

the

remaining

targets

many

of

which

have

thrown

up

new

details

regarding

peptide

selectivity

and

structural

adaptability

of

the

PDZ

domain

when

bound

with

a

peptide

As

expected

for

most

of

these

domains

the

peptide

interaction

was

similar

to

the

standard

configuration

-LSB-

-RSB-

in

that

the

side-chain

of

the

C-terminal

hydrophobic

residue

-LRB-

position

-RRB-

was

bound

in

a

conserved

hydrophobic

pocket

and

that

the

peptide

's

position

Ser/Thr

coordinates

the

His

side

chain

from

the

αB

helix

However

there

were

a

number

of

surprises

of

which

the

biggest

was

for

MPDZ

in

which

a

class

II

mode

of

binding

was

observed

for

a

class

I

peptide

which

involved

a

translation

of

the

αB

helix

-LRB-

Fig.

of

-LSB-

-RSB-

-RRB-

Biology

area

III

Structural

Genomics

of

human

protein

kinases

Kinases

play

an

essential

role

in

most

-LRB-

if

not

all

-RRB-

signalling

pathways

and

dysregulation

has

often

been

linked

to

disease

Several

successful

inhibitors

developed

to

target

kinases

have

shown

that

members

of

this

large

protein

family

are

excellent

targets

for

the

development

of

drugs

Currently

protein

kinases

constitute

about

of

presently

pursued

drug

targets

in

industry

-LSB-

-RSB-

There

are

identified

human

protein

kinases

constituting

of

all

human

genes

which

have

been

grouped

into

families

-LSB-

-RSB-

Despite

the

large

number

of

members

and

their

involvement

in

large

variety

of

pathways

evidence

points

to

a

common

single

ancestral

protein

As

a

result

the

structural

features

as

well

as

key

regulatory

elements

and

catalytic

mechanism

of

phosphate

transfer

are

all

well

conserved

High

resolution

structures

are

therefore

essential

for

the

rational

design

of

potent

and

selective

inhibitors

Before

the

contribution

of

SG

efforts

the

progression

of

publicly

available

kinase

structures

was

linear

with

only

human

kinase

structures

publicly

available

in

-LSB-

-RSB-

Currently

novel

human

kinases

structures

have

been

released

by

the

SGC

-LRB-

from

Oxford

-RRB-

which

started

to

target

this

protein

class

in

This

increased

the

number

of

unique

human

kinase

catalytic

domain

structures

available

in

the

pdb

-LRB-

-RRB-

to

by

the

end

of

Many

structures

released

by

SG

were

only

distantly

related

catalytic

domain

structures

previously

known

and

in

some

cases

provided

the

first

structural

information

for

a

subfamily

Thus

these

structures

significantly

enriched

the

coverage

of

the

three

dimensional

structure

description

of

the

kinome

Among

the

structures

where

the

SGC

determined

the

first

representative

structure

of

a

family

were

the

NEK

-LRB-

never

in

mitosis

NIMA

-RRB-

family

member

the

like

kinases

family

member

and

as

well

as

the

first

structure

of

a

NAK

-LRB-

Numb-associated

kinases

-RRB-

kinase

These

kinases

are

quite

diverse

in

terms

of

primary

structure

and

it

is

therefore

not

surprising

that

many

novel

structural

features

have

been

discovered

For

instance

a

novel

activation

loop

architecture

characterized

by

a

large

helical

insert

has

been

discovered

in

the

structure

of

the

structures

of

and

revealed

a

family

conserved

antiparallel

beta

sheet

flanking

the

kinase

hinge

region

and

the

structure

of

identified

a

short

helix

following

the

activation

segment

DFG

motif

that

may

be

explored

for

the

development

of

specific

inhibitors

-LSB-

-RSB-

Kinases

are

extremely

flexible

proteins

that

may

adopt

a

number

of

distinct

catalytically

active

or

inactive

conformations

during

their

catalytic

cycle

upon

activation

by

phosphorylation

or

by

binding

of

a

regulatory

protein

and

consequently

a

number

of

clinically

successful

inhibitors

have

been

developed

to

target

specifically

the

inactive

state

of

kinases

-LSB-

-RSB-

For

example

the

anti-leukaemia

drug

Imatinib

binds

selectively

to

the

inactive

state

cABL

characterized

by

an

outward

conformation

of

the

DFG

motif

a

conserved

tripeptide

motif

that

ligates

ions

-LSB-

-RSB-

It

is

not

clear

to

date

how

many

kinases

are

able

to

adopt

this

conformation

which

makes

development

of

these

so-called

type

II

inhibitors

possible

In

general

these

are

characterized

by

largely

improved

specificity

For

the

development

of

conventional

inhibitors

that

target

the

active

state

of

kinases

information

about

the

plasticity

of

the

catalytic

domain

greatly

facilitates

the

rational

design

of

inhibitors

Consequently

it

is

desirable

that

several

structures

of

the

same

target

in

complex

with

different

ligands

are

available

Also

here

the

structural

information

content

regarding

ligand

binding

was

significantly

increased

during

the

last

three

years

by

SG

In

only

human

kinases

had

a

structure

available

in

the

public

domain

and

only

publicly

available

structures

contained

non-adenosine

chemotypes

-LSB-

-RSB-

From

the

structures

of

kinase

catalytic

domains

released

by

our

laboratory

were

determined

in

the

presence

of

an

non-adenosine

kinase

inhibitor

and

several

structures

were

determined

in

complex

with

more

than

one

inhibitor

scaffold

-LRB-

Table

and

Fig.

showing

apo/inhibitor

-RRB-

Table

kinase

structures

determined

by

SGCNamePDB

IDResolution

-LSB-

Å

-RSB-

Inhibitor

nameDisease

HymenialdisinePot

nonePot

Compound

52CK1CK1γ22C472.405-IodotubercidinGeneticCK1CK1γ3d2CHL1.95

Triazolodiamine

StaurosporineInflammation

InhibitorPot

BIM

I

Triazolodiamine

StaurosporinePot

Pyridone

GSK

inhibitor

also

deposited

as

an

apo-structure

in

two

different

spacegroups

with

different

inhibitors

and

substrate

BZI

SLK

also

deposited

as

phosphorylated

protein

pdb-code

Detailed

description

of

structures

of

targets

solved

in

Oxford

is

available

in

form

of

iSee

datapacks

freely

downloadable

on

with

different

inhibitors

formal

link

to

the

disease

has

not

been

established

so

far

but

is

likelyfCardiovascular

diseaseFig

of

-LRB-

cyan

-RRB-

and

the

purine

complex

-LRB-

orange

-RRB-

highlighting

the

decomposed

movements

of

the

glycine-rich

loop

-LRB-

flapping

-RRB-

and

the

αC

helix

-LRB-

swinging

-RRB-

-LSB-

-RSB-

In

addition

the

SGC

has

supported

development

of

entirely

new

inhibitor

classes

exemplified

by

co-crystal

structures

with

Ruthenium-half

sandwich

complexes

These

stable

organometallic

compounds

are

extremely

potent

inhibitors

for

kinases

-LSB-

-RSB-

The

co-crystal

structure

of

three

inhibitors

of

this

class

showed

that

the

inert

metal

centre

in

this

scaffold

functions

as

a

hypervalent

carbon

allowing

it

to

occupy

the

binding

pocket

efficiently

with

excellent

shape

complementarity

Contributions

of

NMR

to

Structural

Genomics

NMR

as

a

complementary

method

to

crystallography

for

protein

structure

determination

The

NMR

spectroscopy

can

play

an

important

role

in

structural

genomics

providing

complementary

information

to

that

obtained

from

X-ray

crystallography

Importantly

for

large-scale

structural

genomics

projects

NMR

provides

an

alternative

route

to

solving

the

high

resolution

three-dimensional

structures

of

proteins

that

prove

refractory

to

crystallization

We

were

able

to

use

NMR

to

solve

the

structures

of

a

number

of

relatively

small

protein

domains

-LRB-

kDa

-RRB-

in

which

the

domain

contained

at

least

one

flexible

region

The

RGS

domains

from

the

regulator

of

G-protein

signalling

proteins

were

all

very

good

examples

of

this

Multiple

constructs

of

these

were

designed

which

expressed

to

high

yield

in

stable

highly

soluble

form

yet

did

not

yield

high

quality

crystals

despite

many

months

of

concerted

effort

The

domains

were

therefore

expressed

as

uniformly

proteins

using

standard

growth

methods

in

E.

coli

and

their

spectra

were

recorded

to

assess

the

feasibility

of

structure

determination

by

NMR

In

all

cases

excellent

spectral

dispersion

was

observed

and

we

were

able

to

obtain

almost

complete

assignment

of

the

protein

resonances

We

have

since

deposited

the

high

resolution

NMR

structures

of

three

RGS

domains

in

the

PDB

and

the

resonance

assignments

of

four

RGS

domains

in

the

BioMagResBank

-LRB-

BMRB

-RRB-

The

structures

and

assignments

of

two

further

non-crystallizing

domains

-LRB-

domain

and

Bright/ARID

domain

-RRB-

have

also

been

deposited

and

those

of

several

other

non-crystallizing

domains

rescued

by

NMR

are

currently

underway

-LRB-

Table

-RRB-

Table

NMR

structures

and

assignmentsGenePDB

depositionResonance

assignment

NMR

as

an

assessment

tool

for

the

feasibility

of

structure

determination

Further

examples

where

NMR

has

proven

useful

as

a

rescue

strategy

include

particular

families

of

signalling

domains

which

have

a

known

tendency

to

be

partially

unfolded

in

their

unliganded

states

Some

examples

include

certain

WW

domain

-LSB-

-RSB-

We

successfully

identified

peptide

binding

partners

for

a

WW-tandem

construct

using

the

SPOTs

screening

technique

-LSB-

-RSB-

following

which

the

most

strongly

binding

peptides

were

synthesized

on

a

large

scale

for

NMR

measurements

Although

the

spectra

of

this

pair

of

tandem

domains

in

isolation

were

very

unhopeful

the

spectra

of

their

complexes

showed

significant

improvements

in

signal

dispersion

indicating

that

in

the

complexed

form

the

protein

was

better

folded

At

this

point

the

protein

entered

our

NMR

structure

determination

pipeline

The

recording

of

a

quick

spectrum

has

in

several

cases

allowed

us

to

rescue

protein

constructs

with

promising

but

borderline

behaviour

for

example

proteins

showing

good

signal

dispersion

but

low-medium

levels

of

aggregation

Far

from

abandoning

these

constructs

we

took

these

constructs

back

to

the

drawing

board

and

made

rational

construct

improvements

with

the

help

of

bio-informatic

tools

Successfully

re-designed

constructs

were

then

re-screened

for

fold

quality

by

After

iterations

of

this

procedure

it

was

often

possible

to

refine

promising

constructs

sufficiently

for

structure

determination

For

example

Fig.

shows

the

stepwise

improvement

in

the

spectral

properties

of

a

hopeful

though

initially

problematic

DNA-recognition

domain

from

the

oxygenase

protein

The

NMR

structure

is

now

deposited

-LRB-

PDB

code

-RRB-

In

all

of

the

above

cases

a

quick

showed

immediately

whether

the

structure

determination

of

a

protein

having

to

failed

to

crystallize

should

be

pursued

or

abandoned

hence

reducing

unnecessary

attrition

in

the

structure

determination

pipeline

Fig.

improvement

in

quality

of

spectra

over

two

rounds

of

iterative

construct

re-design

for

the

Bright/ARID

domain

The

leftmost

-LRB-

initial

-RRB-

construct

shows

potential

The

structure

of

the

final

construct

on

the

far

right

was

determined

by

NMR

-LRB-

PDB

code

-RRB-

The

study

of

protein

dynamics

by

NMR

The

use

of

NMR

to

study

the

rotational

correlation

times

and

internal

dynamics

of

the

proteins

offers

good

explanations

as

to

why

crystallization

sometimes

fails

even

for

well-folded

proteins

In

all

of

the

proteins

we

rescued

by

NMR

heteronuclear

NOE

and

relaxation

data

revealed

regions

of

internal

mobility

within

the

proteins

which

would

have

hindered

long-range

order

and

impaired

or

prevented

efficient

crystal

packing

A

striking

example

was

the

case

of

the

RGS

domain

from

in

which

NMR

relaxation

data

confirmed

true

local

mobility

in

a

region

of

the

domain

which

not

only

lacked

in

NMR

restraints

but

also

showed

no

electron

density

in

the

crystal

structure

of

the

complex

of

with

-LRB-

PDB

-RRB-

Comparison

of

mobility

in

RGS

domains

from

different

branches

of

the

phylogenetic

tree

leads

to

clues

about

their

specificity

and

helps

to

guide

further

investigations

In

some

cases

the

and

data

have

also

identified

partial

dimerization

in

proteins

that

fail

to

crystallize

thus

explaining

the

latter

NMR

relaxation

data

were

in

each

case

confirmed

by

analytical

ultracentrifugation

-LRB-

AUC

-RRB-

The

combined

information

allowed

us

to

decide

whether

these

proteins

should

be

highlighted

as

candidates

for

structure

determination

by

NMR

and

to

judge

the

best

conditions

under

which

they

should

be

studied

Future

and

outlook

The

future

role

that

NMR

will

play

in

structural

genomics

will

depend

heavily

on

the

continued

development

and

implementation

of

new

faster

methods

of

data

acquisition

processing

resonance

and

NOE-assignment

and

structure

determination

and

refinement

These

topics

have

been

covered

extensively

in

other

reviews

for

a

concise

summary

see

-LSB-

-RSB-

and

references

therein

The

potential

time

gains

that

could

be

gained

from

these

methods

make

high

throughput

structure

determination

by

NMR

a

realistic

possibility

for

the

future

Structural

bioinformatics

and

rationalisation

of

experimental

results

A

crystal

structure

of

a

protein

in

absence

of

ligand

or

substrate

may

not

always

provide

insight

on

reaction

mechinasms

or

specificity

Ideally

such

information

can

be

derived

from

additional

structures

with

bound

ligands

In

the

absence

of

such

co-crystals

interpretation

of

results

from

biochemical

assays

and

compound

screening

is

more

speculative

However

these

results

can

be

rationalised

with

in

silico

docking

of

potential

ligands

into

the

active

site

of

unliganded

protein

structures

An

example

illustrating

this

point

is

the

analysis

of

the

structure

-LSB-

-RSB-

Analysis

of

the

different

docking

poses

can

be

correlated

with

experimental

results

allowing

direct

visualisation

of

the

putative

protein

ligand

complex

With

these

results

further

modifications

of

the

enzyme

can

be

suggested

more

reliably

allowing

a

faster

progress

towards

the

complete

elucidation

of

the

mechanistics

Dissemination

of

structural

genomics

data

and

knowledge

Structural

genomics

produces

a

wealth

of

information

of

different

types

DNA

and

protein

seqeuences

biochemical

information

coordinates

of

crystal

structures

and

structural

annotation

This

information

is

deposited

in

one

or

more

public

databases

predominantly

the

PDB

in

addition

to

publication

in

journals

This

form

of

data

distribution

does

not

adequately

disseminate

the

full

information

to

a

wide

scientific

audience

The

first

issue

is

the

fragmentation

of

data

between

different

formats

A

user

may

have

to

read

text

information

in

a

journal

paper

which

may

include

a

few

two-dimensional

Figures

then

download

a

PDB

structure

file

and

image

with

a

separate

application

and

then

perform

analysis

and

alignment

of

data

from

say

SNP

database

using

alignment

software

The

second

issue

is

that

non-structural

biologists

do

not

routinely

access

PDB

files

especially

of

structures

that

were

not

published

in

pubmed-indexed

journals

We

have

approached

this

challenge

by

developing

a

new

intuitive

dissemination

concept

in

conjunction

with

Molsoft

LLC

-LRB-

San

Diego

CA

-RRB-

-LSB-

-RSB-

This

concept

-LRB-

which

we

denoted

iSee

-RRB-

integrates

all

the

information

associated

with

any

given

target

solved

by

SGC

into

a

small

self-contained

file

annotated

by

the

authors

-LRB-

Fig.

-RRB-

The

file

not

only

allows

the

direct

visualisation

of

text

information

but

also

offers

an

interactive

visualisation

feature

fully

integrated

to

the

structural

data

being

presented

At

any

stage

the

annotation

written

by

the

expert

can

be

coupled

with

an

interactive

molecular

graphics

scene

Transition

between

each

anotated

viewpoint

is

fully

animated

on-the-fly

to

convey

a

sense

of

three-dimensionality

which

is

vital

for

the

user

to

grasp

the

spatial

relationship

between

different

features

on

a

structure

Fig.

of

iSee

datapack

The

annotation

text

-LRB-

top

left

panel

-RRB-

includes

links

-LRB-

blue

text

-RRB-

which

lead

to

structural

images

focused

at

areas

of

interest

simultaneously

accessing

other

types

of

information

-LRB-

sequence

alignment

small

molecule

formulae

etc.

-RRB-

Each

of

these

files

-LRB-

called

an

iSee

datapack

-RRB-

as

well

as

the

software

needed

to

visualise

them

-LRB-

ICM-Browser

-RRB-

are

available

for

free

download

from

our

website

-LRB-

-RRB-

We

also

maintain

and

curate

each

of

these

files

by

revising

each

datapack

quarterly

to

ensure

that

all

the

recently

disclosed

information

is

added

-LRB-

either

by

ourselves

through

follow-up

experiments

or

by

external

collaborators

working

on

the

same

targets

-RRB-

Each

of

the

datapacks

has

a

built-in

automated

updating

function

that

can

be

executed

on

user

's

request

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

high-throughput
protein kinase
14-3-3
binding specificity
protein crystallography